Immunohistochemical Study of Hexose Transporters GLUT-2 and GLUT-5 in Birds Gastrointestinal Tract

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免疫组化的原理与操作

胞化学分类方法迅速发展； ——2000年各种免疫组化技术更加成熟，使免疫组
化技术成为当今生物医学中形态、功能代谢综合研究的一项有力工具。其应用范围深达医学各个学科，是目前生命科学工作者应该掌握的基本技术之一； ——国内起步晚，从70s开始。
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二、IHC的现状和发展前景
（一）方法学
如将该法中的卵白素（avidin）变换成链霉卵白素(streptavidin)，LAB法即成LsAB法，或称SP法。
（B ★为生物素标记的过氧化物酶）
AB★C复合物是avidin与酶联biotin 在使用前30min 配置，混匀。1个avidin分子结合了3个biotin分子，剩下 1个结合点与2抗的生物素结合，avidin起桥联作用。
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（1）ABC复合物的制备：
（2）ABC法反应原理：
特点：ABC法敏感性更高，比PAP法敏感20~40倍，背景也更清晰。
特点或优点：（1）特异性强、灵敏度高；
（2）可定性、定位、定量观察；
（3）将形态学改变与功能和代谢变化结
合起来；
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IHC不仅具有传统形态学（包括LM和EM水平）能对组织和细胞进行仔细、客观观察的优点（特别是经苏木精或伊红复染后），而且还克服了传统免疫学反应只能定性和定量（离体、液相），而不能定位的缺点。IHC则可在原位和固相对染色结果进行观察。目前IHC的定位可精确到亚微结构水平。
荧光素直接标记特异性抗体（一抗）上，标记抗体与抗原结合（在切片上）荧光显微镜下观察→ 检测抗原。
优点：简单，时间短，特异性强。
缺点：灵敏度低，所需抗体量大（不经济）。
应用：基本不用了！！
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Science：英国科学家揭示神经干细胞重回静默状态的重要机制

Science：英国科学家揭示神经干细胞重回静默状态的重要机制2016年7月18日讯/生物谷BIOON/ --近日，著名国际学术期刊Science刊登了英国弗朗西斯克里克研究所-米尔希尔实验室研究人员的一项最新研究进展，在这篇文章中他们发现了增殖状态下的神经干细胞如何重新回到静默状态，从而维持神经干细胞池的平衡。

海马体是大脑中负责调节记忆和情绪的重要区域，其中的神经干细胞能够产生新的神经元，即使在成年阶段也具有这样的能力。

形成多少个新的神经元以及何时形成新的神经元取决于神经干细胞池的静默和增殖之间的平衡。

那么什么样的信号能够让处于增殖状态的神经干细胞回到静默状态引起了研究人员的兴趣。

研究人员发现一个促进细胞增殖的关键转录因子发生泛素化降解，就能调节神经干细胞回到静默状态，但是这种静默状态与神经干细胞的原始状态并不相同，这种处于静默但又活化状态的神经干细胞维持了神经干细胞池的平衡。

研究人员表示，神经干细胞的静默对于成体神经干细胞的长期维持非常重要，微环境信号能够调节神经干细胞从静默状态变成激活状态，但是增殖状态的神经干细胞如何重新回到静默状态仍未可知。

在这项研究中，研究人员发现E3连接酶Huwe1是增殖状态下的成年小鼠海马体神经干细胞回到静默状态的一个必要分子。

Huwe1能够通过泛素化降解系统使增殖状态下的海马体神经干细胞中促进细胞增殖的转录因子Ascl1变得不稳定，进而阻止细胞周期蛋白D的积累促进增殖细胞回到静默状态。

当神经干细胞回到静默状态，增殖的神经干细胞池会逐渐耗竭。

研究人员认为长期维持海马体神经元生成需要依赖于快速降解这种关键的促激活因子使神经干细胞回到一种暂时性的静默状态。

这项研究发现了促进增殖的神经干细胞回到静默状态的一条重要分子机制，机体通过这种静默状态和增殖状态的神经干细胞池平衡维持正常的神经元生成，该研究对于神经疾病研究和治疗甚至癌症研究都有重要提示意义。

免疫组织化学

核酸分子探针-杂交-免疫组织化学放大和显示杂交信号杂交免疫组织化学
基因重组技术免疫组织化学图像分析
单抗技术
技术
流式细胞术
激光共聚焦显微术
免疫组织化学的全过程：抗原的提取和纯化免疫动物或细胞交融〔单克隆抗体〕抗体效价检测和提取、纯化标志抗体细胞、组织切片标本的制备免疫组织化学反响和显色察看和记录结果
● 切片边缘、刀痕或皱折区域、坏死或挤压的细胞区、胶原结缔组织等，常表现为一样的阳性染色强度，不能用于判别阳性。
第三章免疫组织化学常用方法引见
第一节免疫酶细胞化学
免疫酶细胞化学是免疫组织化学中最常用的方法，它是在抗原抗体特异反响存在的前提条件下，借助于酶细胞化学手段，检测某种物质〔抗原／抗体〕在组织细胞内的存在部位。即预先将抗体与酶连结〔酶标抗体〕，再使其与组织内特异性抗原反响，经细胞化学染色后，在光镜或电镜下察看分析。
后再用显微镜察看〔免疫酶〕
1. 加强特异性染色的方法 ● 蛋白酶消化法胃蛋白酶、胰蛋白酶 ● 适宜的抗体稀释度第一抗体 ● 温育时间 37℃ 30-60 min, 4℃ 过夜 ● 多层染色法〔双层、三层〕
2. 减少或消除非特异性染色的方法加二抗显色剂的浓度、温育时间
二抗 1
一抗
1:50
1:100
1:200
1:400
_____________________________________ ___________
1:50
+++
+++
◆ 以中等阳性稀释度为佳 ◆ 抗体稀释液的配制
0.01mol/L pH7.4 PBS or TBS

chinadaily双语新闻：挑战动物起源理论丹麦海绵需氧极少

★⽆忧考英语资源频道为⼤家整理的china daily 双语新闻：挑战动物起源理论丹麦海绵需氧极少，供⼤家参考。

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One of science's strongest dogmas is that complex life on Earth could only evolve when oxygen levels in the atmosphere rose to close to modern levels. But now studies of a small sea sponge fished out of a Danish fjord shows that complex life does not need high levels of oxygen in order to live and grow.The origin of complex life is one of science's greatest mysteries. How could the first small primitive cells evolve into the diversity of advanced life forms that exists on Earth today? The explanation in all textbooks is: Oxygen. Complex life evolved because the atmospheric levels of oxygen began to rise app. 630 -- 635 million years ago.However new studies of a common sea sponge from Kerteminde Fjord in Denmark shows that this explanation needs to be reconsidered. The sponge studies show that animals can live and grow even with very limited oxygen supplies.In fact animals can live and grow when the atmosphere contains only 0.5 per cent of the oxygen levels in today's atmosphere."Our studies suggest that the origin of animals was not prevented by low oxygen levels," says Daniel Mills, PhD at the Nordic Center for Earth Evolution at the University of Southern Denmark.Together with Lewis M. Ward from the California Institute of Technology he is the lead author of a research paper about the work in the journal PNAS.A little over half a billion years ago, the first forms of complex life -- animals -- evolved on Earth. Billions of years before that life had only consisted of simple single-celled life forms. The emergence of animals coincided with a significant rise in atmospheric oxygen, and therefore it seemed obvious to link the two events and conclude that the increased oxygen levels had led to the evolution of animals."But nobody has ever tested how much oxygen animals need -- at least not to my knowledge. Therefore we decided to find out," says Daniel Mills.The living animals that most closely resemble the first animals on Earth are sea sponges. The species Halichondria panicea lives only a few meters from the University of Southern Denmark's Marine Biological Research Centre in Kerteminde, and it was here that Daniel Mills fished out individuals for his research."When we placed the sponges in our lab, they continued to breathe and grow even when the oxygen levels reached 0.5 per cent of present day atmospheric levels," says Daniel Mills.This is lower than the oxygen levels we thought were necessary for animal life.The big question now is: If low oxygen levels did not prevent animals from evolving -- then what did? Why did life consist of only primitive single-celled bacteria and amoebae for billions of years before everything suddenly exploded and complex life arose?"There must have been other ecological and evolutionary mechanisms at play. Maybe life remained microbial for so long because it took a while to develop the biological machinery required to construct an animal. Perhaps the ancient Earth lacked animals because complex, many-celled bodies are simply hard to evolve," says Daniel Mills.His colleagues from the Nordic Center for Earth Evolution have previously shown that oxygen levels have actually risen dramatically at least one time before complex life evolved. Although plenty of oxygen thus became available it did not lead to the development of complex life.据外媒2⽉17⽇报道，科学界⼀直认定，地球上的复杂⽣物是在⼤⽓中的氧⽓含量达到现在的标准时才逐渐进化出来的。

