永生化人成牙本质细胞样细胞系的建立

・研究原著・文章编号:100022790(2003)1020876203永生化人成牙本质细胞样细胞系的建立王捍国,肖明振,赵守亮,郝建军,张　进　(第四军医大学口腔医学院牙体病科,陕西西安710033)收稿日期:2003202205;　修回日期:2003203207基金项目:国家自然科学基金(39970792,30270374);全军“十五”计划基金(012089);国家教育部回国人员启动基金(2000HG003)通讯作者:肖明振.Tel.(029)3376078　Email.ytsys @ 作者简介:王捍国(19742),男(汉族),重庆市人.博士,讲师,主治医师.Tel.(029)3376078　Email.wanghanguo2000@Establishment of immortalized human odontoblast 2like cell lineW A N G Han 2Guo ,XIA O Ming 2Zhen ,ZHA O S hou 2L iang ,HA O Jian 2J un ,ZHA N G JinDepartment of Operative Oentistry and Endodontics ,Stomato 2logical College ,Fourth Military Medical University ,Xi πan 710033,China【Abstract 】AIM :T o e stablish an immortalized human odonto 2blast cell line.METH ODS :Primary human odontoblast 2like cells were cultured and transfected with human telomerase re 2verse transcriptase (hTERT )gene.Anti 2drug cell clone s were formed by screening with G 418,and were selected using filter paper and expanded for continuous culture.Integration and ex 2pre ssion of hTERT in the transfected cells were detected and the biological characteristics of the cell line were analyzed.RESU LTS:A fter stable transfection ,mRNA and protein of hTERT were expre ssed in transfected cells.Among the 5cell clone s ,cells derived from 5b showed high activity of alkaline phosphatase (A LP ),normal phenotype and expre ssion of dentin sialophosphoprotein (DSPP ),which was the specific marker for odontoblast at mRNA level.The cell line had been in culture for about 6months ,56passage s ,which we named hTERT 2immortalized odontoblast 2like cell line (hTERT 2hOd 2l ).CONC L USION:An immortalized human odontoblast 2like cell line hTERT 2hOd 2l has been e stablished.【K eyw ords 】odontoblasts ;immortalization ;cell line ;telom 2erase 【摘　要】目的:建立永生化人成牙本质细胞系.方法:采用脂质体转染法,将人端粒酶逆转录酶(hTERT )基因导入原代人成牙本质细胞样细胞.培养液加G 418筛选约1mo 后,抗性细胞克隆形成,滤纸片法转移、扩增并连续培养.检测转染细胞内hTERT 的基因整和和表达,初步分析细胞系的生物学特征.结果:hTERT 稳定转染入目的细胞,表达mRNA 及其蛋白.转染后共获得5个细胞克隆,克隆5b 来源的细胞在体外长期连续培养下生长良好,具有较高碱性磷酸酶活性,在转录水平表达成牙本质细胞特异性标志牙本质涎磷蛋白,细胞核型正常.该细胞系至今传代56代,约6mo ,命名为hTERT 2hOd 2l.结论:建立起永生化人成牙本质细胞样细胞系.【关键词】成牙质细胞;永生化;细胞系;端粒,末端转移酶【中图号】R780.2 【文献标识码】A0　引言成牙本质细胞是合成和分泌牙本质基质的唯一细胞,在牙齿发育和损伤修复的过程中发挥着重要作用.人牙乳头组织来源的牙乳头外胚间充质细胞和(或)成牙本质细胞在通常的体外培养条件下经过十余次的传代后,就会停止增殖,发生衰老和死亡.我们将外源性人端粒酶逆转录酶(human telomerase re 2verse transcriptase ,h TER T )基因转染至原代人成牙本质细胞样细胞,以期建立永生化细胞系.1　材料和方法1.1　材料　质粒pCIneo 2h TER T (h TER T 全长cD 2NA 在酶切位点EcoRI 和Sal I 克隆至真核表达载体pCIneo ,美国Weinberg RA 博士惠赠).兔抗人、小鼠和大鼠h TER T 多克隆抗体(sc 27212;Santa Cruz ).1.2　方法　原代培养人成牙本质细胞样细胞后[1],接种24孔板,每孔2×105个细胞.待细胞长到95%时,按Lipofect amine TM 2000转染试剂盒(G ibco )说明书和文献[2]进行pCIneo 2h TER T 的转染.24h 后1∶10传代,次日加0.2g ・L -1G 418(G ibco ),10～14d 后未转染组细胞在G 418作用下全部死亡,此时将G 418浓度降为0.1g ・L -1,直至出现抗性细胞克隆,滤纸片法转移至24孔培养板,扩大培养.