腺病毒载体介导外源性端粒酶在组织工程种子细胞中的表达  

Expression of exogenous telomerase in tissue engineering seed cells mediated by adenoviral vector

作　　者：李卿[1] 陆峻泓[1] 王丹茹[1] 苗春雷[1] 王爱丽[1] 刘伟[1] 曹谊林[1] 

机构地区：[1]上海第二医科大学附属第九人民医院整复外科上海市组织工程重点实验室,200011

出　　处：《中华实验外科杂志》2004年第1期15-16,共2页Chinese Journal of Experimental Surgery

基　　金：国家"973"组织工程研究项目 (G1 9990 5430 4 )

摘　　要：目的　通过腺病毒载体将外源性人端粒酶逆转录酶催化亚基 (hTERT)基因导入组织工程常用种子细胞中 ,探讨其对各细胞生存状况的影响。方法　取第 4代的猪耳软骨细胞、猪骨髓基质干细胞和人皮肤成纤维细胞 ,分别以Adeno hTERT和Adeno lacZ进行基因转染。 5d后 ,取各组细胞样品 ,以逆转录 聚合酶链式反应 (RT PCR)和PCR 酶联免疫吸附法 (PCR ELISA)分别检测各自的hTERTmRNA表达及其hTERT的活性。各组hTERT转染细胞与对照细胞接种于 96孔板中 ,通过MTT法对细胞的生存和活力状况进行检测。结果　各组细胞经hTERT转染后 ,端粒酶的表达和活性均显著上升 (P <0 .0 1)。四甲基偶氮唑盐 (MTT)氧化反应结果显示hTERT转染的细胞中 ,细胞存活和活力状况显著高于对照组的细胞 (P <0 .0 1)。猪耳软骨细胞经hTERT转染后达到空白对照组细胞的 (12 1.75± 5 .3 7) % ;猪骨髓基质干细胞达 (12 5 .2 2± 2 .78) % ;人皮肤成纤维细胞达 (112 .5 7± 3 .99) %。结论　腺病毒载体介导的外源性hTERT基因在组织工程种子细胞中的表达 ,可以提高其在体外的扩增潜力 ,改善组织工程构建中种子细胞来源不足的情况。Objective To determine the effects of telomerase gene transfection on the survival and viability of seed cells for tissue engineering.Methods Porcine auricular chondrocytes,porcine bone mesenchymal stem cells and human dermal fibroblasts in passage 4 were cultured as monolayers and infected with Adeno-hTERT or Adeno-lacZ.RT-PCR and PCR-ELISA were performed 5 days later to detect the expression and activity of telomerase in variant cells.The changes in survival and viability of the infected cells were evaluated by MTT assay.Results All of the cells infected with Adeno-hTERT expressed higher telomerase activity ( P <0.01).MTT assay revealed that the survival and viability of the infected cells were significantly higher than those of the controls ( P <0.01).Porcine auricular chondrocytes were increased to (121.75±5.37)%,porcine bone mesenchymal stem cells to (125.22±2.78)%,and human dermal fibroblast to (112.57±3.99)% of non-infected cells respectively.Conclusion Introduction of exogenous telomerase into seed cells results in an increase in survival and viability,which may represent a way to expand sufficient cells for tissue engineering.

关 键 词：腺病毒载体 介导 外源性端粒酶 组织工程种子细胞 表达 基因转染 PCR-ELISA 

分 类 号：R318.0[医药卫生—生物医学工程]