关于水杨酸研究的英文文献

关于水杨酸研究的英文文献Abstract:Salicylic acid (SA), primarily derived from willow bark, has been extensively studied due to its diverse therapeutic effects on human health and its potential applications in various fields. This review aims to explore the recent advancements in salicylic acid research and summarize the key findings and applications of SA in medicine, skincare, agriculture, and other domains. The paper also addresses the mechanisms of action and potential side effects associated with salicylic acid usage.1. IntroductionSalicylic acid, a monohydroxybenzoic acid, has been used in various forms for centuries due to its analgesic, antipyretic, anti-inflammatory, and antiplatelet properties. This section briefly discusses the history, chemical properties, sources, and forms of salicylic acid.2. Pharmacological Properties of Salicylic AcidThis section focuses on the pharmacological properties of salicylic acid, including its analgesic, anti-inflammatory,anti-platelet, and antipyretic effects. Various studies are discussed to understand the underlying mechanisms of these properties.3. Medical ApplicationsSalicylic acid has been widely used in the medical field for treating various conditions, such as pain management, acne, psoriasis, and warts. This section reviews the clinical efficacy of salicylic acid in these applications based on available studies, and also highlights the challenges and future prospects of salicylic acid-based therapeutics.4. Skincare and Cosmetic ApplicationsThe use of salicylic acid in skincare and cosmetic products has gained significant popularity due to its effectiveness in treating acne, reducing hyperpigmentation, and improving the overall appearance of the skin. This section discusses the formulation and efficacy of salicylic acid formulations in skincare products, including cleansers, toners, creams, and masks.5. Agriculture ApplicationsSalicylic acid has emerged as a potential plant growth regulator, providing enhanced resistance against various biotic and abiotic stresses. This section explores the role ofsalicylic acid in agriculture, including its effects on plant growth, disease resistance, and post-harvest preservation.6. Mechanisms of ActionUnderstanding the mechanisms of action is essential for harnessing the potential of salicylic acid. This sectiondiscusses the cellular and molecular pathways involved in the pharmacological effects of salicylic acid, such as its regulation of cyclooxygenase (COX) enzymes, NF-κB signaling, and reactive oxygen species (ROS) generation.7. Side Effects and PrecautionsWhile salicylic acid has diverse therapeutic effects, it is essential to understand its potential side effects and precautions. This section examines the adverse reactions associated with salicylic acid usage, such as gastrointestinal irritation, bleeding, and allergic reactions. Additionally, it provides guidelines to ensure safe and effective use ofsalicylic acid.8. ConclusionKeywords: Salicylic acid, medical applications, skincare, agriculture, mechanisms of action, side effects.。

单唾液酸四己糖神经节苷脂钠联合脑蛋白水解物治疗帕金森病的疗效观察

单唾液酸四己糖神经节苷脂钠联合脑蛋白水解物治疗帕金森病的疗效观察发表时间：2019-07-24T15:59:14.347Z 来源：《中国结合医学杂志》2019年5期作者：朱少平[导读] 针对临床收治的帕金森病患者给予单唾液酸四己糖神经节苷脂钠联合脑蛋白水解物治疗，可有效增强临床总有效率，缩短症状改善时间，安全性较高，具有临床研究意义。

娄底市双峰县人民医院湖南娄底 417700【摘要】目的：观察单唾液酸四己糖神经节苷脂钠联合脑蛋白水解物治疗帕金森病的临床疗效。

方法：选取临床收治的帕金森病患者56例，均为2017年1月至2018年12月期间收治，就单纯给予单唾液酸四己糖神经节苷脂钠治疗（对照组，n=28）与给予单唾液酸四己糖神经节苷脂钠联合脑蛋白水解物治疗（观察组，n=28）临床疗效、治疗前后UPDRS评分及不良反应展开对比。

结果：在临床疗效、治疗后UPDRS评分的对比上，观察组均优于对照组（P<0.05）；治疗后两组均发生1例不良反应，组间比较无差异（P>0.05）。

结论：针对临床收治的帕金森病患者给予单唾液酸四己糖神经节苷脂钠联合脑蛋白水解物治疗，可有效增强临床总有效率，缩短症状改善时间，安全性较高，具有临床研究意义。

【关键词】帕金森病；单唾液酸四己糖神经节苷脂钠；脑蛋白水解物；疗效【Abstract】Objective：To observe the clinical efficacy of monosialotetrahexosyl ganglioside combined with brain protein hydrolysate in the treatment of Parkinson's disease. METHODS：A total of 56 patients with clinically treated Parkinson's disease were enrolled. All patients were treated with monosialotetrahexosylganglioside sodium from January 2017 to December 2018（control group，n=28）. The clinical efficacy，UPDRS score and adverse reactions before and after treatment were compared with the administration of monosialotetrahexosylganglioside sodium combined with brain protein hydrolysate（observation group，n=28）. RESULTS：In the comparison of clinical efficacy and post-treatment UPDRS scores，the observation group was superior to the control group（P<0.05）. One patient had adverse reactions after treatment，and there was no difference between the two groups（P>0.05）. Conclusion：The treatment of patients with Parkinson's disease treated with monosialotetrahexosylganglioside combined with brain protein hydrolysate can effectively enhance the total clinical effective rate，shorten the symptom improvement time，and have high safety. significance.【Key words】 Parkinson's disease；monosialotetrahexosylganglioside sodium；brain protein hydrolysate；therapeutic effect帕金森病属于神经系统疾病，多发生在中老年人群体，随着我国人口逐渐趋向老龄化，工作生活压力增大，帕金森病逐年上升，成为神经内科较为常见的疾病[1]。

嗅成鞘细胞海马内注射对阿尔茨海默病模型大鼠的作用

阿尔茨海默病（ｚｅｒＳｄｓａｅＡＤ）Ａ１ｈｉｉｅｓ，ｍｅ是
一
Hale Waihona Puke 探讨ＯＣ移植对阿尔茨海默病模型大鼠的治疗Ｅｓ
效果。
组以学习记忆减退和认知障碍为主要特点的临
床综合征，发病机制与神经元中的线粒体结构受其损和能量代谢障碍关系密切；细胞色素氧化酶而（ｙｏｈｏｘｄｓ，ＯＸ）ｃｔｃｒｍｅｏｉａｅＣ又是线粒体呼吸链的
４Ｏ只随机分为４组：康对照组、健ＡＤ模型组、Ｃ移植ＯＥｓ组、ＳＭＣＦ注射组，组１每０只１２建立大鼠Ａ．Ｄ模型将Ａ．于无菌生理盐水（￣／，人３ ℃ 温Ｂ．溶５／ｍＩ）放ｇ７
郧阳医学院（４００４２０）姚淞元姚伟史丹青王金勇王军姚柏春
【摘要】目的探讨嗅成鞘细胞（Ｅｓ海马内注射对阿尔茨海默病模型大鼠的作用。方法Ｓ大鼠双ＯＣ）Ｄ侧海马注射Ａａ，立ＡＤ大鼠模型。实验动物分为４组：康对照组、ｌ。建健ＡＤ模型组、Ｃ移植组、工脑脊液ＯＥｓ人注射组，组ｌ每Ｏ只。体外原代培养嗅成鞘细胞并将其移植至Ａ大鼠海马内。运用行为学测试、织化学、位Ｄ组原杂交，合图像分析以及电镜等技术，察、结观比较各组大鼠学习记忆能力、马Ｃ区线粒体细胞色素氧化酶海Ａ

immunohistochemistry labelling 免疫组化

Fluorescent labels
Advantages

Size Specificity Sensitivity
Disadvantages

Hardware Limited selection Background
IgG
IgG
Confocal
Application in renal diseases
Very high sensitivity Used in research more than routine studies. It is longer and more expensive.
• In order to visualize the enzymes labelling the antibodies with light microscope, enzyme – substrate reactions, which convert colorless chromogens into visible colored endproducts, is used.: • Peroxidase- hydrogen peroxide- diaminobenzidine (DAB): BROWN
IgG
The basic principle of immunofluorescence
To
use a fluorescent compound (usually fluorescein) to detect the binding of antigen and antibody
The Ab is labelled with the fluorescent compound Under a fluorescence microscope, fluorescein

