Mr[1].Chen Application of Biological Pesticides

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同事推荐信英文

同事推荐信英文英文推荐信应该怎么写呢?在考试中，工作中经验要用到推荐信，以下是同事推荐信英文范文，请参考!英文版的工作推荐信【1】Dear Sir/Madam,It has always been a great pleasure to write to you since I can never fail to get help, inspiration or enlightenment from you.Now I am writing to recommend to you Miss Liu Mingwho wants to pursue her graduate study for the Masters Degree under your supervision.Miss Liu Ming was one of my classmates in Tianjin University.She was one of the most excellent students in our university as can be seen from her straight A grades on all subjects.After graduation she worked in Tianyi Biological Company for three years, which considerably added to her practical experience.It is my belief that with her intelligence, diligence and rich experience, Miss Liu Ming has great potential for further development in the field of pharmacology both theoretically and practically.Therefore, I recommend her to you with no reservations. your sincerely，XXXX英文推荐信范文【2】To whom it may Concern,I am writing this letter to attest to Pauls skills in language and public relations.In the four years I have known him, I have been consistently impressed with his ability not only to negotiate complex ideas in other languages, but also to relate these things in a personable, conscientious fashion.His manner in these cases is both professional and personal, two qualities which I find particularly valuable in a professional setting.He has personally helped me in professional negotiation for everything from train tickets to contract information, and I have always been able to count on him.I first met Paul in school, where he was a student at the university at which I taught.He was well-known to most of the westerners in town, who could call from different universities to ask for his help.Sometimes this help involved translation of professional documents, and sometimes it involved personal help in making phone calls.Many of these westerners continue to call him to day, even though they live quite far away from him, because they have come to trust him very much.From this foundation in public relations Paul has found positions in variousprofessional capacities and has been highly-valued in each place.He is generally the sort of employee a company finds most valuable in its dealings with both foreign and domestic clients.He puts people at their ease with hislanguage ability and manner, both of which communicate to people that they can relax and simply communicate.I would highly recommend Paul as an employee.His experience and manner are rare and very valuable.Robert MooreMentor介绍信英语作文【3】Dear，Iwanttotakethisopportunitytoinformyouthat Yjbys1willbejoiningusshortly.Sheholdsadoctor'sdegreeingeneticsandhasworkedinthisfiel dfortwelveyears.Itisexcitingthatshehasdecidedtojoinourpanyinthistransformation period.共2页: 上一页12下一页英文推荐信参考范文推荐信是帮助您更好的获得机会的一种心体，而英文的推荐信书写更加要求语法的严格和准确，请看下面这三篇英文的推荐信参考范文：英文推荐信【一】Dear Colleagues：As a teacher in Department of Biology, Nankai University, a leading university in China, I am very pleased to take this opportunity to recommend one of my favorite students to your PhD program.In September, 2004, Miss Zhang was my students inGeneral Biology, the first professional course they take in the field.She likes it very much.I teach her in an open and interactive manner, she is active and passionate about answering class questions.Miss Zhang is bright, energetic and enthusiastic girl who loves speaking out her own ideas.She never escapes from those points of which she is skeptical.Apart from that, she often puts forward her ideas upon questions and exchanges all of her innovate ideas with me after class.Upon ending of General Biology course, I asked my students to write an essay upon their understanding of biology.I discovered Miss Zhangs essay was so impressive and persuasive; her careful essay comes from dozens of references to Biology literatures.In essay Miss Zhang stretched out her creative thinking upon status quo of biological research and branches in the field of biology.All of this speaks to her creative and logical and scientific thinking capability.I am gladly to say that Miss Zhang has a clear understanding about General Biology, that comprehension has been reflected fully in her essay.Therefore Miss Zhang got a full point for her essay in her class, and other students took it as model essay for granted.I believe Miss Zhangs industriousness, passion and dedication will make her an ideal candidate you are seeking for your program, so I highly recommend her without any hesitation to you.And I will greatly appreciate it if you could accept her into your program.Sincerely yours,Prof.英文推荐信【二】At the request of mr.xizhen chen, my former student in the department of computer science, beijing univ.Of sciences, i am glad to write this letter furnishing my evaluation ofhis academic aptitude for your reference.mr.chen is interested in your graduate program in computerscience.I came to know him in september 1987 when mr.chen enrolled in my class on fortran iv programming, a three semesters course.In the class he was one of the most outstanding students.at the semester final he earned a high grade of 81, which should be "aaccording to our grading system.I also ound him good at other studies.after the class, he had personal talks with me several times.he indicated a great interest in computer hardware, in my opinion, mr.chen has a potential in computer science, which can be further developed.In view of his previous achievement s in this college, I am firmly convinced that mr.chen will make a successful graduate student.Your favorable consideration of his admission will be highly appreciated.英文推荐信【三】Dear Sir:It is my pleasure to recommend Mr.Fuping Wang for his application to graduate studies in your school.Mr Wang has been my research assistant of the National Science Council's project- "The United States and the United Nations' Financial Crisis: The Role of U.S.Congresssince September 1997.His major responsibilities include information collecting, analyzing and translating.He is an enthusiastic and progressive young man with extremely high potentiality.Mr.Wang is not only quick at learning and good at solving difficult problems, but also with a logical mind that enables him to effectively analyze difficulties.All the work handed to him was completed satisfactorily.Actually, he is so reliable that I assign him with heavy responsibilities.With his help, I have been able to spend more time in writing the research paper.I really consider myself very fortunate to have such acapable assistant.I am certain his diligence, coupled with a good competence and pleasant personality, will assure him of academic achievements in his future academic pursuits.I strongly recommend his admission without any reservation.学校的推荐信推荐信应该找一些具有一定知名度且与申请人熟识的人撰写，一般的最佳人选就是申请人的专业课老师、班主任、学校里跟申请人接触较多的比较权威的老师、或者实习公司的领导等等。

烟酰胺磷酸核糖转移酶的调控因素及药物作用

烟酰胺磷酸核糖转移酶的调控因素及药物作用吕小群;张偌瑜;缪朝玉【摘要】烟酰胺磷酸核糖转移酶(Nampt)是生命过程中不可或缺的蛋白,具有多种生理功能.对许多疾病如心脑血管疾病、糖尿病、癌症、自身免疫性疾病等有潜在的作用.阐明各种生理、病理状态以及化合物对Nampt表达水平的改变,可为Nampt的基础性和应用件研究起到推动作用.本文就各种因素对Nampt转录、表达调控的研究进展进行了较为系统的综述.%nicotinamide phosphoribosyl transferase (Nampt) is an essential protein with a variety of physiological functions and performs potential functions on many diseases such as cardiovascular diseases, diabetes, cancer and autoimmune diseases. It is of great significance to clarify the effect of physiological and pathological statuses as well as compounds on the expression level of Nampt, as it will promote the basic and applied researches. A systematic review about various factors regulating the transcription and expression of Nampt were carried out in this paper.【期刊名称】《药学实践杂志》【年(卷),期】2011(029)001【总页数】4页(P11-14)【关键词】Nampt;调控;生理;病理;化合物【作者】吕小群;张偌瑜;缪朝玉【作者单位】第二军医大学药理学教研室,上海,200433;第二军医大学药理学教研室,上海,200433;第二军医大学药理学教研室,上海,200433【正文语种】中文【中图分类】R965烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase, Nampt)，又名内脏脂肪素visfatin，或前B细胞克隆增强因子PBEF，是哺乳动物生命活动中不可或缺的蛋白，Nampt缺陷的纯合子小鼠(Nampt-/-)死于胚胎发育早期[1,2]。

博士入学PPT模板

Results
2.2. Overexpressed of PTBP1 promotes migration of lung cancer cells
Results
2.3. Knockdown of PTBP1 inhibits levels of EMT-related proteins in lung cancer cells
Background
Seven alternative splicing (AS) subgroups: • Exon skipping accounts for nearly 40% of AS events; • alternative 3′ splice site (3′SS) selection (18.4%) and 5′SS
3. Dewei Niu, ******, Shanze Yi, Feng Wang*. Gene cloning, protein expression and functional analysis of a type 3 metallothionein gene from Sonneratia alba with biosorption potential. Polish Journal of Environmental Studies, Accepted. PJOES-00647-2017-02.
A
B
A. PTBP1 expression was elevated in LUAD tissues (N=515) compared with normal lung tissues (N=59) according to TCGA database (p<0.01); B. Kaplan-Meier plots of patients with LUAD according to high (N=127) and low (N=375) PTBP1 expression from the TCGA database and compared by paired t-test, p<0.01.