常规条件下,即DM EM 培养液(G ibco ),100mL ・L -1胎牛血清(G ibco ),50mL ・L -1CO 2和饱和湿度,长期连续培养,汇合前1∶3或1∶4传代.1.2.1　转染细胞h TER T 的表达　根据h TER T 的mRNA 序列(G ene Bank )和文献[3],设计PCR 扩增的两条引物:上游5′2G ACACACA TTC 2CACA GGTCG 23′和下游5′2G ACTCG ACACCGT 2GTCACCTAC 23′,预期产物片段为175bp.采用Ti 2tan TM One Tube R T 2PCR K it (Boehringer Manheim )进行R T 2PCR.反应体系为:2μL 模板RNA ,0.2mmol ・L -1dN TP ,0.4μmol ・L -1引物,5mmol ・L -1D TT,10u RN asi n,1.5mmol・L-1MgCl2,1μL en2 zyme Mix,加无菌双蒸水至50μL.R T2PCR参数为:50℃30min;94℃10s,60℃30s,68℃45s,10个循环;94℃10s,60℃30s,68℃2min,25个循环;68℃,7min.以3种去污剂裂解缓冲液(Tris2HCl 0.05mol・L-1,NaCl0.15mol・L-1,NaN30.2g・L-1,SDS1g・L-1,NP24010mL・L-1,PMSF0.1 g・L-1)裂解细胞并提取细胞总蛋白,经核酸蛋白分析仪定量后,参照文献[4]进行h TER T的Western blot分析,每孔上样量为50μg.显色采用Western blotting luminol reagent(Santa Cruz).1.2.2　细胞系的生物学特征　将细胞按4000个/孔接种于96孔板,培养第3d,吸弃培养液后,用0.01 mol・L-1PBS洗涤细胞3次,加0.1g・L-1TritonX2 10050μL4℃过夜,然后加AL P底物反应液100μL (AL P检测试剂盒,北京中生生物技术公司),37℃孵育60min,0.5mol・L-1NaOH终止反应.酶联免疫检测仪测定410nm波长处吸光度A值,间接反映细胞AL P活性.牙本质涎磷蛋白(dentin sialophos2 phoprotein,DSPP)的表达:参照文献[1]进行R T2 PCR,17g・L-1琼脂糖凝胶电泳后回收PCR产物,连接入p GEM2T Vector(Promega),转化大肠杆菌,蓝白斑挑选阳性克隆.质粒扩增后用限制性内切酶Eco R Ⅰ进行酶切鉴定,目的菌种送上海生物工程有限公司测序;细胞核型由第四军医大学发育和遗传学教研室采用胰蛋白酶2G iemsa染色法分析.2　结果2.1　基因转染和细胞培养　细胞转染质粒pCIneo2h TER T后,几乎无死亡细胞.加G418后第3日,细胞开始死亡.12d,未转染组细胞在G418作用下全部死亡,转染组只剩下数量不多的细胞,同时有新的细胞开始生长.G418浓度降至0.1g・L-1维持筛选, 2wk后出现抗性细胞克隆,滤纸片法转移并扩大培养,共有5个细胞克隆生长良好.3个克隆经过3mo 左右(21～26代)的传代后,细胞发生衰老、死亡.而克隆4a和5b生长状态良好,至今已连续培养约6 mo,分别传代达45和56代(克隆转移至新培养板为第1代).克隆5b较4a具有更高的AL P活性(A值分别为0.34±0.046,0.27±0.035,P<0.01),接近原代成牙本质细胞样细胞(对照组)水平(0.36±0.028,P>0.05),同时表达成牙本质细胞特异性标志DSPP(Fig1),从功能学角度更接近人成牙本质细胞,因此命名克隆5b来源的细胞系为永生化人成牙本质细胞样细胞系h TER T2hOd2l(h TER T2immortal2ized odontoblast2like cell line).h TER T2hOd2l第42代细胞核型,共23对染色体,仍为二倍体细胞,染色体形态结构基本正常(Fig2).M:100bp marker;1:Clone4a;2:Clone5b.图1　RT2PCR产物琼脂糖凝胶电泳(DSPP)Fig1　Agarose gel analysis of RT2PCRproduction图2　第42代hTERT2hOd2l细胞核型Fig2　Phenotype of passage42hTERT2hOd2l cell　×10002.2　转染细胞hTERT的表达　转染后第10和46代细胞的总RNA经R T2PCR后可获得约175bp的特异性片段,表明细胞在mRNA水平上表达h TER T,而原代细胞为阴性(Fig3).Western blot检测结果显示h TER T2hOd2l细胞在125ku左右见特异性条带,为h TER T蛋白(Fig4).3　讨论建立永生化成牙本质细胞系是牙髓生物学的研究热点和难点[5-7].为了克服现有细胞系的缺点,我们将外源性h TER T基因转染至原代人成牙本质细胞样细胞,经G418筛选后获得5个细胞克隆.在体外连续培养下,克隆5b来源的细胞生长良好,保持了许多原代细胞的生物学特征,如AL P 活性高,在mRNA 水平上表达DSPP ,核型正常.牙本质细胞外基质蛋白DSPP 基因,是目前较为公认的成牙本质细胞的特异性标志物[8].该细胞系至今已连续培养56代,约6mo ,命名为永生化人成牙本质细胞样细胞系h TER T 2hOd 2l.而在同样条件下原代细胞可连续培养13～16代,约2mo.图3　RT 2PCR 产物琼脂糖凝胶电泳(hTERT )Fig 3　Agarose gel analysis of RT 2PCRproductionhOd 2l :Primary human odontoblast 2like cells ;P10:hTERT 2hOd 2l cells at passage 10;P46:hTERT 2hOd 2l cells at passage 46.图4　hTERT 蛋白的Western blot 检测Fig 4　Analysis of Western blot with hTERT antibody TER T 是一种永生化基因.