口腔生物学智慧树知到期末考试章节课后题库2024年广州医科大学

口腔生物学智慧树知到期末考试答案章节题库2024年广州医科大学1.骨密质由骨小梁和骨髓构成。

（）答案:错2.组织工程基本要素包括种子细胞、生物支架材料及周围微环境。

（）答案:对3.骨基质中最丰富的非胶原蛋白是骨钙素。

（）答案:对4.在牵张成骨的牵张期形成哈弗系统。

（）答案:错5.巨噬细胞是宿主抵御牙周菌斑微生物的第一道防线。

（）答案:错6.釉原蛋白是成釉细胞最早分泌的釉基质蛋白。

（）答案:错7.转录组学是在RNA水平研究基因表达情况。

（）答案:对8.血链球菌是最初定植在牙菌斑生物膜中的先锋菌之一。

（）答案:对9.在葡萄糖的分解代谢中，EMP途径能产生生物合成嘌呤、嘧啶所必需的前体，但它产生的ATP只有HMP途径的一半。

（）答案:错10.酶联免疫吸附试验、Western印记和免疫荧光技术均能用于检测目标蛋白。

（）答案:对11.在健康人的龈沟液中淋巴细胞主要是T淋巴细胞。

（）答案:错12.皱褶缘是行使骨吸收功能的破骨细胞所特有的。

（）答案:对13.ELISA技术不能用于目标蛋白的定量分析。

（）答案:错14.下颌下腺是唾液分泌量最大的唾液腺。

（）答案:对15.成骨细胞、破骨细胞和骨衬里细胞都存在于骨的表层。

（）答案:对16.龈下菌斑中的主要细菌为微需氧菌和需氧菌。

（）答案:错17.在牙釉质成熟阶段，主要负责降解牙釉质蛋白的酶是基质金属蛋白酶。

（）答案:错18.葡糖基转移酶可通过氨基端的葡聚糖结合区与葡聚糖结合。

（）答案:错19.组织芯片技术结合了分子蛋白质水平研究与组织形态学研究。

（）答案:对20.细胞在-80℃条件下长期冷冻保存对细胞存活率无显著影响。

（）答案:错21.口腔免疫应答的特点包括（）。

答案:口腔健康与口腔黏膜完整性密切相关###唾液腺的局部免疫功能起重要作用###免疫损伤是口腔疾病发生的重要因素###口腔是有菌环境，免疫应答与口腔菌群保持动态平衡22.生物矿化的结晶过程包括（）答案:集聚###成核###固相转换###晶核生长23.以下微生物中有助于抑制龋病的是（）。

子学习情境5

子学习情境5氢化可的松发酵任务一甾类药物甾类药物是指分子结构中含有环戊烷多氢菲核的一类药物，在医学上应用非常广泛，特别是甾体激素类药物，应用在风湿性关节炎，控制炎症，避孕，利尿等各方面的治疗上，对机体起着非常重要的调节作用。

甾类激素根据其生理活性可分为肾上腺皮质激素，性激素和蛋白同化激素三大类。

、肾上腺皮质激素皮质激素两大类。

糖皮质激素如可的松(cortiso ne )和氢化可的松(hydrocortisone )是由肾上腺束状带细胞所合成和分泌，主要影响人体的糖、蛋白质和脂肪的代谢，对水、盐的代谢作用影响较小。

临床上主要用于抗炎、抗毒素、抗休克和抗过敏等。

以醛甾酮和去氧皮甾酮为代表的盐皮质激素是由肾上腺的球状带细胞分泌，主要作用是促进钠离子肾小管的重吸收，从而使钠的排泄量减小，促进钾的排泄。

临床上主要用于治疗慢性肾上腺皮质机能减退症(阿狄森病)及低血钠症。

、性激素性激素的重要生理功能是刺激副性器官的发育和成熟，激发副性特征的出现，增进两性生殖细胞的结合和孕育能力，还有调节代谢的作用。

按其生理功能可分为雄性激素和雌性激素两类。

雄性激素属于C 19类固醇，主要由睾丸和肾上腺皮质所产生，卵巢也有少量合成。

睾丸分泌的雄激素主要有3种：睾酮、脱氢异雄酮和雄烯二酮。

雌性激素包括雌激素和孕激素两类，主要由卵巢合成和分泌，肾上腺皮质和睾丸也能少量合成。

雌激素，真正由腺体分泌，有活性的只有 3种：17P -雌二醇、雌酮和雌三醇。

三种激素生理活性相差很大，其相对比活为100 : 10 : 3。

孕激素属于C 21类固醇，体内真正存在的是孕酮。

三、蛋白同化激素蛋白同化激素是一类从睾丸酮衍生物中分化出来的药物，特点是性激素的作用大为减弱, 蛋白质同化作用仍然保留甚至增强，临床使用比较安全，较少引起男性化症状等不良反应。

如17a-甲基去氢睾丸素(17-methyldehydro-testosterone 商品名为大力补)。

知母乙醇提取物对阿尔茨海默病模型小鼠学习记忆能力的影响

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陕西医学杂志２１００年８月第３卷第８期９
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迷迭香酸对过氧化氢处理下的皮肤黑色素瘤的抗氧化作用(原文翻译)

迷迭香酸（罗丹酚酸）对H2O2处理过的皮肤黑色素瘤细胞的抗氧化作用Sun Mi Yoo1 and Jeong Ran Kang2*1.韩国光州500-741号东冈大学美容系2.韩国首尔143-701号建国大学生物工程系2009.2.6收到 2009.4.17接收本学科旨在检测迷迭香酸对人工孵育的皮肤黑色素瘤细胞在ROS下的抗氧化作用。

通过XTT比色法，以细胞毒性和抗氧化作用来分析细胞粘附活性，DPPH自由基清除活性以及H2O2处理1-10h和未经处理的两种情况下乳酸脱氢酶的活性。

用20-110 μM 的H2O2处理皮肤黑色素瘤细胞5-7h后，细胞活性的降低呈剂量和时间依赖性。

通过XTT比色法测得H2O2的半抑制浓度（IC50 ）为90μM。

同时H2O2增强了LDH细胞的剂量依赖性。

用50-90μM的H2O2处理8h后测得LDH50为60 μM H2O2。

迷迭香酸能增强细胞活性和DPPH自由基清除活性，降低乳酸盐脱氢酶的活性。

细胞的H2O2处理证实了对人工孵育的皮肤黑色素瘤细胞的强抗氧化作用。

通过H2O2的处理，迷迭香酸能在细胞内能增强细胞活性和DPPH 自由基清除活性，降低乳酸盐脱氢酶的活性。

这被认为是迷迭香酸对ROS（ROS）如H2O2的抗氧化作用。

Key words：DPPH-radical scavenging, LDH, rosmarinic acid, XTT assay关键字：DPPH自由基清除活性，乳酸脱氢酶，迷迭香酸，XTT比色法据研究发现，ROS通过氧化应激对细胞的损伤和一些脑部疾病比如帕金森症或心脏疾病例如心肌梗塞之间有很大的关联[Difazio et al., 1992; Delanty and Dichter, 1998].尤其是研究人员认为ROS是皮肤老化的一个主要的因素后，一直试图从ROS方面研究衰老。

[Yokozawa et al., 1998].据研究表明，ROS的氧化应激会通过萎缩细胞引起各种疾病，例如超氧自由基、H2O2（H2O2）或羟基自由基的巯基蛋白反应中断酶的活性，破坏脱氧RMA（DNA）或RMA（RNA），诱导细胞膜脂质过氧化。

Immunohistochemical study of tubular epithelial cells and vascular in glomerulonephritis