木质素过氧化物酶的应用

doi:10.3969/j.issn.2095-1736.2021.03.099收稿日期:2020-07-10;最后修回日期:2020-09-01基金项目:江苏省农业科技自主创新资金项目CX(17)3044;江苏省重点研发计划(现代农业)BE2017355;江苏省自然科学基金项目BK20170541作者简介:梁晓玉,硕士研究生,研究方向为微生物降解和秸秆饲料,E-mail:2211817033@ 通信作者:陈华友,博士,教授,研究方向为微生物降解和秸秆饲料,E-mail:hyc@木质素过氧化物酶的应用梁晓玉,崔周磊,王洪成,陈华友(江苏大学生命科学研究院,镇江212013)摘㊀要㊀综述木质素过氧化物酶的催化机制㊁酶活测定㊁克隆表达㊁协同作用与应用的研究进展㊂木质素过氧化物酶在化学化工㊁生态修复以及生物饲料行业等方面有着广泛的应用,而木质素过氧化物酶工业化应用受酶产量及酶活性影响㊂提高酶表达量㊁改善酶学性质㊁探究木质素过氧化物酶高效利用的方法是促使木质素过氧化物酶工业化应用的关键,也是近年来的研究热点㊂结果为进一步深入研究开发木质素过氧化物酶提供参考,并对木质素过氧化物酶研究前景进行展望㊂关键词㊀木质素过氧化物酶;催化机制;酶活;协同作用中图分类号㊀Q939.9文献标识码㊀A 文章编号㊀2095-1736(2021)03-0099-04Research in lignin peroxidaseLIANG Xiaoyu,CUI Zhoulei,WANG Hongcheng,CHEN Huayou(Institute of Life Sciences,Jiangsu University,Zhenjiang 212013,China)Abstract ㊀This paper reviewed the related research progress of the catalytic mechanism,enzyme activity assay,cloning and expression,synergy,application of lignin peroxidase.Lignin peroxidase has broad application prospects in chemical industries,ecological restoration and biological feed industries,however,the industrialized application of lignin peroxidase is limited by its production and enzyme activi-ty.The key to promote the industrialized application of lignin peroxidase is to increase the enzyme expression,improve the enzymatic properties,and explore the efficient use of lignin peroxidase,which is also a research hotspot in recent years.The results provide refer-ences for further research and development of lignin peroxidase,and a forecast of the research prospect of lignin peroxidase.Key words ㊀lignin peroxidase;catalytic mechanism;enzyme activity;synergy㊀㊀木质素过氧化物酶(Lignin peroxidases,LiP)㊁锰过氧化物酶(Manganese peroxidase,MnP)㊁漆酶(Laccase,Lac)等构成了白腐菌的主要木质素降解酶系,与木质素降解辅助酶系协同降解木质素㊂LiP 主要来源于真菌,是血红素过氧化物酶超家族Ⅱ类过氧化物酶中的一员㊂在木质素降解酶系中,LiP 氧化还原电位比MnP㊁Lac 高,除降解木质素外,还能降解生态系统中其他难降解的高分子化合物,这提高了LiP 的应用价值㊂LiP 在化学化工㊁生态修复㊁生物饲料等行业具有广阔的应用前景,是当前研究的热点,本文综述了近年来LiP 的研究进展,并对其研究前景进行展望㊂1㊀LiP 的催化机制LiP 有一个所谓的配体通道,可使小分子底物通过通道与血红素直接作用㊂底物的大小和理化性质共同决定其能否通过配体通道与血红素反应㊂Ecker 等[1]研究发现,除草剂阿特拉津进入配体通道后与通道中某些氨基酸残基结合,最终无法到达血红素腔活性位点㊂这类物质与配体通道结合后能否再被释放出来,其与配体通道结合时是否会阻碍其他底物对血红素腔的可及性,以及是否会对LiP 的其他活性位点产生影响,值得深入探究㊂目前对配体通道中氨基酸残基了解有限,明确配体通道中关键氨基酸残基㊁分析配体通道的功能是下一步研究的方向,这有利于深入了解LiP 作用机理,并对LiP 改性提供参考㊂木质素等大分子物质无法通过通道与血红素作用,而是通过酶表面氨基酸残基经长电子转移机制最99终与血红素反应㊂Sandra等[2]提出长电子转移机制两种可能的电子转移路径,一种是Phe s路径,按照Trp171-Phe205-Trp251的顺序转移自由基,另一种被称为Phe#2 s路径,按照Trp171-Asp264-Phe265-Phe267的顺序转移自由基,这两种转移路径与多功能过氧化物酶中发现的电子转移路径类似㊂虽然对LiP这两个氧化位点有了初步了解,但它们之间的联系尚不清楚㊂藜芦醇(Veratryl alcohol,VA)既可作为LiP催化底物,也可充当LiP催化反应的介质,提升酶促效率㊂有研究认为LiP氧化VA生成的芳基阳离子自由基VA+㊃可以扩散到周围体系中,拓展LiP作用范围㊂这种假说被Houtman等[3]否定,他们以黄孢原毛平革菌(Phanero-chaete chrysosporium Burdsall)LiP(H8)为研究对象,认为真菌LiP不使用VA+㊃作为扩散的木质素降解氧化剂,但实验中并未涉及其他LiP同工酶,对是否存在LiP以VA+㊃为扩散氧化剂还需进一步研究㊂2㊀LiP酶活测定基于毛细管电泳的胶束电动色谱法可应用于未纯化㊁未浓缩的微量LiP酶活测定㊂胶束电动毛细管色谱法是色谱和电泳分离原理相结合的一种复合方法,首先通过电泳分离目标产物,然后通过色谱进行定量分析[4]㊂Airi等[5]已成功应用该方法测定LiP活性㊂研究发现,某些底物如VA和Mn2+,LiP和MnP均具有氧化能力,但它们对同种底物氧化的能力不同,Sumire等[6]基于此,应用胶束电动毛细管色谱法成功测定了LiP和MnP混合体系中LiP和MnP活性㊂这种基于酶活差异同时测定混合体系中不同酶酶活的方法理论上适用于两种或两种以上酶的混合体系,但使用时体系中酶的种类要与底物种类一致,否则难以计算㊂胶束电动色谱法灵敏㊁准确,且样品无需浓缩,可直接用于酶活测定,但其对设备要求较高,实验条件限制了胶束电动色谱法的广泛推广㊂目前LiP酶活测定主要还是通过分光光度计进行,VA㊁天青B等物质均可作为分光光度法测LiP酶活的底物㊂藜芦醇氧化法测LiP酶活的原理是LiP在H2O2存在时催化VA转化为藜芦醛,根据310nm处吸光度变化值计量LiP酶活[7]㊂天青B法通过监测OD651处吸光度变化计量LiP酶活㊂酶活测定时,310nm处吸光度易受粗酶液中芳香化合物及其他过氧化物酶干扰,651nm处吸光度受此影响较小,但天青B法测定时间较长,因此,当测量体系中干扰较多且时间允许时,可选择天青B法;当测量体系干扰较少时,藜芦醇氧化法更加便捷㊂对比不同研究发现,即使选择同种底物,LiP酶活测定体系中缓冲液㊁pH值㊁反应体系也存在差异[8-10],这使得不同研究中的LiP酶活不具有对比性,因此,亟须制定LiP酶活测定的统一标准㊂3㊀LiP克隆表达天然产LiP菌株产酶不稳定,表达量低,所产LiP 其性质多不适用于工业生产,因此,人们将LiP异源表达以提高LiP的表达量㊁改善LiP的理化性质,使其更满足人们的应用需求㊂LiP主要在大肠杆菌中过表达,获得纯品蛋白后进行酶学鉴定㊂值得注意的是,木质素降解酶类其酶学性质鉴定多以木质素结构类似物为底物,缺乏真正意义上以秸秆中木质素为底物的性能评估㊂崔周磊等[11]将来源于白囊耙齿菌(Irpexlacte-us)的MnP在大肠杆菌中异源表达,以木质素为底物评估所得MnP效能,所得MnP可使玉米秸秆㊁麸皮中木质素降解率分别可达35.8%和27.3%,这种酶活鉴定方法使得酶的应用潜力更加直观㊂虽然已有数个LiP在大肠杆菌中成功过表达,但目的蛋白多以包涵体形式产生,分离纯化复性不便,且其生物安全性存在疑问,工业化尤其是食品工业级应用困难㊂出于工业化应用的目的,LiP克隆表达需要选择合适的受体菌㊂LiP在其他表达系统如黑曲霉㊁杆状病毒等克隆表达的研究也有报道,但应用价值较低[12-13];肖建龙等[14]在食品微生物酿酒酵母中成功表达有活性LiP,但实用性未知㊂枯草芽孢杆菌作为饲用菌种,一般具有生物安全性,且生长速度快,能够产生大量胞外蛋白,因此有望作为LiP胞外表达的工业级宿主㊂目前已筛选得到可以胞外分泌LiP的枯草芽孢杆菌[8],为提升LiP胞外表达量,需要进一步优化启动子和信号肽㊂由于酿酒酵母的ADH7启动子可以在木质素衍生物香兰素浓度达到一定限度时启动目的基因表达降解香兰素[15],香兰素既是诱导物又是底物,这类高效低成本产LiP的启动子有望应用于工业大规模生产LiP㊂工业化应用时,LiP需要耐受环境因素的影响,天然LiP稳定性不高,耐受能力不强,因此,除通过基因工程提升酶的产量外,还应提升酶的稳定性,使其更能耐受环境影响㊂Semba等[16]构建真菌过氧化物酶系统发育树,推断真菌LiP祖先氨基酸序列,并设计多个含有祖先氨基酸残基的突变酶,所得突变酶热稳定性优于野生型;Karla等[9]构建LiP基因的随机诱变文库,所得突变LiP能耐受更高浓度的H2O2;Le等[17]在黄孢原毛平革菌LiP(H8)中引入盐桥,提高了LiP的耐酸性,工程酶在极端酸性条件下的半衰期提高了12.5倍㊂耐受极端环境的酶可以拓宽酶的工作范围,其合成对LiP工业化应用具有重大意义㊂4㊀LiP与木质素降解木质素是由松柏醇㊁芥子醇和对香豆醇3种醇单体聚合而成的高分子化合物,这些醇单体通过碳碳键㊁001醚键互相偶联,然后通过自由基机制聚集形成木质素聚合物[18],这种聚合物包被着纤维素和半纤维素,且本身结构紧密,限制木质纤维素的利用㊂LiP在H2O2存在时可以氧化各种酚类芳香底物㊁非酚类木质素以及一系列氧化还原电位高于1.4V的化合物,被认为是木质素降解最高效的酶[19]㊂LiP催化非酚类木质素降解时,从酚或苯环上提取一个电子生成自由基,然后通过后续的非酶反应经侧链裂解㊁去甲基化㊁分子内加成和重排等方式破坏木质素分子的主要化学键,导致木质素降解[20]㊂LiP催化木质素中的芳香环发生裂解,裂解生成的酚衍生物转化为醌类化合物后,经一系列酶的协同作用,以纤维二糖酸内酯的形式进入三羧酸循环或戊糖磷酸途径,终产物为CO2[21]㊂LiP降解木质纤维素的效果多用木质纤维素降解率衡量,此外,LiP降解木质纤维素后还原糖㊁总酚量㊁酚羟基含量等指标也被用于描述木质素降解程度[22-23],因木质素降解产物多样[24],多指标相互验证才能更准确地评估LiP降解木质素的效果㊂5㊀LiP的协同作用与应用前景5.1㊀LiP用于化工行业㊀㊀LiP可降解黑色素,可替代美白产品中常用的苯二酚[7]㊂LiP的催化活性依赖于H2O2,但高浓度的H2O2会导致LiP失活,因此,美白产品中需要持续存在低浓度的H2O2㊂HO等[25]在美白产品中协同LiP与葡萄糖氧化酶,利用葡萄糖氧化酶生成的H2O2催化LiP对黑色素脱色,避免了高浓度H2O2使LiP失活,提高了黑色素的脱色效率㊂此外,LiP降解木质素产生的二元羧酸㊁小分子酚类物质等是重要化工原料,如: LiP降解木质素产生的香兰素可代替天然香兰素用于食品㊁化妆品行业;LiP作用于木质纤维素,脱木质素后作为原料生产乙醇等㊂LiP在化学化工行业的更多应用潜力尚待挖掘㊂5.2㊀LiP用于生态修复生态系统中的纺织废水㊁农药㊁抗生素等有机化合物严重威胁着人类健康,但环境中这些污染物浓度较低,且种类多样,LiP虽然具有很强的降解这些污染物的能力,但其产量少,难以大规模投放;底物谱有限,单独作用效果不佳[26]㊂Pylypchuk等[27]将辣根过氧化物酶与LiP固定到纳米粒子上,多酶协同固定化不仅使酶作为催化剂可以重复使用,还提高了污染物的降解效率㊂pail等[28]将产LiP的细菌和其他细菌同时投放到纺织废水污染的土地上,发现混合微生物的染料脱色和解毒作用比单菌株更明显㊂多酶或菌酶协同一定程度上提升了酶的利用效率,然而,这种投放方式针对性不强㊂根据底物特征,有规划地投放合理的复合酶系能更有效地提升利用率,这需要我们在投放之前能够明确体系中底物种类及其与酶的作用潜力㊂Berbar 等[29]首次尝试并建立了一套评估底物对酶敏感性的理化指标,评估了25种有机污染物的氧化还原酶敏感性,敏感性评分与降解率基本一致㊂类此,建立LiP对底物敏感性的理化指标,评估底物敏感性后,针对性投放合理的酶处理体系可以避免酶无效投放㊂5.3㊀LiP与生物饲料中国秸秆资源丰富,但没能得到充分利用,木质素降解菌或者酶处理秸秆后转化为生物饲料,既能提高秸秆利用率,又解决了牧区牛羊粗饲料短缺㊁人畜争粮争地的问题㊂秸秆中高含量的木质纤维素成分难以被动物消化,且影响饲料适口性㊂微生物发酵后的秸秆中木质纤维素被降解成易于吸收的含糖物质,既提高了适口性,又提高了营养价值㊂实际应用中,单一菌种底物谱有限,酶活普遍较低,不能满足降解需求,而多菌种混合发酵可以提供较为完整的木质纤维素酶体系,提高降解效率,降低时间成本,提高微生物对大规模应用的适用性[30]㊂粗糙脉孢菌(Neurospora crassa)含有丰富的纤维素降解基因,表达数种纤维素降解酶,能够有效降解纤维素[31]㊂崔周磊[32]将MnP基因克隆于食品安全级粟酒裂殖酵母中,工程菌协同粗糙脉孢菌等发酵菌种制备秸秆生物饲料,与单菌种发酵相比,混菌发酵过程中菌酶协同作用转化了菌种之间对碳源的竞争,提高了发酵的效率和品质㊂除食品级粟酒裂殖酵母外,木质素降解酶在饲用菌种如枯草芽孢杆菌中克隆表达有助于推进秸秆等农业废弃物饲料化㊂6㊀总结与展望LiP具有强大应用潜力,但无法大规模应用,根本原因是产酶菌生长缓慢㊁产酶量低,以及酶本身性质限制,因此,LiP的克隆表达应围绕受体菌㊁表达量㊁酶学性质3个方面㊂对工程菌质量的综合评估,应以应用目的为基准,如在生物饲料行业中,LiP的安全性至关重要,LiP的克隆宿主应选择无抗,生物安全且可用于饲料的菌种,在此基础上进一步优化产酶量及酶学性质;在环境治理中,普遍认为真菌抗逆性优于细菌,因此,受体菌应优先选择真菌,受体菌中酶活力也不是评估工程菌质量的唯一标准,酶的适用性和酶活力共同决定酶的应用价值㊂此外,如何使LiP高效应用是研究的另一个重点㊂小分子介质被认为能够拓宽酶底物范围,提升酶活力,目前对LiP-介质系统的研究落后于MnP和Lac,且相关研究多为ABTS㊁HBT㊁HAA等介质,这类介质价格昂贵,不适合大规模工业化应用,理想介质应该高效提升酶活,且不会给环境带来污染,同101时廉价㊁易于获取且高效,因此,还需进一步筛选优质LiP介质㊂酶与酶之间协同作用已经被证实[24],除木质素降解酶之间的协同外,构建作用范围广且高效的完整酶系可大大提升LiP的应用价值㊂参考文献[1]ECKER J,FÜLOP L.Lignin peroxidase ligand access channel dys-function in the presence of atrazine[J].Scientific Reports,2018, 8(1):1-7.[2]SANDRA A,RUIZ-DUENASF J,MARIO T,et al.Mapping the long-range electron transfer route in ligninolytic peroxidases[J].Jour-nal of Physical Chemistry 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[27]PYLYPCHUK I V,GEOFFREY D,KESSLER V G,et al.Removal of diclofenac,paracetamol,and carbamazepine from sodelaqueous solutions by magnetic sol-gel encapsulated horseradish peroxidase and lignin peroxidase composites[J].Nanomaterials(Basel,Switz-erland),2020,10(2):282.[28]PAIL S M,SURYAVANSHI M V,CHANDANSHIVE V V,et al. Regeneration of textile wastewater deteriorated microbial diversity of soilmicrocosm through bioaugmentation[J].Chemical Engineering Journal,2020,380(1):122533.[29]BERBAR E A,LIU Z Y,SMITH S anic contaminant biodeg-radation by oxidoreductase enzymes in wastewater treatment[J].Mi-croorganisms,2020,8(1):122.[30]JIA J R,CHEN H R,WU B G,et al.Protein production through microbial conversion of rice straw by multi-strain fermentation[J]. Applied Biochemistry&Biotechnology,2019,187(1):253-265.[31]吴邦国.玉米秸秆预处理及粗糙脉孢菌发酵[D].镇江:江苏大学,2018.[32]崔周磊.白囊耙齿菌锰过氧化物酶表达鉴定及在秸秆发酵中的菌酶协同作用[D].镇江:江苏大学,2020.201。