当外源性TER T 被导入目的细胞并有效表达后,端粒酶逆转录活性增加,使端粒酶RNA (TR )的逆转录速率提高;同时,TER T 还可以通过保护或稳定作用延长TR 的半衰期.这样,外源性TER T 激活目的细胞端粒酶,维持端粒长度,细胞因为染色体稳定而逾越衰老期(senes 2cence ,M1期)和危机期(crisis ,M2期),发生永生化.h TER T 转染人乳腺上皮细胞、人肺成纤维细胞系IMR90以及早衰患者的皮肤成纤维细胞后建立起相应的永生化细胞系[7].与传统的永生化基因不同,TER T 激活端粒酶诱导细胞永生化类似生殖干细胞内存在的生理途径,因此h TER T 介导的永生化细胞系保留了更多的正常细胞的生物学特征.如具正常生长速率和核型,呈现接触抑制和锚着依赖性,不具有致瘤性等.同时,TER T 介导的永生化细胞已逾越M2期,同生殖干细胞一样,是真正意义上的永生化.【参考文献】[1]Wang HG ,Xiao MZ ,Zhao SL ,Hao JJ.Primary culture of humanodontoblast 2like cell [J ].Yati Yasui Yaz hou Bi ngx ue Zaz hi (Chi n J Conserv Dent ),2002;12(2):55-58.[2]Liao W J ,Liu YF ,Liu YF ,G ao TW.Stable expression of humaninterlukin 22gene in transfected keratinocytes [J ].Di 2si J unyi Dax 2ue X uebao (J Fourth Mil Med U niv ),2001;22(24):2226-2231.[3]Hahn WC ,Counter CM ,Lundberg AS ,Beijersbergen RL ,BrooksMW ,Weinberg RA.Creation of human tumour cells with defined genetic elements [J ].N at ure ,1999;400(6743):464-468.[4]Ma CL ,Liu YF ,Li ZQ ,Liao W J.Influence of human papillo 2mavirus type 16E6and E7gene on keratinocyte growth and the ex 2pression of CyclinA ,CyclinD1and cdk4[J ].Di 2si J unyi Dax ue X uebao (J Fourth Mil Med U niv ),2000;21(9):1156-1158.[5]Su Y J ,Chen B ,Tang CW ,Hu DH ,Jiang D Y ,Jia CY.In vit rotransformation of human keratinocytes by simian virus 40[J ].Di 2si J unyi Dax ue X uebao (J Fourth Mil Med U niv ),1999;20(5):382-385.[6]Su Y J ,Chen B ,Jia CY ,Tang CW.Biological characteristics oflong 2term in vitro culture of SV40transformed human keratinocyte line [J ].Di 2si J unyi Dax ue X uebao (J Fourth Mil Med U niv ),1999;20(5):386-388.[7]Ouellette MM ,McDaniel LD ,Wright WE ,Shay J W ,Schultz RA.The establishment of telomerase 2immortalized cell lines representing human chromosome instability syndromes [J ].Hum Mol Genet ,2000;9(3):403-411.编辑　王小仲・期刊文摘・　Postoperative endoscopic surveillance of human living 2donor small bow el transplantations[Ding J ,Guo CC ,L i CN ,S un A H ,Guo X G ,Miao J Y ,Pan B R.2003M ar ;9(3):595-598]AIM :To determine the significance of endoscopic surveillance in the diagnosis of acute rejection after human living 2donor small boweltransplantations.METH ODS :Endoscopic surveillance was performed through the ileostomy after human living 2donor small bowel trans 2plantations.The intestinal mucosa was observed and biopsies were performed for pathological observations.RESU LTS :Acute rejection was diagnosed in time by endoscopic surveillance.The endoscopic and pathological manifestations of acute rejection were described.CONC L USION :Endoscopic surveillance and biopsy are reliable methods to diagnose the acute rejection after human living 2donor small bowel transplantations.。