/rnfISSN:0886-022X(print),1525-6049(electronic)Ren Fail,2014;36(8):1208–1214!2014Informa Healthcare USA,Inc.DOI:10.3109/0886022X.2014.929525CLINICAL STUDYImmunohistochemical study of tubular epithelial cells and vascular endothelial cells in glomerulonephritisFlaviu Bob1,Gheorghe Gluhovschi1,Diana Herman2,Ligia Petrica1,Gheorghe Bozdog1,Cristina Gluhovschi1,Silvia Velciov1,Florica Gadalean1,Romulus Timar3,Elena Potencz2,Alis Dema2,and Adalbert Schiller11Department of Nephrology,Emergency County Hospital Timisoara,University of Medicine and Pharmacy,Timisoara,Romania,2Department of Pathology,Emergency County Hospital Timisoara,University of Medicine and Pharmacy,Timisoara,Romania,and3Department of Diabetics and Metabolic Diseases,Emergency County Hospital Timisoara,University of Medicine and Pharmacy,Timisoara,RomaniaAbstractBackground and aims:In order to assess the role played by tubular epithelial cells(TEC)andinterstitial vascular endothelial cells(VEC)in interstitial fibrogenesis in human glomeruloneph-ritis,we studied the expression of markers of activated fibroblasts(a-smooth muscle actin(a SMA)and vimentin(Vim))and of the transforming growth factor b(TGF b),at the level ofthese cells.Methods:We studied retrospectively41renal biopsies from patients with primaryand secondary glomerulonephritis[24males,17females,mean age45.5±12.9years].Immunohistochemistry using monoclonal antibodies(SMA,Vim,TGF b)was assessed using asemiquantitative score,that was correlated with biological and histological data(quantifiedusing a scoring system in order to assess active-inflammatory and chronic–sclerotic/fibroticlesions).Results:The presence of SMA and Vim as markers of myofibroblasts was found in TECsand VECs.TEC Vim expression correlated with interstitial Vim expression(r¼0.38;p¼0.008),interstitial infiltrate(r¼0.31;p¼0.027),interstitial fibrosis(R¼0.25;p¼0.042),GFR(r¼À0.35;p¼0.016),SMA(r¼À0.42;p¼0.015),TGF b(r¼0.25;p¼0.046),and hemoglobin(r¼À0.55;p50.001).VEC Vim expression showed indirect correlations with interstitial infiltrate(r¼À0.32;p¼0.023)and interstitial fibrosis(r¼À0.34;p¼0.017).Conclusion:Our studyreflects the complexity of the involvement of VEC and mainly of TEC in fibrosis.The expressionof mesenchymal markers at the tubular cell level(especially Vim)correlates with histologicalinterstitial changes,with the decrease of renal function and more strongly with anemia.KeywordsAlpha smooth muscle actin,TGF b,tubularepithelial cells,vascular endothelial cells,vimentinHistoryReceived15December2013Accepted17May2014Published online19June2014IntroductionChronic kidney disease regardless of its etiology progressestowards end stage renal disease,and the final commonpathway in this process seems to be fibrosis.1An importantrole is played by tubulo-interstitial fibrosis,which is thestrongest morphological predictor of clinical outcome and ismost tightly linked to progression of disease,even though theprimary disease may be of glomerular origin.2In a previous study,we have shown that the interstitialhistological changes,especially the scores indicating scler-otic/fibrotic lesions,correlate with the presence of interstitialmyofibroblasts,with an important role played by TGF b.3Thus the main effector cell in this process is the interstitialmyofibroblast,being most responsible for interstitial matrixaccumulation.Other cells present at this level(tubularepithelial cells(TEC)and vascular endothelial cells(VEC))are also involved in fibrogenesis.In the current study performed on human renal biopsies,we have set ourselves to investigate by means of immuno-histochemistry,compared with histological and biologicaldata,the possible role of TEC and VEC in interstitial fibrosis.As shown in experimental studies,these cells are con-sidered possible progenitor cells for myofibroblasts.Duringthe course of kidney fibrosis in mice,about30%ofmyofibroblasts are derived via epithelial–mesenchymal tran-sition(EMT)from the TEC of the kidney.In addition,it hasbeen shown that another35%of myofibroblasts are derivedvia endothelial–mesenchymal transition(EndMT)from theendothelial cells normally residing within the kidney.Theremaining portions are speculated to arise via activation ofresident fibroblasts or other mesenchymal cells,such asperivascular smooth muscle cells/pericytes and fibrocytes inthe circulation,or are fibroblasts derived from the bonemarrow.4,5Despite these experimental data,the origin ofinterstitial myofibroblasts remains debatable,because datafrom in vivo studies is scarce.In order to assess the role played by TEC and interstitialVEC in human glomerulonephritis,we studied at the level ofthese cells the expression of mesenchymal markers that are Address correspondence to Flaviu Bob,MD,PhD,Department ofNephrology,Emergency County Hospital Timisoara,University ofMedicine and Pharmacy,2Chopin Str.,300366Timisoara,Romania.E-mail:flaviu_bob@markers of activated fibroblasts(a-smooth muscle actin (a SMA)and vimentin(Vim))and of the transforming growth factor-b(TGF b),a growth factor that is involved in this activation.Materials and methodsPatientsWe studied retrospectively the renal biopsies of41patients admitted at the Department of Nephrology,Timisoara,with chronic glomerulonephritis(17females,24males;mean age 45.5±12.9years,range18–74).Only those cases were included that presented enough paraffin wax embedded in the biopsy material to permit the cutting of additional sections for immunohistochemistry.Cases with fewer than five glomeruli were excluded from the study.6The patients had either primary(30cases)or secondary glomerulonephritis(systemic vasculitis,four cases;infectious, three cases;collagenoses,two cases;neoplasia,two cases).The histopathological diagnoses were mesangial proliferative glomerulonephritis(12cases),mesangiocapillary glomerulo-nephritis(1case),membranous nephropathy(5cases), minimal change disease(5cases),focal and segmental glomerulosclerosis(15cases),and crescentic glomeruloneph-ritis(3cases).We used as controls renal tissue samples obtained from four patients who underwent nephrectomy for kidney tumors. The samples were obtained from the normal renal tissue.All control patients had normal serum creatinine,eGFR460mL/ min,and no proteinuria,at the moment of nephrectomy.All biopsies were performed after obtaining an informed consent from patients regarding the procedure and the possible use of the obtained material for scientific purposes. The present study has the approval of the local ethical committee.HistologyRoutinely fixed and processed sections of kidney were processed for light microscopy and stained with hematoxylin and eosin(HE),periodic acid-Schiff(PAS),and Gomori’s trichrome techniques using routine methods.All stained slides were assessed separately by two pathologists.In order to better quantify the histological lesions,a scoring system adapted by Neumann et al.7for ANCA-associated vasculitis, based on the standardized scoring system for activity and chronicity developed for lupus nephritis,was employed.At the tubulo-interstitial level,inflammatory lesions(edema, interstitial infiltrate)and sclerotic/fibrotic lesions(interstitial fibrosis,tubular atrophy)were assessed.Tubulo-interstitial lesions were assessed semi-quantitatively:530%of tubules or interstitial area affected was considered as mild(1point), 31–60%affected as moderate(2points),and460%affected as severe(3points).7ImmunohistochemistryThe detection of a SMA,Vim,and TGF b was performed on 4m m-thick formalin-fixed,paraffin-embedded sections using a horseradish peroxidase-labeled streptavidin–biotin(LSAB2-HRP)method(a system intended for use with primary antibodies for the qualitative identification of antigens in paraffin-embedded tissues).The primary antibodies used were ready-to-use monoclo-nal mouse anti-Vim(Vim3B4antibody,DAKO,Carpinteria, CA);monoclonal mouse anti-smooth muscle actin(clone 1A4,DAKO,Carpinteria,CA);and concentrated monoclonal mouse anti-TGF b(MCA797,Serotec,Raleigh,NC).Sections were first deparaffinized and rehydrated by routine protocol,then incubated with3%hydrogen peroxide in distilled water for5min and afterwards rinsed with distilled water and placed in Tris-buffered saline(TBS)for5min. The next step was incubation with a primary antibody,diluted 1/75,for10–30min,followed by sequential10-min incuba-tions with a biotinylated link antibody and peroxidase-labeled streptavidin(both purchased ready-to-use,DAKO, Carpinteria,CA).The labeling of TGF b,a SMA,and Vim immunoreactivity, at the level of TEC and VEC,was graded for statistical evaluation using a semi-quantitative intensity scale from0to 3,similar to that used by Alexopoulos et al.8We considered 0¼no labeling(negative),3¼the most intense labeling, whereas1and2are labeling of an intermediate degree.The same semi-quantitative intensity scale was used for assessing interstitial Vim staining.Clinical parametersIn addition to the histological data,we obtained clinical and biological parameters at the time of the biopsy from the patients’files.In all patients,renal function(serum creatinine and glomerular filtration rate(eGFR)),blood pressure, proteinuria(24h urine specimen),and hemoglobin count were taken.GFR was estimated using the MDRD4formula.9 Statistical analysisData were recorded in a file created in Microsoft Excel,which was organized and managed as a database.Correlations between histological and immunohistochemical parameters were performed using the non-parametric Spearman’s rank order test,while correlations among clinical,biological data, and immunohistochemistry were performed using parametric Pearson’s test.Correlation coefficients of linear regression analysis are presented in relation with p values.The significance of the correlation coefficient(r)is as follows:r¼0–0.25indicates little or no correlation;r¼0.25–0.50indicates a fair degree of relationship;r¼0.5–0.75indicates moderate to good correl-ation;r¼0.75–1indicates very good to excellent correl-ation.10In order to perform these tests,we used WinStat for Microsoft Excel and Epi3.2.2.ResultsIn24of the studied cases,we found positive Vim immunostaining at the level of TEC;in5of the cases,Vim staining was present either at a proximal(two cases)or at a distal(three cases)tubular level.The mean expression of Vim in TEC was0.77±0.87in proximal tubules and0.71±0.76in distal tubules.There was,however,no statistically significant difference between the two tubular segments overall.DOI:10.3109/0886022X.2014.929525Immunohistochemical study of TEC and VEC in glomerulonephritis1209Vim expression was low or moderate at the level of TEC in our patients;in only one patient(with mesangiocapillary GN), we had to use the degree3from the semi-quantitative scale to express a strong Vim staining.Vim expression was positive in patients with focal segmental glomerulosclerosis(nine patients at proximal level and seven patients at distal level), minimal change disease(two patients at proximal and three at distal level),membranous nephropathy(three at proximal and four at distal level),mesangioproliferative GN(five patients