3D类器官模型的研究进展及其在化学品毒理学评价中的应用展望

生态毒理学报Asian Journal of Ecotoxicology第16卷第4期2021年8月V ol.16,No.4Aug.2021㊀㊀基金项目:国家重点研发计划项目(2018YFE0110700);国家自然科学基金资助项目(21661142001,21777020);中央高校基本科研业务费(DUT20LAB102,DUT20LK19)㊀㊀第一作者:刘薇(1980 ),女,博士,教授,研究方向为持久性有机污染物毒性效应和机理以及干细胞毒理学模型原理和应用,E -mail:liu_************.cn㊀㊀*通讯作者(Corresponding author ),E -mail:****************.cnDOI:10.7524/AJE.1673-5897.20200817001刘薇,梅玺丽,陈雨萌,等.3D 类器官模型的研究进展及其在化学品毒理学评价中的应用展望[J].生态毒理学报,2021,16(4):32-42Liu W,Mei X L,Chen Y M,et al.Progress in 3D organoid models and application perspectives in toxicity test of chemicals [J].Asian Journal of Ecotox -icology,2021,16(4):32-42(in Chinese)3D 类器官模型的研究进展及其在化学品毒理学评价中的应用展望刘薇1,∗,梅玺丽1,陈雨萌1,向哲源1,高泽欣1,邢立国2,赵慧敏1,刘猛1,陈景文11.大连理工大学环境学院,工业生态与环境工程重点实验室,大连1160242.中化集团沈阳化工研究院有限公司,沈阳110027收稿日期:2020-08-17㊀㊀录用日期:2021-03-22摘要:化学品毒理学评价主要采用2D 细胞培养体系和动物实验模型,但2种模型均存在一定的局限性㊂2D 细胞培养常用特定细胞系,与体内多细胞组织及其生理功能差异较大㊂动物实验耗时㊁昂贵,并受伦理学限制,且存在种属差异问题㊂3D 类器官模型可模拟组织器官的复杂结构和功能,产生细胞间和细胞-基质相互作用,与体内生理反应更为相似,因此能更准确地反映毒性效应和机理㊂类器官模型在化学品毒理学评价领域具有良好的应用前景,将显著提升毒理学基础研究能力以及对化学品风险评价和管理的支撑作用,但相关研究尚处于起步阶段㊂本文从类器官的生理学特性㊁构建方法及组织类型等方面,重点讨论了类器官模型在化学品毒理学研究中的适用性,并提出了存在的挑战和对策㊂关键词:类器官;毒理学替代法;体外模型;高通量测试文章编号:1673-5897(2021)4-032-11㊀㊀中图分类号:X171.5㊀㊀文献标识码:AProgress in 3D Organoid Models and Application Perspectives in ToxicityTest of ChemicalsLiu Wei 1,*,Mei Xili 1,Chen Yumeng 1,Xiang Zheyuan 1,Gao Zexin 1,Xing Liguo 2,Zhao Huimin 1,Liu Meng 1,Chen Jingwen 11.Key Laboratory of Industrial Ecology and Environmental Engineering of Ministry of Education,School of Environmental Science and Technology,Dalian University of Technology,Dalian 116024,China2.Shenyang Research Institute of Chemical Industry of SINOCHEM Group,Shenyang 110027,ChinaReceived 17August 2020㊀㊀accepted 22March 2021Abstract :Toxicity assessment of chemicals predominantly adopted either 2D cell culture or animal models where -as both models have intrinsic flaws.2D cell culture systems are usually derived from a specific set of cell lines,dif -fering from the tissue function encountered in vivo containing multiple cell types.Animal models are limited by the cost and time,associated ethical concerns and xenogeneity.3D organoids achieve a greater level of structural and. All Rights Reserved.第4期刘薇等:3D类器官模型的研究进展及其在化学品毒理学评价中的应用展望33㊀functional complexity more closely analogous to in vivo physiology,showing cell-cell and cell-extracellular matrix interactions,and thus enabling a more accurate reflection of the toxic effects and mechanisms.With promising per-spectives,the progresses in organoid toxicology would significantly promote the fundamental toxicology research, and support the chemicals risk assessment and regulation as well.However,organoid-based toxicological research of chemicals is still in its infancy.In this review,we discussed recent developments and trends in organoid model concerning the physiological features,development systems and methods,and tissue-specific types,focusing on its feasibility in toxicological researches of chemicals,and the challenges and strategies are also proposed. Keywords:organoid;toxicology alternatives;in vitro model;high-throughput test㊀㊀工业革命以来,人类合成了化肥㊁农药㊁药物和各种工业化学品,至今美国化学文摘社(Chemical Abstracts Service,CAS)注册的化学物质有1.5亿种以上(每天约增加15000种),人类在市场上使用的化学品在35万种以上[1]㊂根据2019年3月联合国环境署(United Nations Environment Programme,UN-EP)发布的全球化学品展望Ⅱ(Global Chemicals Out-lookⅡ)[2],欧盟区域2016年使用的3.45亿t化学品中,62%对人体健康有害㊂因此,合成化学品是生态与人体健康的重要风险源,是人类社会可持续发展面临的重大挑战㊂预防和控制化学品对人体健康的风险,需要化学品的毒性数据,以填补关于化学品危害性的信息空白㊂然而,传统的毒性测试方法往往需要消耗大量的测试动物,成本高,耗时长,存在动物实验伦理问题㊂而且,动物实验结果外推至人类具有极大的不确定性㊂新时代的环境毒理学,倡导从以往主要是描述性的科学向更具预测性㊁主要基于人源细胞和组织的体外高通量测试的科学转变;倡导通过对毒性通路(toxicity pathway,TP)和有害结局路径(ad-verse outcome pathway,AOP)的机理认识并构建计算毒理学模型来进行化学品的风险评价和预测,进而减少实验测试动物的数目㊁测试成本和时间㊂因此,发展基于人源细胞和组织的体外高通量毒性测试方法体系,是新时代环境毒理学的前沿方向[3-5]㊂人源细胞和组织用于毒理学测试,先后经历了2D单层细胞培养㊁细胞球模型和类器官模型(表1)㊂2D细胞培养和细胞球模型常用单一的特定细胞系,与体内多细胞组织及其生理功能差异较大㊂所谓类器官(organoid),指的是由诱导多能干细胞(induced pluripotent stem cells,iPSCs)㊁胚胎干细胞(embryonic stem cells,ESCs)或成体干细胞(adult stem cells, ASCs)在体外自组织并经历一定程度的细胞分化形成的3D结构,具备体内组织器官的部分典型功能,具有较为稳定的表型和遗传学特性[6-7]㊂3D类器官模型可模拟组织器官的复杂空间形态,突破了细胞间单纯的物理接触和联系,表现出细胞间和细胞-基质相互作用,与体内组织器官具有更相似的生理反应㊂类器官模型与2D单层细胞模型或细胞球模型相比,能更好地用于模拟器官组织的发育过程及生理病理状态,因而能更好地反映体内毒性效应(表1)㊂关于类器官模型的基础研究以及在再生医学领域的应用进展,已有相关综述[6-13]㊂目前类器官模型在化学品毒理学评价领域的应用仅处于起步阶段[14],本文从类器官的生理学特性㊁构建方法及组织类型等方面,重点综述了类器官模型对化学品暴露的毒性响应特征,及其在化学品毒理学评价中的优势和可行性,并提出了存在的问题和对策,以期为基于类器官模型的化学品毒理学研究提供参考㊂1㊀类器官的特性(Organoid features)类器官由多种细胞自组织形成,在细胞类型㊁结构和功能等方面模拟体内相应组织器官㊂作为实验室培养的器官微缩模型,类器官应用于体外环境中特定靶器官毒理学的研究,具有以下几方面优势㊂(1)细胞形态呈3D模式㊂与传统细胞培养中贴壁生长的2D细胞形态不同,在类器官模型中,细胞在3D空间中生长,形态学特征和生理特性与体内更相似㊂例如,3D毛囊乳头细胞球相较于单层培养的毛囊乳头细胞,其细胞分泌因子能够改善毛囊微环境,促进毛囊中β-catenin和CD133的表达,这2种蛋白与毛囊干细胞功能和毛囊再生密切相关[15]㊂呼吸道上皮细胞在3D培养条件下表达上皮细胞标志物PCK㊁CK5和ZO-1,而且形成纤毛样突起,但在2D条件下培养并无以上特征[16]㊂(2)能够反映细胞间相互作用和细胞-基质相互作用㊂类器官模型包含多种细胞类型,突破了传统2D模型中简单的细胞间物理接触,形成了更加紧密的细胞间生物通信和信号网络,细胞间相互影响㊁反. All Rights Reserved.34㊀生态毒理学报第16卷馈及协作发育㊂细胞间相互作用和细胞-基质相互作用支持细胞微环境稳态的建立,有助于形成和维持具有特定结构和功能的微型组织器官㊂类器官模型可显著促进针对细胞相互作用过程的毒理学研究,例如对细胞粘附㊁迁移㊁分化和愈合等细胞功能的损伤作用[17]㊂(3)具有良好的生理功能㊂例如,皮肤类器官比单层培养的角化细胞㊁成纤维化细胞具有更好的抗氧化应激能力[18-19],与人体表皮脂质成分高度相似,具有较为完善的屏障功能㊂Lancaster等[20]构建的脑类器官模型,不仅符合哺乳动物的基本神经发育机理,还具备了一定的人类大脑发育特征,并且作者利用该脑类器官模型成功构建了在小鼠体内很难模拟的小头畸形障碍疾病模型,有效解决了该疾病实验研究的难题㊂相对于单层细胞培养,3D培养条件下的生殖细胞中自噬体数量明显降低,缓解了细胞在体外培养中产生的应激反应[21]㊂2㊀类器官模型的构建方法(Organoid development methods)㊀㊀根据使用的支架材料和培养设备等,常用类器官构建方法主要包括4类:悬浮培养㊁凝胶支架培养㊁旋转生物反应器和气液界面培养(图1)㊂此外,生物打印技术和器官芯片技术是类器官模型构建的新兴方法㊂2.1㊀悬浮培养使用低附着力表面的培养瓶/微孔板㊁磁悬浮器件或者采用悬滴法,细胞可在液体培养基中悬浮生长并形成类器官㊂悬浮法不使用支架材料,细胞自发团聚,避免了其他因素的干扰㊂悬滴法中细胞悬滴所含细胞数相同,球体大小一致,无需借助额外装置,自发实现细胞成球,局限性主要在于不适于大规模培养[22]㊂磁悬浮方法将具有生物相容性的磁性纳米颗粒引入细胞,将磁性驱动组件盖在培养板顶部,细胞在磁力作用下悬浮到空气-液体界面,并集中在磁铁下,形成3D结构㊂Tseng等[23]利用磁悬浮法实现了4种细胞共培养,包括成纤维细胞㊁内皮细胞㊁上皮细胞和平滑肌细胞,形成了细支气管3D模型㊂2.2㊀凝胶支架水凝胶由交联的多聚链或复杂蛋白分子组成的网络构成,其高含水量和高孔隙性使得氧㊁营养物质和废物的运输更为便利,为细胞提供附着㊁分化和增殖的场所,因而可以作为高效的3D细胞培养基质[24]㊂水凝胶包括天然来源和人工合成材料,其中表1㊀不同体外细胞模型的优缺点Table1㊀Advantages and disadvantages of various in vitro cell models单层细胞模型Monolayer cell model细胞球模型Cell sphere model类器官模型Organoid model优点Advantage1.㊀操作简单Simple operation2.㊀获得结果快Get results quickly3.㊀重复性强Easy to replicate1.保持细胞的基础结构Maintain basic cellular structure2.细胞-胞外基质相互作用Cell-to-extracellular matrixinteractions3.细胞功能较充分且稳定Complete and stable cell function4.与体内实验结果差异小Reduced difference within vivo results1.与来源组织具有相似的结构和功能Similar with in vivo structure and function2.由多种细胞组成Consist of multiple types of cells3.细胞间生物通信和信号网络更加紧密Closer intercellular biological communicationand signal networks4.具有稳定的表型和遗传特性Stable phenotypic and genetic characteristics缺点Disadvantage 1.缺乏细胞-胞外基质的相互作用Lack of cell-to-extracellular matrixinteractions2.细胞功能不完全Incomplete cell function3.细胞类型单一Single cell type4.与体内实验结果差异大Differ from in vivo results1.培养时间长,成功率低Long culture time and low yield2.得出实验结果的过程较慢Get results slowly3.不能体外自组织Inability to self-organize in vitro1.培养困难Cultural difficulties2.缺乏标准方法Lack of standard protocol3.表型不成熟Phenotypic immaturity4.模型血管化困难Difficulty in vascularization. All Rights Reserved.第4期刘薇等:3D 类器官模型的研究进展及其在化学品毒理学评价中的应用展望35㊀图1㊀类器官的构建方法和组织类型注:ASCs 表示成体干细胞,ESCs 表示胚胎干细胞,iPSCs 表示诱导多能干细胞㊂Fig.1㊀Methods for organoid development and tissue -specific typesNote:ASCs stand for adlut stem cells;ESCs stand for embryonic stem cells;iPSCs stand for induced pluripotent stem cells.Matrigel 是一种细胞体外培养最常用的天然基质胶,是从富含胞外基质蛋白的EHS 小鼠肿瘤中分离得到的,其组分与体内大多数活细胞的胞外基质相似[25]㊂Matrigel 在4ħ时为液体,当浓度>4g ㊃L -1时,在24~37ħ时凝胶化[26]㊂Matrigel 主要成分为层粘连蛋白㊁Ⅳ型胶原㊁巢蛋白和硫酸肝素糖蛋白等,具有结构支持和信号转导的作用㊂其中层粘连蛋白含量最为丰富,是主要的凝胶因子㊂Matrigel 还包含FGF ㊁EGF ㊁TGF -β㊁IGF 和PDGF 等生长因子,通过改变生长因子的组成和浓度,可调控特定类型的细胞增殖和分化㊂Matrigel 的局限性主要在于组分不完全明确,存在一定的产品批次差异[11]㊂2.3㊀旋转生物反应器旋转生物反应器解决了类器官构建的主要难题之一,即细胞的营养和氧气供给㊂例如,与静态悬浮培养相比,旋转生物反应器中的神经节细胞和S 型视锥细胞的分化能力增强,促进和改善了视网膜类器官构建中的细胞增殖和分化[27]㊂在旋转生物反应器中建立的小头畸形障碍的大脑类器官模型,尽管6~7个月后尺寸有所皱缩,但可维持活性长达15个月[20]㊂保持适宜的转速对使用旋转生物反应器培养类器官至关重要,搅拌速度过慢会使细胞球沉降,速度过快则会损坏细胞㊂此外,该方法不适用于培养粘附性低或对剪切力敏感的细胞[28]㊂2.4㊀气液界面法气液界面(air -liquid interface,ALI)培养是指细胞的顶层暴露于空气中,底层与液体培养基接触,而不是完全浸入培养基中㊂ALI 培养主要用于构建呼吸道和皮肤模型,还用于肾和脑类器官[12,29-30],有利于呼吸道纤毛细胞和皮肤表皮细胞等细胞的定向分化和增殖㊂ALI 法有利于氧传输,提高了培养体系内的氧浓度,ALI 培养体系中的氧梯度相比于浸没培养体系降低了1.5倍[13]㊂因此,ALI 法改善了细胞氧合作用,进而促进类器官的自组织,有助于其维持更长时间㊂例如,ALI 培养体系中的大脑类器官可以维持更长时间,从而促使模型进一步成熟[29]㊂ALI 培养在操作上存在一定的难度,此外在半透膜上培养的类器官模型难以进行分离和高通量毒性测试[31]㊂. All Rights Reserved.36㊀生态毒理学报第16卷2.5㊀器官芯片技术传统的类器官培养需在特定时间点向培养基中加入外源因子,外源因子和细胞分泌的可溶性因子在干细胞的局部微环境中自发扩散,不容易控制,无法精确模拟体内器官发育关键因子的梯度分布㊂器官芯片技术在体外模拟机械力刺激㊁特定信号通路和化学梯度等复杂条件的组织器官生理微环境,从而精确地调控细胞行为,减少类器官构建的变异性[32]㊂微流控芯片技术能促进内分泌细胞分化和胰岛类器官成熟,Tao等[33]采用人诱导多能干细胞在多层可灌注芯片上动态灌注培养出了胰岛类器官,相比于静态培养形成的胰岛类器官,对于葡萄糖的刺激更敏感,且胞浆中Ca2+通量更高㊂此外,可通过器官芯片技术模拟灌注血管,Shirure等[34]构建的肿瘤类器官芯片器件由3个相互连接的隔室组成,可支持内皮细胞自组装形成血管,是解决3D细胞培养中血管形成难题的重要策略之一㊂Wang等[35]提出了一种在微流控芯片细胞灌注式培养系统中构建肝类器官的方法,通过对微阵列的尺寸进行优化,调控类器官大小与均一性㊂由于微流控芯片技术是按照预先确定的方式设计和构建的,对细胞的大小㊁形状和相对排列的控制存在一定的局限性[32]㊂2.6㊀3D生物打印3D生物打印将生物材料和活细胞在指定的逐层堆叠的组织中同步定位,与传统的组织构造方法相比,采用3D生物打印法构建类器官具有精确㊁可重复及尺寸可控性强的优势[36],能够精确地定位活细胞㊁蛋白质㊁DNA㊁药物㊁生长因子和其他生物活性物质的时空分布,以控制生物组织的形成㊂大部分的3D组织模型缺乏组织界面,如血管内皮组织与周围结缔组织和实质细胞之间的界面,而组织界面对器官功能至关重要[37]㊂King等[38]利用3D生物打印平台开发了近端人肾小管模型,由肾成纤维细胞㊁内皮细胞和原代人肾小管近端内皮细胞形成了组织界面,该模型暴露于顺铂后,组织活性呈剂量依赖性降低,且通过组织病理学观察到上皮细胞数量也呈剂量依赖性降低,通过抑制阳离子摄取转运蛋白OCT2可保护其免受这种损伤㊂Nguyen等[39]报道,通过生物打印的人类肝脏组织在培养4周时仍能维持ATP㊁白蛋白水平以及细胞色素P450酶活性,且对肝毒性药物曲氟沙星和结构类似的左氧氟沙星表现出不同的毒性反应,表明该肝组织模型能够区分结构高度相似的药物毒性㊂3D生物打印的局限性主要在于打印过程中细胞存活率降低,影响类器官活性,此外生物打印的类器官数量较为有限[40]㊂3㊀类器官的组织类型(Tissue-specific organoid types)㊀㊀根据模拟组织器官的类型,类器官模型主要包括皮肤㊁肝㊁肾㊁肺㊁脑㊁心脏和生殖类器官等㊂此外,也有研究报道成功构建了肠㊁乳腺㊁前列腺㊁胰腺和视网膜等类器官模型[10,41]㊂3.1㊀皮肤类器官欧盟提出从2009年3月开始禁止动物试验用于化妆品安全性评价[42],极大地促进了3D皮肤组织模型的发展㊂其中较早投入使用的模型有Episkin TM模型和Epikutis®3D表皮模型㊂Episkin TM 模型将分离的人表皮细胞接种在特定的生物材料上培养,形成具有3D结构的人表皮模型,已经被广泛应用于皮肤腐蚀性体外试验[43]㊂张光甫等[44]依据Epi Derm的构建原理采用人皮肤成纤维细胞与鼠尾胶原混合培养模拟真皮层,在真皮层上接种人角化细胞,经气液培养形成表皮层,构建人皮肤模型,并利用该模型对10种杀菌类农药的皮肤腐蚀性和刺激性进行检测,腐蚀性检测结果与动物评价测试结果的一致率为100%,刺激性结果的一致率达到80%,表明重组人皮肤模型在农药品皮肤毒性评价中具有良好的应用前景㊂Liu等[45]构建的由上皮细胞㊁成纤维细胞和内皮细胞组成的无支架双膜皮肤模型,与成纤维细胞和内皮细胞的共培养以及单一培养相比,3种细胞之间的相互作用上调了血管化相关的生长因子VEGF㊁bFGF和PDGF的表达,形成了更丰富的血管㊂3.2㊀肝脏类器官肝脏是人体最重要的解毒器官,也是很多外源化学物毒性作用的靶器官,构建肝细胞体外3D模型可以对化学物的毒性进行更有效的检测㊂2013年,Takebe等[46]将肝细胞㊁人脐静脉内皮细胞和人间充质干细胞共培养,模拟肝脏发育早期的细胞谱系,并通过内皮细胞和间充质干细胞的相互作用,产生了类似人肝芽组织的3D聚合物,移植入小鼠体内后具有良好的血管生成功能,该研究被Science评为2013年的十大突破之一㊂不足之处是这些体外培养的肝芽组织缺乏胆管结构㊂2017年,Vyas等[47]利用肝细胞胞外基质支架,使人胎儿肝祖细胞自组织形成了肝类器官,提升了对肝胆器官形成过程的模拟效果,同步形成了分化的肝细胞和胆管结构㊂. All Rights Reserved.第4期刘薇等:3D类器官模型的研究进展及其在化学品毒理学评价中的应用展望37㊀Shinozawa等[48]利用多能干细胞构建了具有胆汁转运功能的肝脏类器官,含有具有胆管样结构的极化未成熟肝细胞,建立了胆汁酸的单向转运途径,通过测试存活率㊁胆汁淤积和线粒体毒性,对238种市售药物的肝毒性具有较高的预测能力(敏感性:88.7%,特异性:88.9%),并将其转化为适用于384微孔板的高速实时成像分析平台㊂3.3㊀肾脏类器官原代肾上皮细胞在分离后20min内即丧失细胞特征,在单层培养条件下几周内即完全去分化,因此肾毒性评估通常使用永生化细胞系㊂但永生化细胞系往往不具备诱发毒性的基本分子结构,例如摄取化学物的小分子转运体和部分毒性靶标受体,并且存在药物外排能力低等局限[49-50]㊂在肾小球类器官模型中,可诱导表达裂隙隔膜㊁肾脏滤过功能和肾小球发育相关基因,而且在阿霉素作用48h后, MAFB-BFP2强度呈剂量依赖性下降[51]㊂Astashkina 等[52]报道,庆大霉素等4种肾毒性药物作用于肾小管类器官,药物扩散较好,而且能够诱发和体内毒性相似的效应,包括尿N-乙酰-β氨基葡萄糖苷酶和γ-谷氨酰转移酶升高㊁炎症因子升高㊁肾毒性蛋白和基因标志物升高等,而永生化细胞HEK293和LLC-PK1则无相关毒性反应㊂此外,在单层培养的肾小管细胞中,细胞色素P450活性迅速丧失,而在3D 培养中可持续表达㊂3.4㊀肺类器官肺癌的发病率和死亡率占恶性肿瘤的首位, 2018年全球癌症统计报告显示,185个国家地区的肺癌发病率在36种癌症中占11.6%,有176.64万人因肺癌死亡,约占癌症死亡总人数的18.4%[53],室内外环境空气污染是呼吸道疾病的主要诱因之一㊂人体气道上皮是吸入空气污染物的主要毒性靶区,且人呼吸道上皮基底细胞中表达CYP450代谢酶,基于该细胞的肺类器官成为体外吸入毒理学研究的理想模型[17]㊂气管结构的出现是肺类器官构建成功的重要标志[10]㊂Desai等[54]构建的肺3D模型,可在培养基中维持100d以上,并形成组织良好的近端气道上皮结构,包括基底细胞和纤毛细胞以及少量club细胞㊂Treutlein等[55]建立的3D肺类器官可以表达远端肺上皮细胞的标记物Sftpc/Sox9和Hopx/ Sox9㊂Yamamoto等[56]诱导人多能干细胞培育出肺泡类器官,经GNE7915和胺碘酮处理后Ⅱ型肺泡上皮细胞的板层小体明显增大,与体内毒性反应一致㊂最近,Takayama[57]利用支气管类器官,发现SARS-CoV-2病毒的主要靶标是呼吸道上皮基底细胞,并导致I型干扰素信号升高㊂EpiAirway TM是一个商品化的肺3D组织模型[58],具有良好的均一性和重复性,由正常人气管/支气管上皮细胞培养形成高度分化的假复层上皮组织模型,包含基底细胞㊁杯状细胞和纤毛细胞,其黏液纤毛表型与人类呼吸道上皮组织非常相似[59]㊂EpiAirway TM测试预测强吸入毒性化学物质与动物测试效果相当,预测中/低毒性呼吸道刺激物的毒性效应优于动物测试[60]㊂Chang 等[17]比较了不同多环芳烃类化合物对EpiAirway TM 模型转录组学特征的影响,指出可利用该方法识别多环芳烃类化合物的致癌机理㊂Hild和Jaffe[61]利用Matrigel支架建立了一种不依赖ALI培养的高通量类支气管模型培养方法,使用384孔细胞培养板即可,在该培养体系中人呼吸道上皮基底细胞可分化为杯状细胞和纤毛细胞㊂Liu等[62]进一步考察了该方法的性能,发现在一定的初始细胞接种数量和细胞代际范围内,细胞群落形成效率和支气管模型尺寸变异性较小,具有良好的稳定性㊂3.5㊀脑类器官人脑的复杂性极高,很难采用动物模型研究人类大脑功能,因此迫切需要建立人脑发育体外模型[20]㊂脑类器官模型的转录组和表观遗传组学特征和胎儿大脑相似,有利于研究致畸物对中枢神经系统发育的毒性作用和机理㊂利用单细胞测序比较胎儿大脑和脑类器官皮层细胞组成和谱系关系,发现超过80%的皮层疾病或进化相关的差异表达基因在脑类器官中表达模式相似[63]㊂2013年,Lancaster 等[20]培养出与9~10周胚胎大脑类似的类大脑 ,该模型可以形成分离的但相互依存的不同脑区,如大脑皮层和脑膜等,极少一部分会分化成海马体,该脑类器官表现出人大脑皮层发育的重要特征,即特征性前体区域组织,其中含有大量的放射状胶质干细胞㊂2015年,Kirwan等[64]构建了人大脑皮层神经网络,能够模拟体内皮质网络的发育和功能,可用于人类前脑神经网络生理学机制的研究㊂乙醇作用于脑类器官造成祖细胞过早分化㊁神经突生长抑制及细胞死亡,且转录组学分析发现一系列新的毒性靶基因和信号转导通路,包括GSX2㊁RSPO2和Hippo 信号通路[65]㊂尼古丁损害脑类器官的皮层发育,造成神经元分化和迁移异常[66]㊂类前脑经双酚A暴露,对神经祖细胞增殖和脑区厚度产生剂量依赖性. All Rights Reserved.。

