ts-SV40LT抗原转基因干细胞增殖活性的温度调控机制分析  

The Mechanism Analysis of Temperature Regulating Proliferation of Stem Cells Modified by tsSV40LT Gene

作　　者：刘洪良[1] 刘晓智[1] 刘振林[1] 姜忠敏[2] 盛凤[3] 李罡[1] 

机构地区：[1]天津市第五中心医院神经外科,300162 [2]天津市第五中心医院病理科,300162 [3]天津市第五中心医院中医内科,300162

出　　处：《天津医药》2012年第3期247-250,I0002,共5页Tianjin Medical Journal

基　　金：国家自然科学基金项目(项目编号:30872668)

摘　　要：目的:研究温度敏感性猿猴病毒40大T抗原(ts-SV40LT)转基因干细胞增殖活性的温度调控机制。方法:用含ts-SV40LT抗原的表达质粒转染脐带间充质干细胞(UCMSCs),将其分别置于33℃和37℃培养,细胞免疫荧光方法检测增殖细胞核抗原(PCNA)活性,PCR-ELISA端粒酶检测法检测端粒酶活性及细胞周期,WesternBlot法检测周期素(Cyclin)D1、CyclinE、周期素依赖性蛋白激酶(CDK)2、CDK4、CDK6、P16和P21的蛋白表达情况,免疫荧光方法检测巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)的表达。结果:细胞在33℃时培养24、48、72h的PCNA阳性表达百分率均高于37℃时,差异有统计学意义(P<0.01)。转染tsSV40LT抗原基因后,细胞在33℃培养箱时的端粒酶活性和PIx值高于37℃时,差异有统计学意义(P<0.01)。33℃时的CyclinD1、CyclinE、CDK2、CDK4、CDK6及Nestin表达量高于37℃时,P16、P21、NSE和GFAP的表达量低于37℃时,差异均有统计学意义(P<0.05或P<0.01)。结论:通过tsSV40LT抗原基因的有效转染可实现干细胞在不同温度环境下的增殖调控。Objective：To study the mechanism of temperature control in proliferation of stem cells modified by tsSV40LT gene temperature-sensitive simian virus 40 large T-antigen （tsSV40LT）. Methods： After transfecting plasmid containing tsSV 40 LT into umbilical cord mesenchymal stem cells （UCMSCs）. These UCMSCs modified by tsSV40LT were cultured in 33 ℃ and 37 ℃ incubators respectively. The proliferating cell nuclear antigen （PCNA） activity, telomerase activation, cell cycle, cell cycle regulating proteins, including Cyclin D1, Cyclin E, CDK2, CDK4, CDK6, P16, P21, nestin, neurone specific enolase （NSE） and glial fibrillary acidic protein （GFAP） were detected respectively. Results： When cultured at 33 ℃ for 24, 48 and 72 h, the positive percentage of PCNA expression was significantly higher than that of cells cultured in 37 ℃（P 〈 0.01）. After tsSV40LT gene transfection, telomerase activation and Pix value were significantly higher in cells cultured at 33 ℃ than those of cells cultured in 37 ℃（P 〈 0.01）.The expression levels of Cyclin D1, CyclinE, CDK2, CDK4, CDK6 and Nestin were significantly higher in cells cultured at 33 ℃ than those of cells cultured in 37 ℃（P 〈 0.05 or P 〈 0.01）. Conclusion： The proliferation of stem cells can be regulated by temperature through effective tsSV40LT gene transfection.

关 键 词：间质干细胞 脐带 细胞增殖 转基因 增殖细胞核抗原 低温 周期素依赖激酶抑制剂p16 周期 素依赖激酶抑制剂p21 细胞周期蛋白质依赖激酶类 磷酸丙酮酸水合酶 神经胶质原纤维酸性蛋白质 

分 类 号：R446.62[医药卫生—诊断学]