at distal and proximal levels),and crescentic GN and mesan-giocapillary GN(in both types:one patient at distal and proximal levels)(Figure1).In13cases,a SMA immunostaining was present at the level of the TEC.The mean expression of a SMA was0.55±0.64in proximal TEC and0.57±0.69in the distal ones.This positive a SMA immunostaining occurred in six patients with mesan-gioproliferative GN,in three patients with focal segmental glomerulosclerosis,in two patients with minimal change disease,in one patient with membranous nephropathy,and in another one with crescentic GN(Figure2).TGF b TEC immunostaining was present in29of the studied cases,with no statistically significant difference between the proximal and distal segments of the tubules.The mean expression of TGF b was0.97±0.85in proximal TEC and 1.04±0.86in distal TEC.Similar to Vim,TGF b tubular immunostaining was moderate or low,with the exception of two patients in whom we found a strong immunostaining (degree3).A positive TGF b immunostaining at TEC level was found in10patients with mesangioproliferative GN,in 9patients with focal segmental glomerulosclerosis,5patients with minimal change disease,in3patients with membranous nephropathy,in1patient with crescentic GN,and in1patient with mesangiocapillary GN(Figure3).At the level of the VEC,Vim staining was present in27 cases,while TGF b in35cases.The mean expression of Vim was0.78±0.58and,for TGF b,it was0.92±0.46.At the level of VEC,Vim was positive in11patients with mesangiopro-liferative GN,in10patients with focal segmental glomerulo-sclerosis,in5patients with minimal change disease,in 4patients with membranous nephropathy,in1patient with mesangiocapillary GN,and none with crescentic GN.TGF b was present in11patients with mesangioproliferative GN,in 13patients with focal segmental glomerulosclerosis,in 4patients with minimal change disease,in3patients with membranous nephropathy,1patient with mesangiocapillary GN,and in3patients with crescentic GN.In the normal control patients,immunostaining was negative for Vim,a SMA,and TGF b at the level of TEC and VEC.In these renal samples(from normal controls),wefound a SMA in the media of the interstitial vessels.As mentioned above in the patients with glomeruloneph-ritis,the presence of a SMA and Vim as markers of the myofibroblasts,as well as of TGF b involved in this process with the formation of active fibroblasts,was found at the level of TEC and VEC.The process shows a great variability in each patient,fact that makes a statistical correlation with biological and histological data difficult;however,some correlations have been found.First we studied the correlations with tubulo-interstitial histological data assessed by light microscopy.The tubulo-interstitial lesions were studied on standard stains in light microscopy(HE,PAS,and Gomori’s trichrome)using the scoring system adapted from Neumann et al.As already mentioned in the Material and methods section,the following tubulo-interstitial lesions were assessed semi-quantitatively: inflammatory lesions(edema and interstitial infiltrate)and sclerotic/fibrotic lesions(interstitial fibrosis and tubular atrophy).We found a statistically significant small degree of correlation between the scores for Vim staining at the level of the proximal TEC and interstitial infiltrate(r¼0.31, Figure2.Positive a SMA immunostaining in the cytoplasm and nuclei of tubular epithelial structures.a SMA stain LSAB2-DABÂ200.Figure1.Intense positive vimentin immunostaining at the level of the interstitium,peritubular,and periglomerular.Vimentin stain LSAB2-DABÂ100.Figure3.Interstitial capillary and arteriolar endothelium positive for TGF b stain.TGF b stain LSAB2-DABÂ200.1210 F.Bob et al.Ren Fail,2014;36(8):1208–1214p50.05),on one hand,and interstitial fibrosis(r¼0.25, p50.05),on the other hand.As shown in Table1,there are no other statistically significant correlations between immuno-histochemical parameters at the level of the TEC and interstitial histological scores.When comparing the different immunohistochemical par-ameters,we found the following correlations:Vim staining at the tubular level(both proximal and distal)correlated with interstitial Vim staining(r¼0.38,p50.05)and with TGF b tubular staining(only for the distal tubules)(r¼0.24, p50.05).There was also an indirect correlation between a SMA and Vim at the level of the tubules:distal(r¼À0.42, p50.05)and proximal(r¼À0.30,p50.05).We compared immunohistochemical data in all patients at the tubular level with clinical data(serum creatinine,eGFR, proteinuria,and hemoglobin)and we found that Vim staining at the level of distal TEC correlated statistically significantly with renal function:serum creatinine(r¼0.32,p50.05)and eGFR(r¼À0.36,p50.05).Proteinuria correlated indirectly with the proximal TEC Vim(r¼À0.27,p50.005)and TGF b (r¼À0.30,p50.05)staining.We found also a moderate indirect correlation between the hemoglobin and the Vim staining at the level of the proximal (r¼À0.51,p50.001)and distal tubules(r¼À0.55, p50.001).Due to the relatively small number of patients with the different histological types of glomerulonephritis,we were not able to find some immunohistochemical expression patterns at the level of TEC.We studied,however,the two subgroups of patients that were better represented:patients with focal segmental glomerulosclerosis(FSGS,15patients) and patients with mesangioproliferative glomerulonephritis (MPGN,12patients).In MPGN patients,we found a strong indirect correlation between tubular Vim and renal function:proximal TEC Vim with serum creatinine(r¼0.6,p50.05)and with eGFR (r¼À0.6,p50.05);distal TEC Vim with serum creatinine (r¼0.6,p50.05)and with eGFR(r¼À0.6,p50.05).For TGF b at the level of distal TEC,we found the same correlation with eGFR(r¼À0.51,p50.05).Surprisingly in the same group of patients(MPGN),a SMA expression in TEC showed a direct correlation with eGFR both in distal (r¼0.59,p50.05)and in proximal tubules(r¼0.52, p50.1).In FSGS patients,we found that proximal tubular Vim expression correlates directly with the score for interstitial fibrosis(r¼0.4,p50.1),while proximal tubular a SMA expression correlates indirectly with the scores for interstitial infiltrate(r¼À0.62,p50.1)and with interstitial fibrosis (r¼À0.58,p50.1).In this subgroup of patients,we found that TGF b at the level of proximal TEC correlated indirectly with proteinuria(r¼À0.39,p50.1).Regarding VEC,Vim staining at this level correlated indirectly with interstitial infiltrate(r¼À0.32,p50.05)and interstitial fibrosis(r¼À0.34,p50.05).Concerning clinical data,we found only an indirect correlation between protein-uria and TGF b staining at the level of the VEC(r¼À0.25, p50.05)(Table2).No statistically significant correlations have been found between Vim and TGF b at VEC level with renal function or anemia.Table1.Correlation between immunohistochemical staining scores at the level of tubular epithelial cells(TEC)and interstitial histological scores and clinical data(prox,proximal;dist,distal).Pearson correlation Interstitial edema Interstitial infiltrate Interstitial fibrosis Tubular atrophies Proteinuria Serum creatinine eGFRa SMA prox.TECr0.21009156À0.082270980.038980.0850556À0.0757À0.123230.221282 p0.146443910.341652630.423460.33658000.35660.270140.133666 a SMA dist.TECr0.1994508À0.16858008À0.0587À0.13024330.063622À0.153510.32015p0.15928390.200297690.385480.25865170.3787480.2222970.051761 Vimentin prox.TECr0.0998127980.3131628260.259436030.06529312À0.27865780.21132465À0.34791524 p0.2755122360.027781710.042799960.348466250.04745000.101399550.01616026 Vimentin dist.TECr0.2561343170.2232580530.05042303À0.00074431À0.146341970.32781289À0.36793017 p0.0603077150.0889322340.381845850.498230700.193717770.022259470.01152254 TGF b prox.TECrÀ0.189164364À0.172968478À0.1383094À0.1537955À0.3027728À0.026824170.01553297 p0.1181044710.1397452430.194239110.168516100.028780590.433890920.46160562 TGF b dist.TECrÀ0.083709401À0.183802808À0.1107815À0.1665446À0.237490430.05062329À0.06327586 p0.3014131530.1250000990.24524230.149003290.070026060.376637310.34714952 Table2.Correlation between immunohistochemical scores for vascular endothelial cells(VEC)staining and interstitial histological scores and clinical data.Interstitial edema Interstitial infiltrate Interstitial fibrosis Tubular atrophies Proteinuria Serum creatinine eGFR Vimentin VECrÀ0.199299803À0.329175908À0.3469841À0.4079876À0.05671652À0.179012340.20386943 p0.1184851140.023*******.017687220.00609390.37124640.144547500.11308439 TGF b VECr0.2032516640.173681350.193158470.16755051À0.258512340.10722386À0.23323048 p0.1012341270.1387415440.11313870.147528260.04362870.25230200.07111387 DOI:10.3109/0886022X.2014.929525Immunohistochemical study of TEC and VEC in glomerulonephritis1211DiscussionsThe results of our study showed at the level of tubules and interstitial vessels a great variability of the expression of the studied immunohistochemical markers(Vim and a SMA),and also of the growth factor TGF b.Despite this great variability, some facts regarding a certain pattern of staining in these cell types can be discussed.At the level of TEC,the expression of the mesenchymal markers a SMA and Vim could indicate a process of EMT,a biological process in embryological development.11In differ-ent studies(especially in vitro or in experimental animal models),it has been tried to establish whether EMT also occurs in renal epithelial cells,following kidney injury,and to show that the mesenchymal cells formed could give rise to myofibroblasts which populate the renal interstitium,causing fibrosis within it.12,13In the cases studied by us,the expression of Vim as the mesenchymal marker at the level of proximal TEC correlated with histological interstitial markers of activity(interstitial infiltrate)and of chronicity(interstitial fibrosis—especially in our FSGS patients).We found also correlations between tubular and interstitial Vim expression.It has already been shown by other authors that in human biopsies of kidneys with fibrotic lesions,Vim is positive at the tubular level.14 Rastaldi et al.have also shown in human biopsies that tubular Vim correlated with the interstitial infiltrate and with interstitial fibrosis,on one hand.In the same study,it was shown that,on the other hand,tubular a SMA was rare,despite the fact that its interstitial expression is a good marker of renal disease progression.15Our results were consistent with these data,a SMA was present in only13cases at the tubular level; moreover,when was assessed quantitatively,it correlated indirectly with Vim expression.We could also observe that especially in the subgroup of FSGS patients,tubular a SMA showed an indirect correlation with interstitial fibrosis, showing thus an opposite pattern to the tubular Vim expression.TGF b,the other marker used by us,has the ability to induce the expression of extracellular matrix proteins in mesenchymal cells and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM.Elevated TGF b expression in affected organs,and subsequent deregulation of TGF b functions,correlates