硫化砷渣的碱性浸出及浸出动力学

在不同温度下，测得砷浸出率随反应时间的关系如图６所示。
从图６可知，砷浸出率随反应时间延长和反应温度增加而增加，两者呈线性关系；当反应时间为６０ｒａｉｎ，反应温度分别为２５，４０，５５，７０和９０℃时，砷浸出率分别达到９２．７２％。９４．５０％，９５．００％，９５．５０％和９６．４０％。２．２．２浸出动力学
硫化砷渣中固体Ａｓ２Ｓ３与ＮａＯＨ溶液反应，属于液固相反应，而且Ａｓ２Ｓ３与ＮａＯＨ反应无固相产物生
万方数据
第２期
白猛，等：硫化砷渣的碱性浸出及浸出动力学
２７１
近似不变时，，ｆ可识为常数，ｌ诉则可表示为目其中ｋ为表观反应速率常数１。
根据图６，作１－（１一ｒ／（Ａｓ））２／３与反应时间的曲线，结果如图７所示；根据图７求出不同温度下各直线斜率即ｋ，作Ｉｎｋ与ｌｉＴ关系图，如图８所示。
０．０８７％ｗｈｅｎｔｈｅｒｅａｃｔｉｏｎｔｅｍｐｅｒａｔｕｒｅｉＳ９０℃，ｔｈｅｒａｔｉｏｏｆｓｏｌｉｄｔｏｌｉｑｕｉｄｉＳ１：６，ｔｈｅｒｅａｃｔｉｏｎｔｉｍｅｉｓ１．Ｓｈ，ｔｈｅｍｏｌｅｒａｔｉｏ
ｏｆＮａＯＨｔｏＡｓ２．Ｓ１ｉｓ７．２：１．ｎｅｍａｓｓｆｉ＇ａｃｔｉｏｒ琏ｏｆＣｕａｎｄＢｉｉｎｔｈｅｌｅａｃｈｅｄｓｌａｇｉｎｃｒｅａｓｅｆｒｏｍ１０．９０６／０ａｎｄ１．８５％ｔｏ５０．００％
氢氧化钠浸出硫化砷渣动力学进行探讨。研究结果表明：当反应温度为９０℃，固液比为ｌ：６，反应时间为１．５ｈ，
ＮａＯＨ与Ａｓ２Ｓ３的摩尔比为７．２：ｌ时，氢氧化钠浸出硫化砷渣。砷浸出率达到９５．９００，铜浸出率仅为０．０８７％；经
过氢氧化钠浸出，渣中Ｃｕ和Ｂｉ质量分数分别从１０．９０％和１．８５％增加到５０．００％和１０．６３％，Ｃｕ和Ｂｉ得到高度富

杨梦苏教授1984年毕业于厦门大学,1988年获

简介杨梦甦教授1984年毕业于厦门大学化学系，1993年获得加拿大多伦多大学博士学位，1993-1994期间在美国加州生物医学机构Scripps Research Institute从事研究工作。

1994年底加入香港城市大学，现任香港城市大学生物及化学系讲座教授和香港城市大学深圳生物医药中心主任，并兼任英国中兰开夏大学、浙江大学、解放军第三军医大学、沈阳药科大学、及解放军总医院客座教授。

在行政工作方面担任香港城市大学校董会荣誉学位委员会、大学顾问委员会、大学教务会、研究生院理事会等多个委员会成员。

杨教授主要研究领域为生物芯片技术的开发与应用和纳米生物领域的基础与应用研究, 1995年迄今成功获得包括国家高科技发展计划（863计划）、香港特区政府创新科技基金和研究资助局、及香港赛马会基金等逾6千万港元研究基金的资助，负责管理多个大型科研项目，亦是香港特区政府大学资助委员会之“药物发现与合成分子技术研究”、“中药基础研究与开发”、“海洋环境研究与创新技术”三个卓越学科的主要成员之一。

迄今在国际权威杂志共发表论文160余篇，专著章节17篇，在国内核心杂志发表论文50余篇，在大型国际会议共发表论文80余篇，应邀在各大学和研究机构做过逾70场学术报告，在相关领域申请/获得20多项中国及美国专利。

杨教授实验室已培训18名博士、8名硕士及16名博士后。

杨教授为香港特区政府创新科技基金评审委员会和健康防护基金评审委员会成员，及香港特区政府创新科技署纳米技术和先进材料研究院(NAMI)技术顾问，并兼任英国皇家化学会专业杂志《The Analyst》和中国化学会专业杂志《生命科学仪器》编辑委员会成员，德国《Microchimica Acta》国际顾问委员会成员，以及国际生物芯片大会学术委员会和世界华人高科技化学大会学术委员会成员。

杨教授从2002年主持筹建香港城市大学深圳生物医药中心，领导该中心成功获得包括国家高科技发展计划（863）课题及广东省科技重点攻关项目的资助，并得到深圳市/广东省政府的多次奖励，被授予深圳市生物芯片重点实验室的称号。

生物酶在制浆造纸过程中的应用及研究进展

摘要：基于生物酶的专一性、高效性和环境友好性，生物技术在制浆造纸工业已有一些成熟应用，并取得了良好的经济和环境生态效益。

本文介绍了纤维素酶、半纤维素酶、木素降解酶、果胶酶等生物酶在生物制浆、生物漂白、酶促磨浆/打浆、酶法脱墨、纤维酶法改性、生物法树脂障碍控制、生物法处理制浆废水等制浆造纸各单元中的应用和研究进展。

关键词：生物酶；生物制浆；酶法脱墨；生物漂白Abstract: Based on the specif icit y, high eff iciency and environmental friendliness of biological enzymes, biotechnology has been applied in pulp and paper industry and achieved good economic and environmental ecological benefits. The application and research progress of cellulase, hemicellulase, lignin degrading enzyme, pectinase and others in various units of pulping and papermaking, such as biological pulping, biological bleaching, enzymatic refining/beating, enzymatic deinking, cellulase modification, biological resin barrier control and biological treatment of pulping wastewater, were introduced in this paper.Key words: biological enzyme; bio-pulping; enzymatic deinking; bio-bleaching生物酶在制浆造纸过程中的应用及研究进展⊙ 陈嘉川1贾倩倩1李凤凤1薛玉1王东兴2胡长青3杨桂花1,*（1.齐鲁工业大学（山东省科学院）生物基材料与绿色造纸国家重点实验室/制浆造纸科学与技术教育部重点实验室，山东济南 250353；2.山东世纪阳光纸业集团有限公司，山东昌乐 262400；3.山东晨鸣纸业集团股份有限公司，山东寿光 262700）Application and Research Progress of Biological Enzymes in Pulp and Paper Making Process⊙ Chen Jiachuan 1, Jia Qianqian 1, Li Fengfeng 1, Xue Yu 1, Wang Dongxing 3, Hu Changqing 3, Yang Guihua 1,*(1.State Key Lab of Bio-based Materials and Green Papermaking /Key Lab of Pulp & Paper Science and Technology of Education Ministry of China, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, Shandong, China; 2.Shandong Century Paper Group Co., Ltd., Changle 262400, Shandong, China; 3.Shandong Chenming Paper Group Co., Ltd., Shouguang 262700, Shandong, China)□ 基金项目：国家自然科学基金（31770628，31901273）；山东省科教产融合创新试点工程（2020KJC-ZD14）。

生物反应器工程国家重点实验室-华东理工大学

2010年学术活动第169次SKLBE学术论坛作者：管理员发布：2010年12月16日浏览次数: 172报告人：中国科学技术大学生命科学学院合肥微尺度物质科学国家实验室（筹）刘海燕教授报告题目：分子间相互作用的再造与合成生物学元件摘要：合成生物学的重要理念之一是用模块化、标准化的单元构建有功能的生物系统。

在生物系统中，不同单元之间的“接口”主要需通过分子间相互作用实现，相互作用的特异性决定了不同“接口”之间的绝缘性。

由于生物大分子序列、结构和功能具有模块化的特点，通过分子间相互作用的设计和再造可作为一种通用的元件设计策略，包括：通过对酶-底物结合特异性的改造获得新的催化分子；通过对信号传导分子间结合特异性的改造改变信号通路、通过再造蛋白质-DNA、小分子-RNA相互作用获得通用的转录调控元件等。

本文将结合我们在计算方法、人工转录元件设计方面的工作讨论蛋白质参与的分子间相互作用再造。

时间：2010-12-16 下午2∶00地点：实验十八楼315会议室第170次SKLBE学术论坛作者：管理员发布：2010年12月15日浏览次数: 96报告人：复旦大学生科院副院长、复旦大学遗传工程国家重点实验室副主任王红艳教授报告题目：出生缺陷的遗传基础研究时间：2010-12-15下午3∶30地点：实验十八楼315会议室第168次SKLBE学术论坛作者：管理员发布：2010年12月2日浏览次数: 168报告人：生物芯片上海国家工程研究中心副主任张庆华研究员报告题目：蛋白磷酸化抗体芯片技术及在生命科学研究中的应用报告内容：1. 蛋白芯片技术的发展状况 2. 抗体芯片技术应用案例 3. 蛋白磷酸化抗体芯片的技术与产品特点 4. 蛋白与抗体芯片技术的应用与服务时间：2010-12-2 下午3：15地点：实验十八楼315会议室第167次SKLBE学术论坛作者：管理员发布：2010年11月29日浏览次数: 101报告人：日本九州工业大学清水和幸教授(Prof. Kazuyuki Shimizu)题目: Metabolic reguration analysis of a cell focusing on the relationship between global regulators and the metabolic pathway genes together with 13C-metabolic flux analysis时间: 2010年11月29日上午9:00地点: 实验十八楼315室第166次SKLBE学术论坛作者：管理员发布：2010年11月25日浏览次数: 104报告人：华东理工大学资源与环境工程学院危险化学物质风险评价与控制中心教授、博士生导师冯耀宇教授题目: 水回用和水安全—新发水源性病原微生物的检测和污染源追踪时间: 2010年11月25日下午2:00地点: 实验十八楼315室第165次SKLBE学术论坛作者：管理员发布：2010年10月27日浏览次数: 101报告人：Hesham M. Amin, Assiociate Professor, The University of Texas M. D. Anderson Cancer Center, Houston, TX题目: Novel oncogenic systems in lymphoma时间: 2010年10月27日星期三9:30地点: 实验十八楼315室第164次SKLBE学术论坛作者：管理员发布：2010年10月19日浏览次数: 105报告题目：1.含纤维原料制取生物友好型蛋白-糖饲料添加剂2.石油污染土壤生物修复方法,高效应用和反应条件报告人：俄罗斯科学院通讯院士、微生物学家、门捷列夫化工大学生物工程教研组教授妮娜.葛拉朵娃教授时间：2010年10月19上午9:45地点：实验18楼315室第163次SKLBE学术论坛作者：管理员发布：2010年10月18日浏览次数: 103报告题目：嗜热酶结构、功能及分子进化报告人：上海交通大学生命科学技术学院冯雁教授时间：2010年10月18日上午10:30-11:30地点：实验18楼315室第162次SKLBE学术论坛作者：管理员发布：2010年10月18日浏览次数: 119报告题目：Microbial Synthesis of Drugs and Fuels via Synthetic Biology报告人: 美国伊利诺伊大学厄巴纳香槟分校赵惠民教授时间：2010年10月18日上午9:00-10:30地点：实验18楼315第161次SKLBE学术论坛作者：管理员发布：2010年10月16日浏览次数: 106报告题目：Inorganic Surfaces that Selectively Bind Phosphate: Applications in Bioarrays, Biotargeting, and Supported Lipid Bilayers报告人：美国Florida Univeristy化学系主任Dan Talham教授时间：2010年10月16日上午9:30地点：一教101室第160次SKLBE学术论坛作者：管理员发布：2010年10月14日浏览次数: 101报告题目：Growth and Lipidomics Characterization of Nannochloropsis salina for Biofuels报告人：Prof. Peter Lammers, New Mexico State University, USA时间：2010年10月14日上午9:00地点：实验十八楼315室第159次SKLBE学术论坛作者：管理员发布：2010年10月13日浏览次数: 106报告题目：Refining the Vision for Cost-Competive Biofuel Production from Microalgae报告人：Prof. Bryan Willson, Colorado State University, USA时间：2010年10月13日上午9:00地点：实验十八楼315室第158次SKLBE学术论坛作者：管理员发布：2010年9月26日浏览次数: 107报告题目：极端环境微生物资源与利用报告人：浙江大学生命科学学院微生物研究所博士生导师吴敏教授报告时间：2010年9月26日下午2:30地点:实验18楼315室第157次SKLBE学术论坛作者：管理员发布：2010年9月20日浏览次数: 121报告题目：纤维素乙醇糖平台技术的研究进展和展望报告人：山东大学微生物技术国家重点实验室、国家糖工程技术研究中心方诩教授报告时间：2010年9月20日下午2:00地点:实验18楼315室第156次SKLBE学术论坛作者：管理员发布：2010年9月6日浏览次数: 110报告人：我校特聘讲座教授、日本立命馆大学今中忠行教授题目：Application of nano-bubble to biological systems时间：2010年9月6日上午9:00地点：实验十八楼315室第155次SKLBE学术论坛作者：管理员发布：2010年9月1日浏览次数: 93报告人：庞正宇博士，通用电气公司全球研发中心生命科学技术部，高级研究员题目：molecular pathology时间：2010年9月1日下午1:30地点：实验十八楼315室第154次SKLBE学术论坛作者：管理员发布：2010年8月25日浏览次数: 113报告人：Junyong Zhu, Ph.D Scientific Team Leader USDA Forest Products Laboratory题目：Studies on Cellulase Adsorption in Lignocellulosic Suspensions时间：2010年8月25日星期三下午2:00地点：实验十八楼315室第152次SKLBE学术论坛作者：管理员发布：2010年7月23日浏览次数: 106报告人：Dawei Lin, Ph.D. Director, Bioinformatics Core, UC Davis Genome Center题目：Next Generation Biology Through Next Generation Sequencing时间：2010.7.23日(星期五)下午3:00-4:30地点：实验十八楼315室第153次SKLBE学术论坛作者：管理员发布：2010年7月23日浏览次数: 104报告人：国家自然科学基金会审专家、校学位评定委员会委员、生物工程学院院长张元兴教授题目：国家自然科学基金和职称晋升的申请、答辩与评审时间：2010.7.23日(星期五)上午9:00地点：实验十八楼315室第151次SKLBE学术论坛作者：管理员发布：2010年7月21日浏览次数: 120报告人：美国佛罗里达大学化学系V.T and Louis Jackson杰出讲座教授谭蔚泓教授题目：Elucidation of the Molecular Foundation of Cancer:A Chemical Biology Approach时间：2010.7.21日(星期三)下午14:00地点：实验十八楼315室第150次SKLBE学术论坛作者：管理员发布：2010年7月9日浏览次数: 125报告人：华东理工大学药学院杨有军副教授题目： A Highly Selective Low-Background Fluorescent Imaging Agent for NO using Chemodosimeter Involving *in situ* Fluorophore Synthesis (CIFS) Approach时间：2010-7-9(周五)上午9:00地点：实验十八楼315室第149次SKLBE学术论坛作者：管理员发布：2010年7月1日浏览次数: 1811.报告人：Kal Karim 博士，英国克兰菲尔德大学题目：Advancing a healthy life- Education and business activities in Cranfield Health时间：2010-7-1（星期四）上午10:30地点：实验十八楼315室2.报告人：葛屹博士，英国克兰菲尔德大学题目：Exploring unique and holistic solutions to facilitate and promote good health - Cranfield Health’s cuttin g edge researches & technologies时间：2010-7-1（星期四）下午14:00地点：实验十八楼315室3.报告人：李松军博士，英国克兰菲尔德大学题目：智能分子印迹聚合物设计、合成及其可控制、可调节的催化作用时间：2010-7-1（星期四）下午15:30地点：实验十八楼315室第148次SKLBE学术论坛作者：管理员发布：2010年6月29日浏览次数: 122报告人：Dr. Zhen-Yu CHEN (陳振宇教授)Associate Editor, Journal of Agricultural and Food Chemistry题目：How to Write a Manuscript如何撰写高水准的学术论文时间：2010-6-29（星期二）下午2:00地点：实验十八楼315室第147次SKLBE学术论坛作者：管理员发布：2010年6月9日浏览次数: 104报告人: Department of Pharmaceutical Sciences, University of Tennessee Health Science Center Ram I Mahato, PhD题目: Polymeric Nanomedicines and Combination Therapy for treating Prostate Cancer时间: 2010年6月9日星期三下午3:00地点: 实验十八楼315室第146次SKLBE学术论坛作者：管理员发布：2010年6月7日浏览次数: 107报告人:瑞典Attana生物传感器公司高级研究员\生物芯片研发部经理裴志超题目: QCM生物传感器和微阵列技术研究糖-蛋白，蛋白-蛋白相互作用时间:2010年6月7日星期一下午1:30地点:实验十八楼315室第145次SKLBE学术论坛作者：管理员发布：2010年6月1日浏览次数: 107报告人: 中国药科大学化学生物学研究室主任、特聘教授陈依军题目: 诺卡沙星衍生物及其生物合成途径时间: 2010年6月1日星期二下午2:00地点: 实验十八楼315室第144次SKLBE学术论坛作者：管理员发布：2010年5月28日浏览次数: 111报告人: 美国德州农工大学刘文设教授题目: Expanding the Genetic Code时间: 2010年5月28日星期五上午10:00地点: 实验十八楼315室第143次SKLBE学术论坛作者：管理员发布：2010年5月26日浏览次数: 118报告人: 美国弗吉尼亚理工大学、美国能源部生物能源科学中心张以恒教授(我校校友) Y.-H. Percival Zhang, Associate Professor, Virginia Polytechnic Institute and State University, DOE BioEnergy Science Center (BSEC)题目: Constructing the Carbon-Neutral Carbohydrate Economy for Sustainability时间: 2010年5月26日星期三下午3:00地点: 实验十八楼315室第142次SKLBE学术论坛作者：管理员发布：2010年5月24日浏览次数: 214报告人: 乔治亚医学院生物化学和分子生物学系Huidong Shi（石慧东）教授题目: Next-Generation Sequencing: Applications Beyond Genomes时间: 2010年5月24日星期一上午10:00地点: 实验十八楼315室第141次SKLBE学术论坛作者：管理员发布：2010年5月21日浏览次数: 239报告人：日本京都大学Sugiyama教授题目:Observation and Control of Enzymatic Reaction in DNA Nano Frame时间: 2010年5月21日星期五下午3:30地点: 实验十八楼315室第140次SKLBE学术论坛作者：管理员发布：2010年5月10日浏览次数: 101附件为第140次SKLBE学术论坛信息,请阅。

MLST在乳酸菌鉴定及其多样性分析中的应用

2021年5月第42卷第10期食品研究与开发专题论述~=~218DOI : 10.12161/j.issn.l005-6521.2021.10.034MLST 在乳酸菌鉴定及其多样性分析中的应用赵尘培,姜琳琳* ，张建龙,陈国忠，于馨，朱洪伟,张兴晓*基金项目:山东省自然科学基金项目(ZR2020QC227)；烟台市重点研发计划(2020YT06000171)作者简介:赵尘培(1996-),女(汉)，硕士研究生,研究方向:乳酸菌筛选及鉴定。

*通信作者凄琳琳(1986-),女(汉)，讲师，博士，研究方向:益生菌功能研究;张兴晓(1973-)，男(汉)，教授，博士，研究方向:微生物与免疫学研究。

(鲁东大学生命科学学院，山东烟台264025)摘要:乳酸菌是健康人类及动物肠道中重要的益生菌群，在维持肠道微生态平衡方面发挥重要作用。

由于乳酸菌的益生功能具有菌株特异性，因此•准确、灵敏的分子鉴定技术对乳酸菌功能的研究十分重要。

多位点序列分型(multilocus sequence typing , MLST )是一种基于核酸序列测定的细菌分型方法,能够准确地从亚种水平进行菌株分类，在鉴定物种的遗传多样性方面发挥重要作用。

该文概述MLST 技术的原理、方法、优缺点及其在乳酸菌分类鉴定及遗传多样性方面的研究现状,为乳酸菌株的鉴定提供理论依据。

关键词：乳酸菌；多位点序列分型;鉴定;遗传多样性Application of MLST in Identification and Diversity Analysis of Lactic Acid BacteriaZHAO Chen-pei, JIANG Lin-lin *, ZHANG Jian-long, CHEN Guo-zhong, YU Xin, ZHU Hong-wei,ZHANG Xing-xiao *(School of Life Sciences , Ludong University , Yantai 264025, Shandong , China )Abstract : Lactic acid bacteria is a type of important probiotic bacteria found in the intestines of healthy humansand animals. It plays a crucial role in maintaining the dynamic equilibrium of the intestinal microecology. Because their functions are strain -specific , it is essential to develop a rapid and reliable molecular markertechnique for the identification of different species of lactic acid bacteria. Multilocus sequence typing (MLST) isa method employed for bacterial gene typing and is based on DNA sequencing. Moreover, MLST can simultaneously identify strains at the subspecies level and has been used for the analysis of the genetic diversityof lactic acid bacteria. The aim of the present study was to describe the basic principle , methods , advantagesand disadvantages of MLST, while highlighting the suitability of MLST for the identification and classification oflactic acid bacteria.Key words : lactic acid bacteria; multilocus sequence typing; identification; genetic diversity引文格式：赵尘培，姜琳琳，张建龙，等.MLST 在乳酸菌鉴定及其多样性分析中的应用[J].食品研究与开发，2021, 42(10):218-224.ZHAO Chenpei , JIANG Linlin , ZHANG Jianlong , et al. Application of MLST in Identification and Diversity Analysis ofLactic Acid Bacteria[J]. Food Research and Development , 2021, 42( 10) ： 218—224.乳酸菌(lactic acid bacteria,LAB)是一类能够发酵糖类物质最终产物为乳酸的革兰氏阳性细菌。

蚕丝丝素蛋白材料的生物降解性能研究进展

蚕丝丝素蛋白材料的生物降解性能研究进展徐亚梅;李明忠【摘要】Silk fibroin has good biocompatibility. However, being used as tissue engineering scaffold, one of the key factors should be considered, which is the biodegradation rate of the silk fibroin should match the formation speed of a new tissue. This article reviewed the research development of biodegradation properties of silk fibroin biomaterials at home and abroad in recent years. The results show that influence factors of materials degradation performance include material form, structure, mechanical and physiology environment of the implant point, etc. The detailed process and mechanism of degradation behavior influenced by these parameters will be the research emphasis in the future.%丝素蛋白具有良好的生物相容性,但其用于制备组织工程支架等生物材料时,制成的材料还需具备的一个重要条件是其降解速率与组织新生的速率相匹配.近年来国内外对丝素蛋白材料生物降解性能的研究进展表明,影响材料降解性能的因素包括材料的形态、结构、植入点的机械和生理环境等.这些参数影响降解行为的具体过程和机理将是今后的研究重点.【期刊名称】《丝绸》【年(卷),期】2011(048)005【总页数】4页(P19-22)【关键词】蚕丝;丝素;生物材料;降解【作者】徐亚梅;李明忠【作者单位】苏州大学纺织与服装工程学院,江苏苏州 215021;现代丝绸国家工程实验室,江苏苏州 215123【正文语种】中文【中图分类】T5149生物医用材料是用于对生物体进行诊断、治疗、修复或替换其病损组织及器官，或增进其功能的新型高技术材料[1]。