with the abnormal connective tissue deposition observed during the onset of fibrotic diseases.16Therefore,we used TGF b antibodies in our study to find out if there is an involvement of this growth factor in the activation of TEC.According to Fragiadaki et al.,there is no doubt that proximal TEC can undergo EMT in vitro in response to TGF b-1and also other inflammatory stimuli.12The consequence of TGF b stimulation,due to injury,is an increased expression of Vim in the tubular epithelium.17 In our patients,TGF b staining correlated with Vim staining at the level of the distal tubules.If we maintain the hypothesis,proven in experimental studies,that TGF b can promote EMT at the tubular level,then the positive correl-ation between these two stainings(Vim and TGF b)can be explained.The expression of the mesenchymal markers at the tubular level could be correlated with the renal function at the moment of the renal biopsy.In the study performed by Rastaldi et al.,tubular Vim correlated with serum creatin-ine.15In another study performed by de Matos et al.on49 kidney transplant recipients,it was shown that a high expression of tubular Vim was associated with a reduction of graft function.18All these data are consistent with our results concerning the correlation between tubular Vim and renal function(serum creatinine and eGFR)at the moment of renal biopsy.This fact was especially true in the subgroup of patients with MPGN.In this same subgroup of patients, however,a SMA showed,surprisingly,a direct correlation with eGFR.Urinary proteins from patients with minimal change disease and,especially FSGS,induce in cell cultures the expression of a SMA and Vim.19In the aforementioned study performed on human renal biopsies by Rastaldi et al.,there was a strong correlation between proteinuria and tubular Vim.15Surprisingly proteinuria showed in our patients an indirect correlation with tubular Vim.Similar to our results, Yonemoto et al.found in patients with diabetic nephropathy that newly acquired Vim(at the mesangial level in their study) decreased in patients with heavy proteinuria.20Also,in a study performed on patients with congenital nephrotic syndrome of the Finnish type(NPHS1),it was shown that heavy proteinuria did not lead to the transition of the TEC into myofibroblasts,as shown by the expression of tubular Vim and a SMA.21Moreover,in our patients(especially the FSGS patients),there was also an indirect correlation between tubular TGF b and proteinuria.We found an interesting indirect correlation between Vim at the TEC level and hemoglobin.This could be due to the fact that EPO-producing fibroblasts transform into myofibro-blasts at the cost of EPO production.This finding could clarify the link between renal fibrosis and anemia.Studies using in situ hybridization and the transgenic mice approach indicate that EPO is mainly produced by the interstitial fibroblasts in the deep cortex and the outer medulla in the kidney.Asada et al.demonstrate the occurrence in the kidney interstitium of fibroblasts that produce EPO,and can transdifferentiate into scar-producing myofibroblasts,thus losing their EPO-producing activity after kidney injury.22 The fact that in the patients studied by us there was an indirect correlation between tubular Vim and hemoglobin could indicate the fact that EPO-producing fibroblasts have been replaced by myofibroblasts(with Vim expression).It is, however,interesting that for SMA(the other myofibroblast marker),these correlations have not been found.The few studies performed using human renal biopsies, including the present one,show a correlation of tubular Vim staining with interstitial infiltrate and fibrosis,with renal function and with anemia.Our results regarding TEC could support the hypothesis shown mainly in experimental(in vitro)studies that these cells may acquire markers of mesenchymal cells,a process known as EMT. This possible origin of interstitial myofibroblasts remains debatable.Humphreys et al.have shown in vivo,but in a mouse model of ureteral obstruction that after injury TEC do not migrate and do not transform into myofibroblasts.23In another study performed using proximal TEC cultures,it has1212 F.Bob et al.Ren Fail,2014;36(8):1208–1214been shown that shear stress,as it occurs in hyperfiltration, leads to matrix generation and thus fibrogenesis,but inhibits the motility of tubular cells,thus excluding EMT.In contrast,incubation with TGF b induces cell motility and Vim expression in the cultured tubular cells.The authors conclude that renal fibrosis and EMT could exclude each other.24This could be a possible explanation of the fact that in our patients with FSGS tubular a SMA correlated indirectly with interstitial fibrosis,or that it correlated directly with eGFR(in patients with MPGN).It remains, however,difficult to explain why in our patients Vim and a SMA showed different patterns of expression and correl-ations in TEC.It is possible that the two markers are not present at the same time in the different TEC that did undergo a transformation.The fact that we were not able to perform double staining(Vim and a SMA)could be considered a limitation of this study.It is also possible that there are different patterns of expression of these markers in TEC in different histological types of glomer-ulonephritis,as we have shown for FSGS or MPGN,but the reduced number of cases did not permit the drawing of a pertinent conclusion.EndoMT,a newly recognized type of cellular transdiffer-entiation,has emerged as another possible source of tissue myofibroblasts.EndoMT is a complex biological process in which endothelial cells lose their specific markers and acquire a myofibroblastic phenotype and express mesenchymal markers.Similar to EMT,EndoMT can be induced by TGF b.25It has been shown in experimental diabetic nephropathy in mice that endothelial–myofibroblast transition occurs and contributes to the early development and progres-sion of renal interstitial fibrosis.26In the cases studied by us, however,we found that,unlike in TEC,Vim expression in VEC showed an indirect correlation with interstitial histo-logical scores.This surprising fact together with the absence of other correlations between the expression of Vim or TGF b at the level of VEC with histological and clinico-biological parameters could lead to the conclusion that these cells do not undergo a transition into mesenchymal cells,but the presence of these markers at this level opens the perspective to new studies in this field.ConclusionsOur study performed in human biopsies reflects the com-plexity of the involvement of VEC and mainly of TEC in fibrosis.It cannot be stated that TEC are,without doubt, transformed into myofibroblasts during renal injury,but it has been shown that the expression of mesenchymal markers at the tubular cell level(especially Vim)correlates with histological interstitial changes,with the decrease of renal function and more strongly with anemia.The possible link between renal fibrosis and anemia could be important in developing new treatment strategies that are aimed to target both the renal anemia and the reversibility of fibrosis.Declaration of interestThe authors report no conflicts of interest.The authors alone are responsible for the content and writing of the paper.References1.Okada H,Strutz F,Danoff TM,Neilson EG.Possible pathogenesisof renal fibrosis.Kidney Int.1996;49(Suppl.54):S37–S38.2.Barnes JL,Glass2nd WF.Renal interstitial fibrosis:a criticalevaluation of the origin of myofibroblasts.Contrib Nephrol.2011;169:73–93.3.Bob FR,Gluhovschi G,Herman D,et al.Histological,immuno-histochemical and biological data in assessing interstitial fibrosis in patients with chronic glomerulonephritis.Acta Histochem.2008;110(3):196–203.4.Kalluri R,Neilson EG.Epithelial-mesenchymal transitionand its implications for fibrosis.J Clin Invest.2003;112: 1776–1784.5.Kalluri R,Weinberg RA.The basics of epithelial–mesenchimaltransition.J Clin Invest.2009;119(6):1420–1428.6.Ponticelli C,Mihatsch MJ,Imbasciati E.Renal biopsy:indicationsfor and interpretation.In:Davison AM,Cameron JS,Grunfeld JP, et al.,eds.Oxford Textbook of Clinical Nephrology.3rd ed.Oxford: Oxford University Press;2005.7.Neumann I,Kain R,Regele H,et al.Histological and clinicalpredictors of early and late renal outcome in ANCA-associated vasculitis.Nephrol Dial Transplant.2005;20:96–104.8.Alexopoulos E,Stangou M,Papagianni A,Pantzaki A,Papadimitriou M.Factors influencing the course and response to treatment in primary focal segmental glomerulosclerosis.Nephrol Dial Transplant.2000;15:1348–56.9.Levey AS,Eckardt KU,Tsukamoto Y,et al.Definition andclassification of chronic kidney disease:a position statement from kidney disease:improving global outcomes(KDIGO).Kidney Int.2005;67:2089–2100.10.Dawson B,Trapp RG.Basic and Clinical Biostatistics.New York:McGraw-Hill;2004.11.Eriksson JE,Dechat T,Grin B,et al.Introducing intermediatefilaments:from discovery to disease.J Clin Invest.2009;119(7):1763–1771.12.Fragiadaki M,Mason RM.Epithelial-mesenchymal transition inrenal fibrosis–evidence for and against.Int J Exp Pathol.2011;92(3):143–150.13.Iwano M,Plieth D,Danoff TM,Xue C,Okada H,Neilson EG.Evidence that fibroblasts derive from epithelium during tissue fibrosis.J Clin Invest.2002;110(3):341–350.14.Essawy M,Soylemezoglu O,Muchaneta-Kubara EC,et al.Myofibroblasts and the progression of diabetic nephropathy.Nephrol Dial Transplant.1997;12:43–50.15.Rastaldi MP,Ferrario F,Giardino L,et al.Epithelial–mesenchymaltransition of tubular epithelial cells in human renal biopsies.Kidney Int.2002;62(1):137–146.16.Verrecchia F,Mauviel A.Transforming growth factor beta andfibrosis.World J Gastroenterol.2007;13(22):3056–3062.17.Kaneyama T,Kobayashi S,Aoyagi D,Ehara T.Tranilast modulatesfibrosis,epithelial–mesenchymal transition and peritubular capil-lary injury in unilateral ureteral obstruction rats.Pathology.2010;42(6):564–573.18.de Matos AC,Caˆmara NO,Tonato EJ,et al.Vimentinexpression and myofibroblast infiltration are early markers of renal dysfunction in kidney transplantation:an early stage of chronic allograft dysfunction?Transplant Proc.2010;42(9):3482–3488.19.Wen Q,Huang Z,Zhou SF,Li XY,Luo N,Yu XQ.Urinary proteinsfrom patients with nephrotic syndrome alters the signalling proteins regulating epithelial–mesenchymal transition.Nephrology (Carlton).2010;15(1):63–74.20.Yonemoto S,Machiguchi T,Nomura K,Minakata T,NannoM,Yoshida H.Correlations of tissue macrophages and cytoskeletal protein expression with renal fibrosis in patients with diabetes mellitus.Clin Exp Nephrol.2006;10(3):186–192.21.Kuusniemi AM,Lapatto R,Holmberg C,Karikoski R,Rapola J,Jalanko H.Kidneys with heavy proteinuria show fibrosis,inflam-mation,and oxidative stress,but no tubular phenotypic change.Kidney Int.2005;68(1):121–132.22.Asada N,Takase M,Nakamura J,et al.Dysfunction of fibroblastsof extrarenal origin underlies renal fibrosis and renal anemia in mice.J Clin Invest2011;121(10):3981–3990.DOI:10.3109/0886022X.2014.929525Immunohistochemical study of TEC and VEC in glomerulonephritis1213。