Chen_et_al-2013-Journal_of_Pharmacy_and_Pharmacology

Enhanced brain targeting of curcumin by intranasaladministration of a thermosensitive poloxamer hydrogelXi Chen a,b *,Feng Zhi a,c *,Xuefeng Jia c ,Xiang Zhang c ,Rohan Ambardekar e ,Zhengjie Meng d ,Anant R.Paradkar e ,Yiqiao Hu c and Yilin Yang aaModern Medical Research Center,Third Afﬁliated Hospital of Soochow University,Changzhou,b College of Pharmacy,China PharmaceuticalUniversity,c State Key Laboratory of Pharmaceutical Biotechnology,School of Life Sciences,Nanjing University,d Biotechnology and Pharmaceutical Engineering,Nanjing University of Technology,Nanjing,China and e Institute of Pharmaceutical Innovation,University of Bradford,Bradford,UKKeywordscurcumin;nasal delivery;thermosensitive hydrogelCorrespondenceYilin Yang,Modern Medical Research Center,Third Afﬁliated Hospital of SoochowUniversity,#185Juqian Road,Changzhou,Jiangsu 213003,China.E-mail:yilinyang.czfph@ Yiqiao Hu,Room 1416,MengminweiBuilding,Nanjing University,Nanjing,Jiangsu 210093,China.E-mail:hu_yiqiao@Anant R.Paradkar,Norcroft Building (ex IPI),3.17,School of Life Sciences Engineering Design and Technology,University of Bradford,Bradford BD71DP ,UK.E-mail:a.paradkar1@ Received September 19,2012Accepted January 6,2013doi:10.1111/jphp.12043*These authors contributed equally to this work.AbstractObjectives The aim of this study was to develop a curcumin intranasal thermo-sensitive hydrogel and to improve its brain targeting efﬁciency.Methods The hydrogel gelation temperature,gelation time,drug release and mucociliary toxicity characteristics as well as the nose-to-brain transport in the rat model were evaluated.Key ﬁndings The developed nasal hydrogel,composed of Pluronic F127and Poloxamer 188,had shorter gelation time,longer mucociliary transport time and produced prolonged curcumin retention in the rat nasal cavity at body tempera-ture.The hydrogel release mechanism was diffusion-controlled drug release,evaluated by the dialysis membrane method,but dissolution-controlled release when evaluated by the membraneless method.A mucociliary toxicity study revealed that the hydrogel maintained nasal mucosal integrity until 14days after application.The drug-targeting efﬁciencies for the drug in the cerebrum,cerebel-lum,hippocampus and olfactory bulb after intranasal administration of the cur-cumin hydrogel were 1.82,2.05,2.07and 1.51times that after intravenous administration of the curcumin solution injection,respectively,indicating that the hydrogel signiﬁcantly increased the distribution of curcumin into the rat brain tissue,especially into the cerebellum and hippocampus.Conclusions A thermosensitive curcumin nasal gel was developed with favour-able gelation,release properties,biological safety and enhanced brain-uptake efﬁciency.IntroductionThe blood–brain barrier (BBB)prevents the transport of almost 98%of all small-molecule and 100%of large-molecule pharmaceuticals from the bloodstream into the central nervous system (CNS).[1]Many drug-delivery strate-gies have been developed to overcome this barrier.Recently,the nasal mucosa has been investigated as a route for direct delivery of therapeutics to the CNS,circumventing the BBB.In addition to that,it enhances the systemic concentration of the drug by avoiding ﬁrst-pass elimination.[2–5]Unlike most conventional liquid formulations for nasal delivery,bioadhesive nasal gels have high viscosity,which prolongs the drug contact time and releases the drug in a controlledmanner,which results in improved local and systemic bio-availability,reduced dose requirements,and improved patient safety and acceptability.Consequently,bioadhesive nasal gels could be very useful for efﬁcient delivery of drugs used in the treatment of CNS disorders such as brain tumour and Alzheimer’s disease.[6–12]Curcumin,a naturally occurring o-methoxyphenol derivative,extracted from the rhizome Curcuma longa ,has a long history of use in Asia as a spice as well as in traditional therapies.[13]Curcumin has an outstanding safety proﬁle and a number of pleiotropic actions,including anti-inﬂammatory,antioxidant,antitumoural and antimicrobialAnd PharmacologyJournal of Pharmacy Research Paperactivities,with the potential for neuroprotective activ-ity.[14,15]It has shown excellent efﬁcacy in counteracting neu-ronal dysfunction[16]and in eliminating chemoresistance by sensitizing brain tumours to chemotherapy and radia-tion.[17,18]However,its clinical application has been limited due to its poor aqueous solubility,photosensitivity,rapid hydrolysis at alkaline pH and fast systemic elimination.[19] To be effective as a drug therapy for CNS disorders,curcu-min must be combined with other drugs or new delivery strategies must be developed.[20]In this regard,intranasal delivery seems to be an attractive alternative.[21]The physiological characteristics of the nasal mucosa and nasal mucociliary clearance are the two main considerations in designing nasal formulations.[2,22,23]The strategy to improve nasal drug bioavailability is to increase the drug absorption rate via permeation enhancers[24]or by prolong-ing the drug residence time at the nasal absorption site through hydrogels.[25]Biodegradable,thermosensitive poly-mers have been extensively studied for their utility in for-mulation of thermoresponsive intranasal hydrogels.[8,26–28] Such hydrogels can be dripped or sprayed into the nasal cavity as low-viscosity solutions at room temperature,con-sequently forming more viscous gels when in contact with nasal mucosa.[29]Pluronic F127(PF-127)is one of the most important thermoresponsive hydrogel-forming poloxamers and is widely applied in the biomedicalﬁeld as a topical drug-delivery carrier.[30,31]The block copolymer poloxamer is an important component in thermosensitive hydrogels. Poloxamer consists of blocks of poly(ethylene oxide)(PEO) and propylene oxide(PPO)with a triblock structure:PEO x–PPO y–PEO x.A moderately concentrated solution of PF-127 forms a free-ﬂowing solution at or below ambient tempera-ture and is able to form a gel at body temperature.It can thus be localized in the contact site with sustained release of the drug.PF-127alone has an incipient gelation tempera-ture below25°C and a long gelation time due to the low ratio of PEO to PPO(2.93,w/w,weight/weight),while Poloxamer188(P188)has a higher ratio(3.47,w/w).The addition of P188results in more hydrophilic PEO in the micelle,which increases the incipient gelation temperature and reduces the gelation time.Furthermore,the gelation temperature,also referred to as the sol–gel transition tem-perature,is strongly dependent on the poloxamer type and concentration,the type of solvent and other materials involved in the formulation.[32]The purpose of the present study was to develop a curcu-min mucoadhesive thermosensitive nasal hydrogel capable of undergoing sol–gel transition in the temperature range 32–35°C,thereby allowing a stable liquid state to be main-tained at storage temperature.The hydrogel gelation tem-perature,gelation time,drug release,mucociliary toxicity characteristics and nose-to-brain transport in the rat model were also evaluated.Materials and MethodsAnimalsMale Sprague-Dawley rats weighing about250g were pur-chased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences(Shanghai,China)and main-tained under standard environmental conditions(tempera-ture25Ϯ1°C;relative humidity70%;12-h light/dark cycle with lights on at6:00am)with free access to food and water.Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals(Ministry of Science and Tech-nology of China,2006)and the related ethical regulations of our university.All the animal experiments were approved by Soochow University Animal Care and Use Committee (SDU-ACUC)and were arranged to minimize suffering and to reduce the number of animals used.The approval was given on30March2012,number SCXK2008_0016. MaterialsCurcumin was purchased from Nanjing Zelang Medical Technology Co.Ltd(Jiangsu,China).All the procedures related to curcumin were performed under dim light in order to prevent its degradation.Emodin was supplied by the National Institute for the Control of Pharmaceutical and Biological Products(Beijing,China).PF-127and P188 were obtained from BASF(Hanover,Germany).PEG400, sodium chloride and benzalkonium bromide(BKB)were purchased from Sigma-Aldrich(St Louis,MO,USA).Except for the acetonitrile and methanol(of HPLC grade;Merck, Darmstadt,Germany),all other chemicals used were ana-lytical reagent grade.All the solutions were prepared using water from a Milli-Q Gradientﬁltration system(Millipore, Billerica,MA,USA).Thermosensitive hydrogel preparationThe‘cold’method for preparation of Pluronic gels was adopted.[33]Brieﬂy,different amounts of PF-127and P188 (Table1)were dissolved in sterile distilled deionized water and gently mixed with magnetic stirrers for24h at4°C until all of the Pluronic granules were completely dissolved and a clear solution was obtained.Curcumin(5g)was dis-solved in20ml of an ethanol/PEG400mixture(eth-anol:PEG400=1:1)to form a curcumin solution.The PEG400was part of the curcumin control solution.Then 2ml of curcumin solution was added slowly to the Pluronic solution with continuous stirring.All other excipients such as BKB(0.02%,weight/volume,W/V)and sodium chloride 0.9%(w/v)were added to the mixture.The volume was adjusted with sterile distilled deionized water to achieve a 0.5%(w/v)concentration of curcumin.Xi Chen et al.Enhanced curcumin by hydrogelGelation temperature and gelation time studyThe sol–gel transition temperature (T sol–gel )of the prepared hydrogel was recorded using the test-tube-inverting method.[34,35]Brieﬂy,2ml of hydrogel solution was added to a test-tube (10ml)with a diameter of 1.0cm in a digital circulating water bath (IKA,ETS-D5,Staufen,Germany)at 15°C and sealed with paraﬁlm.The hydrogel was slowly heated,at a rate of 1.0°C/min,from 15°C to the temperature at which the meniscus would no longer move on tilting through 90°.The gelation time of samples at 32°C was determined using the same method.Brieﬂy,the hydrogel solution (2ml)was added to a test-tube (10ml),incubated in a water bath at 32°C and time measurements initiated.The ﬂowability of the sample was observed every 10s by tilting the tubes.The time at which ﬂowing of the samples stopped was taken as the gelation time and the values were recorded.In-vitro release evaluation using the dialysis membrane method and the membraneless diffusion methodThe in-vitro drug release from the hydrogel was studied using the dialysis membrane method and the membraneless diffusion method.For the dialysis membrane method,200m l of liquid hydrogel was introduced into a dialysis membrane bag (Spectrapore,cutoff 1.2–1.4kD,Sigma-Aldrich)and the sealed dialysis bag was incubated in 400ml of release medium (0.8g/l NaCl,3g/l KCl and 0.45g/l CaCl 2,pH 6.8,20%ethanol v/v),maintained at 32°C Ϯ0.5°C and con-stantly stirred at a speed of 50rpm.At 5,10,15,20,25,30,40,50,60,80,120,150,180,210,240,300and 360min,3ml of dissolution medium was withdrawn and the same volume of fresh medium was added.For in-vitro release evaluation using the membraneless diffusion method,a volume of 1ml of the liquid hydrogel at room temperature was put into a pre-weighed empty glass tube (12mm ¥75mm)and then placed in a 32°C water bath until a clear gel formed.The initial weight of each tube plus the gel was recorded.A volume of 1ml ofthe medium (pre-equilibrated at 32°C)was carefully layered over the surface of the gel.The tube was then shaken in a thermostatic shaker (THZ-300,Yiheng,Shang-hai,China)at 32°C and 100rpm.At 10,20,40,60,80,100,120,140and 160min,the release medium was completely replaced by fresh medium,and the weight of the tube plus the gel was recorded to calculate the weight of gel dis-solved.The samples were suitably diluted and measured spectrophotometrically at 426nm (SHIMADZU UV-2450spectrophotometer,Koyoto,Japan).The concentration of the drug was determined from a previously constructed calibration curve.The release experiments were run in triplicate using plain formulations as a blank;the results were averaged.In-vivo mucocilliary transport time evaluationThe in-vivo nasal mucociliary transport time was adopted as per previous reports.[36]For the intranasal administra-tion,the rats were anesthetized with an intra-peritoneal injection of 10%chloral hydrate solution,and 50m l of the hydrogel solution was administered via a polyethylene (PE)10tube attached to a microlitre syringe inserted 0.5cm into right nostril of the rats.The pharyngeal remains of the yellow curcumin were detected by swabbing the oral cavity of the rat with moistened cotton-tipped applicators for 1min at regular time intervals.Curcumin solution (50m l)served as a control and was detected similarly.In-vivo morphological studyThe in-vivo morphological study was carried out as previ-ously described.[37]Brieﬂy,the rats were sedated with an intraperitoneal injection of 10%chloral hydrate solution before nasal administration,and then each rat’s right nostril was treated with 50m l of thermosensitive hydrogel every day.Fourteen days later,the rats were sacriﬁced and the nasal septum,with the epithelial cell membrane on each side,was carefully separated from the bone.The left nostril was used as a control.The samples were ﬁxed,sectioned and stained by hematoxylin and eosin,and were examined under light microscopy (IX71,Olympus,Tokyo,Japan).Table 1The gelation temperature (T )and gelation time (t )of different hydrogelformulationsXi Chen et al .Enhanced curcumin by hydrogelPharmacokinetic analysis and brain tissue distribution studyFor the intranasal administration,the rats were anesthetized with an intra-peritoneal injection of10%chloral hydrate solution,and the calculated volume of hydrogel solution was administered via a PE10tube attached to a microlitre syringe inserted0.5cm into the right nostril of the rats at a dose of250m g/kg.For the i.v.administration,the curcumin solution was injected(250m g/kg)through the caudal vein. The animals were decapitated and the blood was collected from the trunk.The skull was cut open and the cerebrum, cerebellum,hippocampus and olfactory bulb were carefully excised.The brain tissues were quickly rinsed with saline and blotted withﬁlter paper to remove the blood taint and macroscopic blood vessels as much as possible.After weigh-ing,the cerebrum,cerebellum,hippocampus and olfactory bulb samples were homogenized.Blood samples were anti-coagulated with sodium citrate and centrifuged at12000g for10min to obtain the plasma.Both plasma and brain tissue homogenates were stored in a deep freezer at-20°C until HPLC analysis.Measurements were repeated on three rats at each time point.Sample preparation and HPLCTwo hundred microlitres of emodin ethanol solution (1mg/l),0.5ml of ethyl acetate and0.5ml of hexane were added to both the500m l plasma samples and500m l brain tissue homogenates(brain tissue:saline=2:1,w/v).The mixture was vortexed for5min and centrifuged at12000g for10min.The organic phase was transferred to a conical tube and evaporated to dryness under a gentleﬂow of nitrogen at40°C.For the plasma sample,the residue was reconstituted in200m l of acetonitrile and20m l of superna-tant was injected onto the HPLC system.For the brain tissue samples,the residue was reconstituted in200m l of acetonitrile and20m l of supernatant was injected onto the HPLC system after centrifugation at12000g for5min. Samples were quantiﬁed using the peak area ratio of curcu-min to emodin.Curcumin content was measured by HPLC (LC-20A and SPD-20A,Shimadzu,Japan)at a wavelength of426nm,and a Hypersil C18ODS column(5m m, 250mm¥4.6mm;Elite,Dalian,China)was used.The mobile phase(acetonitrile:methanol:water,v/v/ v=60:20:20)was delivered at aﬂow rate of1ml/min at ambient temperature.Statistical analysisStatistical analyses of differences between the various treat-ments were performed using the Kruskal–Wallis test.A0.05 level of probability(P<0.05)was taken as the level of signiﬁcance.ResultsGelation temperature and gelationtime studyThe gelation temperature(T)and gelation time(t)are two important parameters for in-situ forming of hydrogels.The performance criteria of the nasal-delivery formulations are imposed by the physiological temperature of the nasal cavity(32–35°C[38])and by the mucociliary clearance time (half-life~21min[39]),which correspondingly specify the temperature range and time limits for the sol–gel transition. As displayed in Table1,the gelation temperature and gela-tion time varied with the concentration of both PF-127and P188.Increasing PF-127concentration led to a decrease in gelation temperature and an increase in gelation time,while increasing P188concentration had the opposite effect.The gelation temperatures of batches F2and F6were32Ϯ0.5°C and31Ϯ1°C,respectively,close to the physiological tem-perature of the nasal cavity.The gelation times of F2and F6 were0.2Ϯ0.2and0.4Ϯ0.1min,respectively,well below the time for mucociliary clearance.Since F2had a shorter gelation time,it was selected for further analysis.In-vitro release evaluation using the dialysis membrane method and the membraneless diffusion methodThe drug-release behaviours of curcumin solution and F2 wereﬁrstly measured in vitro by the dialysis membrane method.As evident from Figure1a,the release of curcu-min solution was rapid and almost complete within2h, while the incorporation of drug into the hydrogel signiﬁ-cantly retarded drug release to about80%after6h,sug-gesting that most of the drug remained incorporated in the hydrogel under the study conditions.Consequently,the data obtained from the in-vitro release experiments were analysed by the commonly used Peppas exponential equation:[40]log log log.MMk n tt=+where M t/M is the fraction of released drug at time t,k is a release constant and is dependent on structural and geo-metric characteristics of the drug/polymer system,and n is the release exponent and is indicative of the release mechanism.If n<0.45,the drug is released from the polymer with a Fickian diffusion mechanism.If 0.45<n<0.89,this indicates anomalous or non-Fickian release.If n>0.89,the main mechanism is matrix erosion. The results revealed that the hydrogel had a value of n=0.27,indicating that the major mechanism was diffusion-controlled drug release or Fickian diffusion kinetics.When the curcumin-loaded hydrogel was sepa-Xi Chen et al.Enhanced curcumin by hydrogelrated from the release medium by a dialysis membrane,the dissolution of PF-127and P188wase almost prevented,and diffusion dominated the drug-release mechanism.In contrast,the membraneless model,also used in our study,allowed the release medium solution to directly contact the gel surface and thereby dissolve the gel.As shown in Figure 1b,in the membraneless dissolution experiment the volume of gel gradually decreased and completely disap-peared by the ﬁnal time point.Several mathematical models,including the Higuchi release model,zero-order release model and ﬁrst-order release model,were used to describe the kinetic behaviour of curcumin released from the hydrogel in the membraneless diffusion system.The drug-release proﬁle followed the zero-order release model better (R 2=0.9965)than the other two release models.The correlation between gel dissolution and drug release was also investigated.The results showed that the remaining weight percentage of gels also followed zero-order kinetics (R 2=0.9985),and there was a good linear correlation between the percentage of curcumin released and the per-centage of gel dissolved,indicating a distinct polymer dissolution-controlled release mechanism (R 2=0.9910).In-vivo mucoadhesiveness of the nasal in-situ gelsThe hydrogels’in-vivo mucoadhesiveness ability was evalu-ated by measuring the mucociliary transport time,taken as the time for the passage of a coloured dye through the nasal cavity of rats until its appearance in their oropharynx.Cur-cumin is a naturally yellow drug and it could also be used as a dye.Figure 2illustrates that the mucociliary transporttime of the thermosensitive hydrogel is about 67min,which is almost 10-fold higher than that of curcumin solution,which served as a control.In-vivo morphological studyThe safety of the optimized in-situ gel formulation was evaluated by studying the histological changes in the nasal mucosa after administration into the right nostril of experi-mental rats for 14consecutive days (Figure 3).Examination of the dosed (right)and undosed (left)sides of the nasal cavity showed intact ciliated respiratory epithelium and normal goblet cell appearance in both cases.Signs of irrita-tion,such as vascular congestion and subepithelial edema,(a)(b)1008060402001008060402001008060402000040408012016080120160200240280320360Time (min)Time (min)D r u g r e l e a s e d (%)D r u g r e l e a s e d (%)Gel remaining weight (%)hydrogel solutionFigure 1(a)In-vitro drug release kinetics from curcumin thermosensitive hydrogel and curcumin solution using the dialysis-membrane model at 37°C.Data are expressed as mean Ϯstandard deviation (n =3).(b)In-vitro drug release (%)and in-vitro gel dissolution (remaining wt %)from ther-mosensitive hydrogel as a function of time (min),using the membraneless model at 37°C.Data are expressed as mean Ϯstandard deviation (n =3).100806040200SolutionHydrogelT i m e (m i n )P < 0.001Figure 2In-vivo mucociliary transport time of the thermosensitive hydrogel and curcumin solution.Xi Chen et al .Enhanced curcumin by hydrogelwere not observed.Moreover,no severe signs such as the appearance of epithelial necrosis,sloughing of epithelial cells and haemorrhage were detected in any of the rats.These observations validated the safety of the formulation.Drug brain distribution andpharmacokinetic analysis of curcumin The mean brain tissue and plasma concentration–time pro-ﬁles of curcumin in male rats following a single dose of the nasal in-situ gel and the i.v.injection of curcumin solution are illustrated in Figure 4.Following administration of the nasal in-situ gel at a dose of 250m g/kg,the respective AUC 0→6h values of curcumin in the cerebrum,cerebellum,hippocampus,olfactory bulb and plasma were 288.50Ϯ25.95ng h/g,295.37Ϯ11.23ng h/g,577.03Ϯ30.29ng h/g,1058.67Ϯ51.73ng h/g and 167.03Ϯ16.57mg h/l (means ϮSE).The AUC 0→6h values of curcumin in the cerebrum,cerebellum,hippocampus,olfactory bulb and plasma fol-lowing an i.v.dose of 250m g/kg were 263.87Ϯ9.82ng h/g,238.37Ϯ7.95ng h/g,464.10Ϯ20.01ng h/g,1159.00Ϯ105.23ng h/g,and 281.97Ϯ50.30mg h/l respectively.The curcumin concentration in the brain by intranasal treat-ment was similar to that by i.v.treatment in the ﬁrst 2h,but it was higher in the following 4h.The AUC 2→6h values of curcumin in the cerebrum,cerebellum,hippocampus and olfactory bulb were 189.23Ϯ12.34,192.26Ϯ26.47,367.84Ϯ18.64and 698.85Ϯ25.72ng·h/g for intranasal administration,while the AUC 2→6h values of curcumin in the cerebrum,cerebellum,hippocampus and olfactory bulb following i.v.administration were 121.83Ϯ6.45,122.61Ϯ8.84,260.75Ϯ13.87and 572.88Ϯ22.64ng·h/g,respectively.Although the amount of curcumin reaching the brain via intranasal administration was slightly higher than that by i.v.administration after 6h,it was signiﬁcantlyhigher in the last 4h.The drug-targeting efﬁciency (DTE)of the i.v.injection or the nasal in-situ gel,calculated as AUC brain /AUC plasma for curcumin,is illustrated in Figure 5.The intranasal administration of the in-situ gel produced signiﬁcantly higher ﬁgures for the DTE in the cerebrum cer-ebellum,hippocampus and olfactory bulb over 0.167–6h than i.v.injection.For example,the DTE for the drug in the cerebrum,cerebellum,hippocampus and olfactory bulb after intranasal administration of the curcumin hydrogel at 6h were 1.82,2.05,2.07and 1.51times that after intrave-nous administration,respectively,which indicated that the hydrogel signiﬁcantly increases curcumin delivery into the brain.DiscussionIn the past decade,intranasal thermosensitive hydrogels have garnered increasing attention for biomedical and phar-maceutical applications,mainly because of their therapeutic convenience.Hydrogels are liquid-like in vitro ,which sim-pliﬁes accurate dose measurement and nasal administration as a drop or by a spray device.Interestingly,they become semisolid as soon as there is contact with the mucosa,con-sequently prolonging the drug residence time and drug absorption in the nasal cavity.Therefore,determination of the proper gelation temperature and a rapid gelation time are the two main aspects of designing a biocompatible ther-mosensitive hydrogel.The physiological range of the nasal mucosa temperature lies between 32and 35°C,which matches the observed T sol–gel (32°C)for the prepared gel.[38]Considering this,the prepared gel is likely to show a clear viscosity rise following intranasal administration.It is pos-sible to modulate the gelation properties of the hydrogels for intranasal drug delivery by varying the combination of poloxamer mixtures.A thermosensitive and mucoadhesive(a)(b)Figure 3Microscopic photos of normal nasal mucosa (a)and nasal mucosa after treatment with thermosensitive hydrogel for 14days (b).Xi Chen et al .Enhanced curcumin by hydrogelin-situ gel for nimesulide has been developed for rectal administration by mixing poloxamer PF-127and polyethyl-ene glycol (PEG),[41]while a temperature-responsive and biodegradable hydrogel for controlled delivery of human growth hormone was obtained by mixing PV A,PVP k30and PF-127together.[42]It is also crucial to measure the gelation time and mucociliary transport time.The shorter the gelation time,the longer will be the mucociliary trans-port time and hence more drug will be retained on the nasal mucosa.Our results suggest that the hydrogel formulation has a suitable gelation time,which enhances drug retention on the nasal mucosa over plain curcumin solution.Further250200150100500Plasmain ivinivinivin ivin iv0123456C o n c e n t r a t i o n (m g /l )C o n c e n t r a t i o n (n g /g )C o n c e n t r a t i o n (n g /g )C o n c e n t r a t i o n (n g /g )C o n c e n t r a t i o n (n g /g )Time (min)123456Time (min)123456Time (min)0123456Time (min)0123456Time (min)806040200806040200CerebrumHippocampusOlfactory bulbCerebellum150100505004003002001000Figure 4Mean concentration–time proﬁles of curcumin in the plasma and various brain regions after intranasal and i.v.administration to male rats (n =3).in,in-situ gel;iv,intravenous injection.Concentrations corrected for the differences in doses.Xi Chen et al .Enhanced curcumin by hydrogelto this,histological assessment validated the safety of the formulation over 2weeks;its suitability for long-term clini-cal use still needs to be assessed.The physicochemical properties of the hydrogel network and the drug determine the mechanism(s)by which the loaded drug is released from the cross-linked matrix.Drug release from the hydrogel occurs by two principal mecha-nisms:(i)drug diffusion from the hydrogel during the initial release phase and (ii)release of drug by the erosion of the hydrogel matrix during the later release phase.The diffusion-controlled release mechanism is that the ﬂux goes from regions of high concentration to regions of low con-centration,with a magnitude that is proportional to the concentration gradient.Once a matrix delivery device comes in contact with a surrounding bioﬂuid,a concentra-tion gradient will exist between the dispersed drug within the hydrogel and the ambient ﬂuid.Thus the gradual release of drug from the hydrogel depends on the incorporating efﬁciency,and its diffusion to the external medium depends on the concentration gradient.In the dissolution-controlled drug-release model,the drug is homogeneously distributedthroughout the polymer matrix.As the polymer matrix dis-solves,drug molecules are released regardless of drug solu-bility in the dissolution media.In the dialysis membrane method,the hydrogel was totally immersed in the stirred release medium,which would facilitate drug release.However,in the membraneless method,the solid hydrogel was at the bottom of a glass tube,with only a small surface area in contact with the release medium.As the polymer matrix dissolved,curcumin was released.Thus the drug-release rate in the membraneless method was lower than that in the dialysis membrane method,especially in the initial phase.The value of n =0.27indicated that the major mechanism was diffusion-controlled drug release or Fickian diffusion kinetics when the dialysis membrane method was used.However,drug release might be hindered by the addi-tional barrier provided by the dialysis membrane,which restricts the dissolution of PF-127and P188.The mem-braneless model allowed the release medium to come into direct contact with the gel surface and thereby dissolve the gel.The drug-release proﬁle in this case followed the zero-order release model (R 2=0.9965).In the nasal cavity,the2.52.01.51.00.50.02.52.01.51.00.50.010864205432100.1670.512460.1670.512460.1670.512460.1670.51246CerebrumCerebellumOlfactory blubHippocampusin ivin ivin ivin ivabbbbaaaaaaaaaaaaaaaaaa aA U C b r a i n /A U C p l a s m aFigure 5Mean brain-to-plasma AUC ratios of curcumin in various brain regions after intranasal and i.v.administration to male rats (250m g/kg.n =3.in,in-situ gel;iv,intravenous injection,b P <0.05,a P <0.01).Xi Chen et al .Enhanced curcumin by hydrogel。

老年患者梅毒螺旋体特异性抗体生物学假阳性的原因及临床意义

老年患者梅毒螺旋体特异性抗体生物学假阳性的原因及临床意义作者：陈娟缪小花吴春梅王信国来源：《中国医药科学》2022年第17期[摘要]目的研究老年患者梅毒螺旋體特异性抗体生物学假阳性的原因及临床意义。

方法选择2021年1—6月在福建医科大学附属闽东医院输血科就诊的老年患者7306例，均应用化学发光微粒子免疫分析法（CMIA）、梅毒甲苯胺红不加热血清试验（TRUST）检测梅毒螺旋体特异性抗体，二者均阳性且 CMIA 的S/CO 值≥10者265例，其余 CMIA 检测结果为阳性者应用梅毒螺旋体明胶颗粒凝集试验进行验证。

观察阳性率、假阳性率。

计算 CMIA 的检测效能。

结果7306例老年患者的初筛（CMIA+TRUST）阳性率为3.63%。

419例的梅毒螺旋体明胶颗粒凝集试验复筛阳性率、假阳性率分别为89.02%、10.98%。

60～69岁患者的 CMIA 单独检测阳性率、单阳进行梅毒螺旋体明胶颗粒凝集试验复筛比例高于70～79岁、≥80岁患者，差异有统计学意义（ P <0.05）；≥80岁患者的复筛阳性率低于60～69岁、70～79岁患者，假阳性率高于其他年龄段，但差异无统计学意义（ P >0.05）；CMIA 检测的敏感度、特异度、准确性、误诊率、漏诊率分别为100.00%、99.31%、99.37%、0.69%、0。

结论老年患者的梅毒螺旋体特异性抗体检测结果易出现生物学假阳性情况，临床应进一步分析相关原因，以便在今后的检测中采取相应的预防措施，达到提到结果准确的目的，避免因误诊出现不良事件。

[关键词]老年；梅毒螺旋体特异性抗体；生物学；假阳性[中图分类号] R759.1 [文献标识码] A [文章编号]2095-0616（2022）17-0149-04Causes and clinical significance of biological false positive of treponema pallidum specific antibody in elderly patientsCHEN Juan MIAO Xiaohua WU Chunmei WANG XinguoDepartment of Blood Transfusion， Mindong Hospital Affiliated to Fujian Medical University，Fujian， Fuan355000，China[Abstract] Objective To investigate the causes and clinical significance of biological false positive of treponema pallidum specific antibody in elderly patients. Methods A total of 7306 elderly patients admitted to and treated in the Department of Blood Transfusion of Mindong Hospital Affiliated to Fujian Medical University from January to June 2021 were selected. Chemiluminescence microparticle immunoassay （CMIA） and tolulized red unheated serum test （TRUST） were both used to detect treponema pallidum specific antibody， and 265 cases were all positive in both tests and the CMIA S/CO value ≥10. The rest of CMIA positive cases were confirmed by treponema pallidum gelatin particle agglutination test. The positive rate and false positive rate were observed. The detection efficiency of treponema pallidum gelatin particle agglutination test was calculated. Results The positive rate of primary screening （CMIA+TRUST） in 7306 elderly patients was3.63%. The positive rate and false positive rate of re-screening in 419 cases of treponema pallidum gelatin particle agglutination test were 89.02% and 10.98% respectively. The positive rate of CMIA alone and the rescreening rate of Treponema pallidum gelatin particle agglutination test in patients aged 60-69 years were higher than those in patients aged 70-79 and ≥80 years old， and the differences were statistically significant （P <0.05）; and the positive rate of re-screening in patients ≥80 years old w as lower than those in patients aged 60-69 and 70-79 years old， and the false positive rate was higher than those in the above two age groups， with statistically significant differences （P <0.05）. The sensitivity， specificity， accuracy， misdiagnosis rate and missed diagnosis rate of treponema pallidum gelatin particle agglutination test were 100.00%， 99.31%， 99.37%， 0.69% and 0 respectively. Conclusion The detection results of treponema pallidum specific antibody in elderly patients are prone to be biological false positive. Clinical analysis should be made to furtheranalyze the related causes， so as to take corresponding preventive measures in future detection，achieve the purpose of accurate results and avoid adverse events due to misdiagnosis.[Key words] The elderly; Treponema pallidum specific antibody; Biology; False positive梅毒是一种具有高度传染性的性传播疾病[1-2]，会对机体多个脏器和系统造成损害，导致临床症状体征复杂多样，具有较大的危害性[3]。

生物携氧治疗剂的研究：作用与应用

Abstract BACKGROUND: Although blood donation by healthy people can relieve the urgent need of clinical blood to some extent, it is impossible to fundamentally solve the problem of blood shortage and blood safety by relying solely on blood donation by healthy people. OBJECTIVE: In combination with the significance of the development of biological oxygen-carrying therapeutics, to expound the unique characteristics of biological oxygen-carrying therapeutics, and to
《中国组织工程研究》 Chinese Journal of Tissue Engineering Research
生物携氧治疗剂的研究：作用与应用
·综述·
娜飞沙·斯马义，陈莉智，王相朦，黄文华(南方医科大学基础医学院人体解剖学国家重点学科，广东省广州市 510515)
DOI:10.3969/j.issn.2095-4344.2575
Biological oxygen-carrying therapeutic agents: roles and applications
NafeishaSimayi, Chen Lizhi, Wang Xiangmeng, Huang Wenhua (Department of Human Anatomy, School of Basic Medical Science, Southern Medical University, Guangzhou 510515, Guangdong Province, China)

博士申请用研究计划英文模板

pH-Responsive Biodegradable Polymers for Intracellular Drug DeliveryA. Proposed area of researchThe aim of this proposed PhD project is to develop and evaluate pH responsive, endosomolytic polymers for efficient intracellular delivery of biological drug payloads.There is a need to better understand the mechanisms of entry into the cell cytoplasm and nucleus in order to design optimal delivery systems for biological molecules. On the one hand, this would open up significant opportunities to deliver potent drug payloads against intracellular targets to positively impact human health. In addition the project aims to develop a more general understanding of the rules governing the uptake of biological molecules into cells.This project proposes to investigate the use of synthetic, biodegradable polymers for intracellular delivery of drug payloads (including siRNA, therapeutic peptide and antibody) against a well-validated intracellular drug target, such as Bcl-2. The novel pH- responsive polymers have been designed by Dr Rongjun Chen’sLab to mimic the activity of viruses, both in their cell entry and endosomal escape mechanisms. Using cancer cell lines (Jurkat or HL-60 cells) as a model system, the polymers would be tested with a variety of different biological payloads in a quantitative comparison of their ability to enter the cell and trigger apoptosis and subsequently cell death. With an efficient model system established, there would then be scope to optimize the systemin terms of the kinetics and mechanisms of cell entry, cytoplasmic and nuclear localization, and the biodegradation of the polymers. There would also be scope to explore the efficiency in other cell systems and with further intracellular targets. This multidisciplinary project is at the interface of Chemistry, Biology and Medicine, and will provide the student with a real opportunity to be involved in the development and evaluation of new nanomedicines.B. BackgroundAdvances in genomics and proteomics have enabled the development of macrodrugs, such as nucleic acids and proteins, with potential for the treatment of a wide variety of diseases. Amongst other problems, their clinical applications may be greatly impairedby low cellular uptake and lysosomal degradation before they can reach their target organelles or cell nuclei. In order to achieve efficient intracellular delivery of such biological molecules, delivery systems are required to enable high cell entry via endocytosis and efficient release into the cytoplasm by endosomal membrane disruption under mildly acidic conditions.Recombinant viruses and fusogenic viral peptides have been used to mediate gene transfection, but their clinical use is potentially limited by safety issues and difficultiesin large-scale production. A variety of synthetic polymers have therefore been developed as non-viral vectors. Cationic polyethyleneimine, poly(2- (dimethylamino)ethyl methacrylate) and polyamidoamine dendrimers mediate gene delivery through the ‘proton sponge ’effect, but suffer from c y y a t o n t d o x r e i c l i a t ivelylow transduction efficiencies. The intensively studied vinyl-based anionic polymers, poly(a-alkylacrylic acid)s, display pH-responsive membrane disruptive behavior, butthey are not biodegradable, thus low molecular weights have to be strictly required toallow renal excretion and their clinical applications are seriously limited.Dr Rongjun Chen’sLab has recently developed a class of novel, biodegradable, pH- responsive polymers to mimic factors that enable efficient viral transfection, but theyare safe, easy to manufacture and have more controllable structures. The parent polymer is a polyamide, poly(L-lysine isophthalamide), which was based on polycondensation of diacyl chlorides and natural metabolite tri-functional amino acids containing both - αa nd ω-amine groups. Hydrophobic amino acids and/or poly(ethylene glycol) were grafted onto its pendant carboxylic acid groups to manipulate its amphiphilicity and structure. The metabolite-derived biomimetic polymers can undergopH-mediated coil-globule changes in conformation. This property enables these polymers to be significantly membrane-disruptive within pH range typical of endosomal compartments, but necessarily non-toxic at physiological pH. Based on previous successful intracellular delivery of the model-drugs such as calcein, dextran (with molecular weight ranging from 3kDa to 70kDa), and therapeutic protein apoptin and siRNA, it is thought that these polymers may be able to deliver a wide variety of different biological molecules (nucleic acids and proteins) into cells for the treatment of various diseases including cancers.C. Applicant ’s work preparation in ChinaThe applicant is an expected bachelor majoring in Polymer Science and Engineering from Beijing University of Chemical Technology (BUCT). After four years of undergraduate studies (2007-2011), I have obtained a strong research background in organic chemistry, polymer physics and chemistry, physical chemistry, etc. Working inthe State Key Laboratory of Polymer Physics and Chemistry in the Institute of Chemistryof Chinese Academy of Science f or more than half of a year has set up my mind in researching polymer drug carriers. Our group cast our eyes towards synthesizing graft copolymers with amino acids as the main monomers, to create a novel carrier which isboth pH and temperature sensitive.We had synthesized a polymer brush from Z-lysine and 2-Bromoisobutyryl bromidethrough ring-opening polymerization. Then we grafted specific temperature sensitive residues onto the polymer brush via atomic transfer radical polymerization, followed by characterization and theoretical analysis of the polymers.In addition, I was a Research Assistant in the state key laboratory of Beijing University of Chemical Technology, working on the characterization of copolymers by NMR. I was alsoa Research Assistant in the Environmental Materials Laboratory of China Building Materials Academy, working on synthesis of FEVE coating. These research experiences have enriched my knowledge and experimental skills for polymer synthesis and enabledme to operate many facilities deftly, such as NMR, GPC,FTIR,vacuum glove box and rotary evaporator.In the summer of 2010, I was selected to attend the program -Cambridg“e S B u U m C m T e r School ”in the Ui v nersi t y of Cambridge. During the three weeks in the UK, I visited the Department of Chemical Engineering and Biotechnology and did experiments relevantto my research its labs. In Cambridge, I also did a case study about the biopharmaceutical market. This deepened my understanding of commercial prospectsof drug delivery technologies, such as the demands of different patients for drug delivery systems and competitiveness of different health testing equipments. Besidesthe University of Cambridge, I also visited the University of Oxford, Imperial College London, University of Birmingham, and University of Loughborough. I also establishedthe contact with Dr Rongjun Chen who is the Group Leader of Biomaterials and Drug Delivery Group at the University of Leeds when I was in the UK, and have been communicating with him via emails since then, discussing about polymer synthesis and characterization and drug delivery research.I believe the above mentioned academic backgrounds and various relevant experiences have prepared myself well for the PhD study on polymer drug delivery research for the treatment of various diseases including cancers in Dr Rongjun Chen’sLab at the University of Leeds.D. Aim of overseas studyThe aim of my PhD study is to apply polymer nanotechnology to drug delivery in orderto improve the safety and pharmaceutical efficacy of drugs that need precise intracellular delivery. I will design and synthesize biodegradable amino acid-based polymer vectors, which are efficient, safe, cost-effective and amenable to large-scale manufacturing. I will then evaluate the polymer-based targeted intracellular delivery of biological drug payloads (including siRNA, therapeutic peptide and antibody) against awell-validated intracellular drug target, such as Bcl-2, for cancer treatment. The intention is to combine the highly novel chemistry expertise surrounding the deliverypolymer with the biological expertise around the discovery and development of a variety of drug payloads. The novel polymer delivery technology to be developed will open the door to a wide variety of cytoplasmic and nuclear targets, previously thoughtto be inaccessible to biological therapy for various disease including cancers.In addition, I will investigate the fundamental mechanisms of the interaction between polymers/polymer-drug entities and different membrane models (artificial lipid membranes, erythrocytes and more complex nucleated mammalian cells) and obtain a better understanding of the rules controlling the uptake of macromolecules into cells.E. Research methodsThe project would break down into discrete stages as described below:(1) Polymer synthesisAmino acid derivatives (e.g. lysine derivative) will be used to carry out the N- Carboxyanhydride ring opening polymerization in order to obtain a polymer brush, which could be the backbone of target polymer. Then environmentally (e.g. pH and temperature) responsive groups will be grafted to the main chain by ATRP or Michael Addition, etc. Fluorescent polymers would be prepared by coupling organic fluorophores (e.g. fluorescein isothiocyanate and Cy5) onto the polymers. Cleavable linker chemistry (e.g. disulfide bond) would be introduced onto the polymer backbonefor drug conjugation. Polyethylene glycol would be added to the polymers to increase their biocompatibility and bioavailability.(2) Characterization of polymersThe structures, molecular weights and compositions of the synthesized peptide polymers will be characterized by NMR, mass spectrometry, GPCand HPLCetc. Their physicochemical properties tested by UV-visible spectroscopy, fluorescence spectroscopy, dynamic light scattering, zeta potential analysis and electron microscopy, and their membrane disruptive activity tested in a well established haemolysis model system.(3) Testing of biological payloads in the model cell systemsiRNA, therapeutic peptide and antibody payloads against Bcl-2 would be tested in the model cellular system for their biological potency. There are known model systemsin which Bcl-2 antagonists are active, such as in Jurkat or HL-60 cells, where the assays for apoptosis and cell proliferation are well established. For this stage of testing, the siRNA payload would be delivered by standard cationic lipid transfection, while the peptide and antibody would be encoded on expression plasmids, delivered via lentiviral transfection.(4) Combination of polymer delivery and biological payloadsBiological payloads such as siRNA, therapeutic peptide and antibody would be directly conjugated onto the polymer delivery system through cleavable linkage. siRNA payload could also be complexed with the stimuli-responsive anionic polymers via the use of cationic packaging modules (e.g. cationic polymers, lipids or ions). At this stage different chemistries could be explored and the combination efficiency and reproducibility assessedby biophysical analysis. Control reagents will also be prepared, e.g. using existing delivery technology systems such as TAT peptides, for later comparison.(5) Testing of polymer-payload conjugates/complexes in the model cell systemIn the model cell system the polymer-payload entities would be compared for their biological potency in dose titration and time course studies to compare the different payloads. Confocal microscopy would be used to study the mechanisms and kinetics of cell uptake and to visualise the distribution of polymers and payloads within the cell compartments. Flow cytometry would be used for quantitative analysis of cell uptake.(6) Biomembrane activity and cytotoxicityThe distinct but closely related research is to understand the activity of nano-systems at the biological membrane level. Dr Rongjun Chen has established production collaborations with Prof Nelson (Centre for Molecular Nanoscince/School of Chemistry, University of Leeds) on interaction of polymers with electrochemical lipid model membrane systems. Cytotoxicity of the polymers and/or polymer-payload entities towards model cell lines will be tested by MTT assay for metabolic activity, propidium iodide assay for membrane integrity, TEM for morphology of intracellular compartments and Comet assay for DNA damage.(7) Extending the polymer systemWith the model delivery system established, the aim is to optimise delivery to both cytoplasm and nucleus, in particular optimising endosomal release through careful study of the fate of polymers with different chemical modifications by confocal microscopy. There are further opportunities to optimise the biodegradation of the polymer through chemical changes as well as to look within different cellular systems and to look at other intracellular and nuclear targets.F. Schedule of researchPeriod of Study: 36 months from 1st October, 2011Programme: Full-timeMaximum time limit for submission of PhD thesis 30th September, 2015.The detailed schedule for different research stages shown in the above Section E is shown in the Table 1 Work plan.G. Introduction of work in China after graduationAfter obtaining my PhD degree from University of Leeds, I will return to Beijing University of Chemical Technology starting my academic career in the Department of Biomaterials of the College of Materials Science and Engineering. I will maintain my collaborative network including my PhD supervisor Dr Rongjun Chen in Leeds and other scientists/engineers/clinicians within and outside of the University Leeds. I will devote myself in the research area of drug delivery and biological therapy for various diseases including cancers. This area of research in nanomedicine aligns well with the research strategies of Beijing University of Chemical Technology, and will also have considerable potential to make a very positive impact upon the development of biotechnology in China. After I return to China at the end of my studies, the multidisciplinary knowledge and skills at the interface of Chemistry, Biology and Medicine, and excellent scientific capabilities that I will have developed will make me succeed in my academic career to the great benefit of Chinese Biotechnology and develop new potential nanomedicinesto positively impact human health in China and worldwide.Specifically speaking, the PhD research will enable me to have a good understanding of the basic science surrounding drug delivery through a systematic assessmentof both the delivery component and the payload component in a general biological system. Current knowledge of intracellular drug delivery is not well-developed and this project aims to shed light on this area. This PhD project fits the concept of taking novel tools into biological systems to advance the fundamental understanding of complex biological processes and translate basic research into new and improved healthcare qualities and practice. It supports knowledge exchange between academia and industry. In addition, the area of research in nanomedicine within Beijing University of Chemical Technology would help catalyze/enhance cross-faculty interactions/collaborations with researchersin the areas of drug delivery, molecular imaging, polymer chemistry, drug design, nanoparticle synthesis and characterisation, computational modeling of membrane activity, nanotoxicology, physics, cell biology, and medicine, therefore facilitating translational research from basic sciences into healthcare initiatives.The projected total market for nanotechnology-enabled drug delivery is predicted to rise to $26 billion by 2012. The area of research in drug delivery and therapy is of strategic interest to biopharmaceutical and healthcare sectors as it would enable new opportunities to make potent and specific drugs to intracellular targets which are currently inaccessible with existing technologies. The key knowledge and research skills developed from my PhD project will be of interest to all nanotechnology, drug delivery and medical scientists anxious to generate much superior disease treatments leading to substantial savings in healthcare budget, better health and improved quality of life.Table 1. Work planTASK Oct 2011–Sep 2012 Oct 2012–Sep 2013 Oct 2013–Sep 2014 Oct 2014–Sep 2015Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q1 Q3 Q4 Q1 Q2 Q3 Q4(1) Polymer synthesis(2) Characterization of polymers(3) Testing of biological payloadsin the model cell system(4) Combination of polymerdelivery and biological payloads(5) Testing of polymer-payloadconjugates/complexes in themodel cell system(6) Biomembrane activity andcytotoxicity(7) Extending the polymer system(8) Writing up PhD thesisApproval by PhD supervisorSignatureName Dr Rongjun ChenPosition Group Leader of Biomaterials and Drug Delivery Group, BHRC Senior Translational Research Fellow,Centre for Molecular Nanoscience, School of Chemistry, University of Leeds, UKTime 12 March 2011Page of 778。