结直肠癌分子标志物临床检测中国专家共识【36页】

检测方法
▪ Checkmate142研究表明，纳武单抗有效率为31%[26]。而非MSI-H/dMMR患者有效率则显著较低。KEYNOTE-177研究表明，MSI-H/dMMR患者姑息一线应用帕博利珠单抗，客观缓解率为 43.8%，而标准化疗靶向组显著较低，为33.1%[27]。
▪ MSI/MMR状态对于遗传性结直肠癌的诊断也具有较大的意义，尤其是林奇综合征的诊断， MMR基因的胚系突变是确诊的金标准。
检测方法
▪ NCCN指南和CSCO指南对BRAF V600E突变mCRC患者的二线治疗均推荐西妥昔单抗+伊立替康+维莫非尼（BRAF抑制剂），或者西妥昔单抗+BRAF 抑制剂±MEK抑制剂的联合方案[17,18]。BRAF基因状态对结直肠癌患者的预后评估也具有指导意义[18,19,20]。BRAF V600E突变患者相比其他患者预后更差，生存时间更短[21]。
▪ 而对于RAS基因突变患者，应用抗EGFR单抗则无明确获益，一般采用化疗联合VEGF单抗治疗。因此，推荐在mCRC患者开始治疗前，应进行RAS 突变的检测，有助于帮助患者选择最佳的个体化治疗方案。
检测方法
2．BRAF基因点突变： ▪ BRAF基因作为RAF原癌基因家族的成员，位于RAS基因下游，是RAS-RAF-
结直肠癌分子标志物临床监测中国专家共识
前言
▪ 结直肠癌是人类最常见的恶性肿瘤之一，全球发病率居恶性肿瘤第3位，死亡率居第2位。在我国，结直肠癌发病率亦呈现逐年上升趋势。根据 2019年国家癌症中心数据显示，2015年中国结直肠癌新发病例38.8万，死亡病例18.7万[1]。
▪ 结直肠癌的早期筛查及预防可以降低发病率、提高治愈率，相关分子标志物的检测是结直肠癌筛查的有效补充，同时对个体化方案的判定、预后判断及疗效预测等方面起到重要作用。

Nature医学：运动或预防阿尔茨海默症，是因为它……

Nature医学：运动或预防阿尔茨海默症，是因为它……图⽚来源： CC0 Public Domain众所周知，体育锻炼可以改善记忆⼒。

研究表明，体育锻炼还可以降低患阿尔茨海默⽒症的风险。

但是研究⼈员⼀直不明其中缘由。

⼏年前，研究⼈员发现了⼀种名为鸢尾素（irisin）的激素，这种激素在运动时被释放到⾎液循环中。

起初，⼈们认为它只在能量代谢中起作⽤。

然⽽，最新的研究发现，这种激素还可能促进⼤脑海马区的神经⽣长。

海马是位于⼤脑内侧颞叶的⼀个器官，与学习和记忆（尤其是长期记忆）有关。

因此，研究⼈员推测，正是运动释放的鸢尾素对这个器官的影响，解释了运动在阻⽌记忆丧失⽅⾯的作⽤。

最新的这项研究由哥伦⽐亚⼤学Vagelos学院的病理学和细胞⽣物学和医学教授Taub 研究所研究员Ottavio Arancio博⼠所领导，结果发表在《Nature Medicine》杂志上。

“这项研究结果有助于解释，为什么体育锻炼改善记忆，并且似乎在阿尔茨海默症等脑部疾病中发挥保护作⽤。

” Arancio说道。

阿尔茨海默症患者的⼤脑中鸢尾素含量降低在最新的研究中，Arancio和同事们⾸先观察鸢尾素和阿尔茨海默症之间的联系。

他们利⽤脑库的组织样本发现，鸢尾素存在于⼈的海马体中，阿尔茨海默症患者的海马体中这种激素的⽔平会降低。

为了进⼀步探索鸢尾素在⼤脑中的作⽤，研究⼩组将⽬光转向了⼩⿏。

实验表明，鸢尾素在⼩⿏体内保护⼤脑突触和动物记忆：当健康⼩⿏海马区鸢尾素丧失功能时，其突触和记忆就会减弱。

同样，提⾼⼤脑中鸢尾素的⽔平也能改善改善⼤脑健康状况。

游泳可以增强鸢尾素⽔平和保护记忆⼒研究⼈员随后观察了有氧运动对鸢尾素和⼤脑的影响。

令⼈惊讶的是，研究⼈员发现，连续五周⼏乎每天游泳的⼩⿏，尽管注射了与阿尔茨海默症有关的β-淀粉样蛋⽩(-淀粉样蛋⽩是⼀种阻塞神经、破坏记忆的蛋⽩质)，但并未出现记忆障碍。