基于CiteSpace_的PHA_研究进展与热点分析

第45卷第1期包装工程2024年1月PACKAGING ENGINEERING·81·基于CiteSpace的PHA研究进展与热点分析杨雪，魏风军*（河南科技大学艺术与设计学院，河南洛阳471023）摘要：目的直观把握PHA相关领域的研究进展和热点，推动PHA领域的发展。

方法以CNKI与Web of Science数据库中近10年的相关文献为对象，采用文献计量方法，使用CiteSpace软件绘制PHA研究知识图谱。

结果相关文献的年度发文量不断上升，国内外学术界对PHA领域的关注度越来越高，国际PHA领域的跨单位合作比国内更广泛。

该领域的研究力量遍布全球，其中清华大学、葡萄牙里斯本新大学、马来西亚理科大学、昆士兰大学、布尔诺理工大学等机构的贡献突出、影响较大，陈国强是该领域发文量最多的学者。

通过分析关键词可知，目前对PHA的关注重点主要集中在力学性能、生物降解、混合菌群、活性污泥、除磷脱氮等方面。

结论在未来的PHA研究中，PHA的增强改性、在活性污泥中提取PHA及生物法合成PHA仍是研究热点。

关键词：聚羟基脂肪酸酯；知识图谱；文献计量法；可视化分析中图分类号：TB324；G353.1 文献标志码：A 文章编号：1001-3563(2024)01-0081-10DOI：10.19554/ki.1001-3563.2024.01.010CiteSpace-based PHA Research Progress and Hot Spot AnalysisYANG Xue, WEI Fengjun*(School of Art and Design, Henan University of Science and Technology, Henan Luoyang 471023, China)ABSTRACT: The work aims to intuitively master the research progress and hot spots in PHA-related fields and promote the development of PHA field. With the related literature in CNKI and Web of Science in the past ten years as the objects, the knowledge map of PHA research was drawn by the CiteSpace software with the bibliometric method. The annual number of publications of related literature increases continuously, the academic communities in China and abroad are paying more and more attention to the PHA field, and the cross-unit cooperation in the international PHA field is more extensive than that in China. The research power in this field is spread all over the world, among which Tsinghua University, NOVA Univ Lisbon, University of Science Malaysia, University of Queensland, and Brno University of Technology are prominent in their contribution and influence. CHEN Guoqiang is the scholar with the largest number of publications in this field. Key word analysis shows that the current focus of attention on PHA is mainly concentrated on mechanical properties, biodegradation, mixed flora, activated sludge, phosphorus and nitrogen removal. In the future research, enhanced modification of PHA based on increasing its structural diversity, extraction of PHA in activated sludge and biological phosphorus removal and nitrogen removal, biosynthesis of PHA, and mixed bacterial colony fermentation will be the future research trends in the field of PHA.KEY WORDS: polyhydroxyalkanoate; knowledge map; bibliometric method; visual analysis收稿日期：2023-10-30基金项目：国家自然科学基金（51675162）·82·包装工程2024年1月高分子材料具有质轻、加工性优、阻透性好、成本低等优势，与金属材料、无机非金属材料并称为三大材料。

抗HPV生物蛋白敷料联合保妇康栓治疗

抗HPV生物蛋白敷料联合保妇康栓治疗宫颈糜烂合并高危型HPV感染的效果及对阴道微生态和炎症因子的影响明小琼①　陈莉莉① 【摘要】　目的：分析抗HPV生物蛋白敷料联合保妇康栓治疗宫颈糜烂合并高危型人乳头瘤病毒（HPV）感染的效果及对阴道微生态和炎症因子的影响。

方法：对2020年3月-2021年5月华润武钢总医院收治的92例宫颈糜烂合并高危型HPV 感染患者展开研究，根据随机数字表法分两组，各46例。

对照组采用保妇康栓治疗，观察组采用HPV生物蛋白敷料联合保妇康栓治疗。

对比两组的治疗效果，比较治疗前及治疗后不同时间段两组人乳头瘤病毒的遗传物质（HPV-DNA）值变化、HPV转阴率、阴道微生态恢复情况，比较治疗前后两组白细胞介素-4（IL-4）、白细胞介素-12（IL-12）、干扰素-γ（IFN-γ）变化情况。

结果：观察组总有效率为95.65%，高于对照组的71.74%（P<0.05）；治疗后3、6个月两组HPV-DNA 值均较治疗前降低，且观察组的HPV-DNA值低于对照组（P<0.05）；治疗后6个月观察组HPV转阴率为89.13%，高于对照组HPV的63.04%（P<0.05）；治疗后观察组阴道微生态恢复正常率为93.48%，高于对照组的76.09%（P<0.05），阴道分泌物pH值、Nugent评分低于对照组（P<0.05）；治疗后两组IL-4、IL-12、IFN-γ均较治疗前改善，但观察组IL-4低于对照组，IL-12、IFN-γ高于对照组（P<0.05）。

结论：抗HPV生物蛋白敷料联合保妇康栓治疗宫颈糜烂合并高危型HPV感染效果确切，能显著提升转阴率，使宫颈局部微生态环境保持良好状态，降低炎症因子刺激作用，增强局部免疫功能，具有较高应用价值。

【关键词】　抗HPV生物蛋白敷料　保妇康栓　宫颈糜烂　高危型HPV感染　阴道微生态 doi：10.14033/ki.cfmr.2022.30.012 文献标识码　B 文章编号　1674-6805（2022）30-0048-05 Efficacy of Anti-HPV Biological Protein Dressing Combined with Baofukang Suppository in the Treatment of Cervical Erosion Complicated with High-risk HPV Infection and Its Effect on Vaginal Microecology and Inflammatory Factors/MING Xiaoqiong, CHEN Lili. //Chinese and Foreign Medical Research, 2022, 20(30): 48-52 [Abstract]　Objective: To analyze the efficacy of anti-HPV biological protein dressing combined with Baofukang Suppository in the treatment of cervical erosion complicated with high-risk human papillomavirus (HPV) infection and its effect on vaginal microecology and inflammatory factors. Method: A total of 92 patients with cervical erosion combined with high-risk HPV infection who were admitted to China Resources WISCO General Hospital from March 2020 to May 2021 were studied. They were divided into two groups by random number table method, with 46 cases in each group. The control group was treated with Baofukang Suppository, and the observation group was treated with anti-HPV biological protein dressing combined with Baofukang Suppository. The therapeutic effects of the two groups were compared. The changes of HPV-DNA values, HPV negative conversion rate, vaginal microecology recovery of the two groups at differert the points before and after treatment were compared, and interleukin-4 (IL-4), interleukin-12 (IL-12), interferon-γ (IFN-γ) were compared between the two groups before and after treatment. Result: The total effective rate in the observation group was 95.65%, which was higher than 71.74% in the control group (P<0.05). At 3 and 6 months after treatment, the HPV-DNA values in the two groups were lower than those before treatment, but the HPV-DNA value in the observation group was lower than that in the contral group (P<0.05). At 6 months after treatment, the negative conversion rate of HPV in the observation group was 89.13%, which was higher than 63.04% in the control group (P<0.05). After treatment, the recovery rate of vaginal microecology in the observation group was 93.48%, which was higher than 76.09% in the control group (P<0.05), and the pH and Nugent score of vaginal secretions were lower than those in the control group (P<0.05). After treatment, the IL-4, IL-12 and IFN-γ in the two groups were improved compared with those before treatment, but IL-4 in the observation group was lower than that in the control group, IL-12 and IFN-γ were higher than those in the control group (P<0.05). Conclusion: Anti-HPV biological protein dressing combined with Baofukang Suppository is effective in the treatment of cervical erosion combined with high-risk HPV infection, which can significantly increase the negative conversion rate, keep the local cervical microecological environment in a good state, reduce the stimulation of inflammatory factors, and enhance local immune function. It has high application value.①华润武钢总医院　湖北　武汉　430080 宫颈糜烂属于一种正常生理现象，为宫颈柱状上皮异位表现，通常无临床特征，少数女性有接触性出血情况，当合并人乳头瘤病毒（HPV）感染时，可使宫颈发生炎症损伤情况，症状表现有月经异常、疼痛、白带增多等，可引起子宫颈癌前病变，甚至演变为子宫颈癌，对患者身心健康造成严重影响[1]。

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enhanced the efficiency.
农药剂型博客农药剂型博客 NPFC 15, Oct. 2006 P.8 P.8
Application of Biological Pesticides
2. Present Bio-Pesticide Formulations
Application of Biological Pesticides
Nantong Pesticide Formulation Development Center (NPFC) Chen Xiaobo
NPFC 15, Oct. 2008 P.1 P.1 农药剂型博客农药剂型博客
At present, there are many kinds of biopesticides are used in the pest control. Normally which can be divided into two categories, the first one is biotic organism pesticide, such as Bt. and Beauveria bassiana. The second one is bio-chemical pesticide, such as Pyrethrum, Abmectin and Emamectin. In this paper, we will choose Bt. and Abmectin as the samples to introduce some common formulations for biopesticides.
农药剂型博客农药剂型博客 NPFC 15, Oct. 2006 P.10 P.10
Application of Biological Pesticides
2.2 Water Dispersible Granule
This product is dustless and protected environment, and greatly eliminated the damage to human bodies during its production and application. Which is regarded as one of the most important environment friendly pesticide formulations. Developed countries are commercialized already. Production unit is over ten thousands tons. To biopesticides formulation, the most important thing is to control the temperature in the formulation process, to avoid the decomposition of A.I. or deactivation of microoganisms.
Application of Biological Pesticides
NPFC 15, Oct. 2006
P.3 P.3
1. Selection Formulations for Biopesticide Production
The same as chemical pesticides, for efficiency and the convenience of application, biopesticides need to choose a suitable target formulation, and produce the final pesticide products for customers. The formulation of biopesticide must consider its own characteristic, such as physical and chemical character, the request of the light, the temperature, the humidity and so on, besides unifying with the application method.
Application of Biological Pesticides
NPFC 15, Oct. 2006
P.6 P.6
1. 2 According to the Application Target of A.I. to Choose
For different targets, different type of formulations would be chosen on the base of one A.I., since different targets have different environment conditions, and have different requirement to the physical and chemical character of final pesticide products. Normally, we should choose suitable formulation on the base of the concrete requirements.
NPFC 15, Oct. 2006
P.2 P.2
Part I The Biopesticide Formulations
1. Requirement of Selection of Biopesticide Formulations
2. Present Biological Pesticide Formulations
NPFC 15, Oct. 2006 P.9 P.9
Application of Biological Pesticides
2.1 Suspension Concentrates Formulation
SC is the environment friendly water base formulation as well as one of Bt. important formulation. It is a big problem of Bt. SC formulation as a low stability in production. It is less than half year to stock in room temperature. An ideal Bt. liquid insecticide should possess good antiseptic, UV repellent, surface spreading agent, wetness agent, insect feeding accelerant to promote pesticide effect.
NPFC 15, P.4 P.4 农药剂型博客农药剂型博客 Oct. 2006
Application of Biological Pesticides
1. Selection Formulations for Biopesticide Production
Application of Biological Pesticides
NPFC 15, Oct. 2006
P.7 P.7
1. 3 To Choose the Formulations on the Base of Efficiency
To choose the type of formulations on the base of efficiency, since different environment conditions will impose different influence on the efficiency. For example, we choose CS formulation for Natural Pyrethrum, the problem of sensibility to light can be solved, the release speed of A.I. can be controlled, the persistence duration can be prolonged, and finally
Hale Waihona Puke Similar like chemical pesticide, when we choose the type of formulation for biopesticide, the physical and chemical character of A.I. such as solubility, melting point, stability conditions should be considered. Different formulations have different manufacturing process, and the process will have different requirement to the character of A.I.
NPFC 15, Oct. 2006 P.11 P.11
Application of Biological Pesticides
2.3 Microcapsule Formulation
Microcapsule formulation is that the effective components are wrapped in the pouch wall to make several microns to several hundred microns small spherule and the pesticide release rate is controlled depending on changing pouch wall thickness and the aperture dimension. After 80’s, 20 century, the study of Bt. microcapsule is active day by day, and starch capsule which Bt. A.I. was wrapped by the moisture powder with starch or the bread flour matrix, the inorganic salt as a dispersing agent .