研究⼈员还发现，⽤药物阻断鸢尾素完全消除了游泳的益处。

瘾疹一消散对急性荨麻疹动物模型血清IgE及Cap通透性的影响

度计６１０ｈｍ处比色，记录其０Ｄ值。
结果：１．与正常组比较，模型组大鼠血清中ＩｇＥ水平升高，差异有显著性（尸＜０．０５）；与模型组比较，瘾疹一消散大、中剂量组及马来酸氯苯那敏片组大鼠血清中ＩｇＥ水平均降低，差异均有显著性（Ｐ＜０．０５）；瘾疹一消散小剂量组大鼠血清中ＩｇＥ水平降低，差异无显著性（尸＞０．０５）；与马来酸氯苯那敏片组比较，瘾疹一消散大、中剂量组大鼠血清中ＩｇＥ水平含量无显著性差异（尸＞０．０５）；瘾疹一消散大、中剂量组间比较，差异无显著性（尸＞０．０５）。２．与正常组比较，马来酸氯苯那敏片和瘾疹一消散大剂量组均可不同程度地缩小小鼠腹部皮肤风团面积，差异均有显著性（尸＜Ｏ．０５）；瘾疹一消散小剂量组小鼠腹部皮肤风团面积缩小，差异无显著性（尸＞０．０５）；与马来酸氯苯那敏片组比较，瘾疹一消散大剂量组小鼠腹部皮肤风团面积缩小无显著性差异（尸＞０．０５）。
论文作者签名蘧矍盎Ｉ－ｔ
论文导师签
同
期：如哆年髟月１
７Ｒ
期：ｊ咿哆年四月三ｌ同
痘疹一消散对急性荨麻疹动物模型血清ＩｇＥ及Ｃａｐ通透性的影响
引
言
荨麻疹（ｕｒｔｉｃａｒｉａ）俗称“风疹块＂，祖国医学称为“瘾疹’’，是由于皮肤、粘
膜小血管反应性扩张及渗透性增加而产生的一种局限性水肿反应Ⅲ。大量的流行病学研究资料表明，荨麻疹的发生率差异很大，它和调查的地区和
陕西中医学院学位论文独创性声明及使用授权声明
学位论文独创性声明
本人郑重声明：所呈交的学位论文，是个人在导师的指导下，独立进行研究工
作所取得的成果，无抄袭及编造行为。除文中已经特别加以注明引用的内容外，本论文不含任何其他个人或集体己经发表或撰写过的作品成果。对本文的研究做出重要贡献的个人和集体，均已在文中以明确方式标明并致谢。本人完全意识到本声明的法律结果并由本人所承担的法律责任。

知母及其提取物改善学习记忆和治疗阿尔茨海默病研究进展

知母及其提取物改善学习记忆和治疗阿尔茨海默病研究进展标签：知母；中药提取物；学习记忆；阿尔茨海默病；综述阿尔茨海默病（Alzheimer disease，AD）主要表现为以学习记忆为主的认知功能障碍。

目前，引起AD发病的因素有多种假说，如胆碱能假说、早期能量代谢障碍假说、自由基代谢异常假说、β类淀粉样蛋白（Aβ）级联反应假说、神经元凋亡假说、突触功能障碍假说以及炎症假说等，虽然对其发病的根本原因和病理机制还未达成共识，但多数研究者认为AD是受各种致病因素共同作用的结果。

目前使用的针对改善学习记忆能力及治疗AD的药物大多是针对单一靶点或改善某些症状，疗效存在短期性、暂时性及不良反应多等局限性，在全面改善AD主要症状方面药效不够明显。

中药以传统中医理论为指导，具有应用历史悠久、来源丰富、疗效广泛、靶点多样、不良反应少等特点，正契合了AD复杂多样的发病机制，为研发AD新药提供了一条新的途径，已引起国内外神经科学研究者的密切关注[1]。

知母（Anemarrhena asphodeloides Bge.）为百合科多年生草本植物，在我国主要分布于河北、安徽、山西、河南、内蒙古、甘肃、陕西及辽宁等地，由于野生资源产量有限，目前已形成了河北安国和安徽亳州2个全国知名的药材种植基地。

中药知母（Anemarrhenae Rhizoma）为百合科植物知母的干燥根茎，《神农本草经》将其列为中品。

作为一种传统中药应用已有两千多年历史，其中以河北易县所产品质为佳，称为“西陵知母”。

现代药物化学研究表明，知母中主要含有菝葜皂苷元（sarsapogenin）、马尔可皂苷元（markosapogenin）、新吉托皂苷元（negitogenin）、薯蓣皂苷元（diosgenin）、F-芰脱皂苷（F-gitonin）、芒果苷（mangiferin）、宝藿苷-Ⅰ（BaohuosideⅠ）、顺-扁柏树脂酚（cis-himokiresinol）、β-谷甾醇（β-sitosterol）、烟酸（Nicotinic acid）及微量元素（铁、锌、铜、锰、钴）等活性成分。

生物化学的发现英文

生物化学的发现英文In the realm of biochemistry, the discovery of DNA's double helix structure stands as a monumental breakthrough.It revolutionized our understanding of genetic informationand paved the way for modern molecular biology.The intricate dance of enzymes and substrates, orchestrating the metabolic pathways within cells, is amarvel of nature's design. Each enzyme, with its unique shape, ensures the specificity and efficiency of biochemical reactions.Another significant revelation in biochemistry is therole of amino acids in protein synthesis. The sequence ofthese building blocks determines the structure and functionof proteins, which are the workhorses of the biological world.The exploration of lipid bilayers and their role in cell membranes has deepened our comprehension of how cellsmaintain their integrity and selectively interact with their environment.The study of biochemistry also unveils the mysteries of cellular energy production. The citric acid cycle andoxidative phosphorylation are processes that convertnutrients into the energy currency of the cell, ATP.Understanding the molecular mechanisms of disease hasbeen greatly advanced by biochemistry. For instance, the identification of the molecular basis of cystic fibrosis has led to more targeted and effective therapies.The emerging field of epigenetics, where biochemistry intersects with genetics, has shed light on how environmental factors can influence gene expression without altering the DNA sequence itself.Finally, the ongoing quest to decode the human proteomeis a testament to the vastness of biochemical knowledge. Each protein's unique function contributes to the symphony of life, and understanding them is key to unlocking the mysteries of health and disease.。

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268 To resolve this problem, we recently established a new antibody against rat GR, using the recombinant GR protein produced in a baculovirus/insect cell system (Fujii et al. 2000, 2001a). Here, we describe the application of the antibody to an eye tissue preparation and show that it is abundantly distributed in the epithelial cells of the cornea, iris, and ciliary body. In addition, it is present in high levels in ganglion cells and in the pigment epithelium of the retina. To gain insight into the role of GR in those cells, its expression in rat eyes at the pre- and postnatal stages was investigated. Materials and methods Materials GSSG was obtained from Sigma. NADPH was obtained from Oriental Yeast Co., LTD. Other reagents were of the highest grade available. Wistar rats were purchased from Japan SLC, maintained under 12 h of light and 12 h of darkness schedule at temperature of 21–23 ◦ C, and fed and given water ad lib. Enzyme assay GR activity was assayed spectrophotometrically by measuring the rate of oxidation of NADPH at 340 nm (Carlberg & Mannervik 1985). The reaction mixture consisted of 0.1 M potassium phosphate, (pH 7.0), 1 mM EDTA, 0.1 mM NADPH, and 1 mM GSSG and the decrease in absorbance at 340 nm at 30 ◦ C was recorded. One unit of GR activity was deﬁned as the amount of the enzyme that catalyzes the oxidation of 1 µmol of NADPH per minute. Assays were performed in duplicate. Preparation of tissue homogenates and protein assay Experiments were performed under the protocol approved by the Animal Research Committee, Yamagata University School of Medicine. Rats were sacriﬁced by decapitation under anaesthesia with diethyl ether. Dissected eyes were either ﬁxed immediately in Bouin solution for immunohistochemical analysis or frozen in liquid nitrogen and preserved at −80 ◦ C until used for enzyme assays. For protein analysis, tissue samples were homogenized in small volumes of phosphate buffered saline containing 10 µg/ml pepstatin, 10 µg/ml leupeptin, 100 µM p -amidinophenylmethylsulphonyl ﬂuoride, and 1 mM benzamidine with a polytron homogenizer. After centrifugation at 10,000 × g for 20 min, the supernatants were collected. Protein concentrations were determined using a BCA kit (Pierce) employing bovine serum albumin as the standard. SDS-PAGE and immunoblot analysis Protein samples were subjected to 10% SDS-PAGE (Laemmli 1970) and then transferred to a Hybond-P membrane