天津医药

• 实验研究 •    

ts-SV40LT抗原转基因干细胞增殖活性的温度调控机制分析

刘洪良,刘晓智,刘振林,姜忠敏,盛凤,李罡   

  1. 天津市第五中心医院
  • 收稿日期:2011-06-02 修回日期:2011-09-15 出版日期:2012-03-15 发布日期:2012-03-15
  • 通讯作者: 刘洪良

The mechanism analysis of temperature regulating proliferation of stem cells modified by tsSV40LT gene

  • Received:2011-06-02 Revised:2011-09-15 Published:2012-03-15 Online:2012-03-15

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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，为实现脑创伤病人在亚低温治疗期间实施干细胞移植打下基础。方法：用含ts-SV40LT抗原的表达质粒转染人源性脐带间充质干细胞（UCMSCs），PCR方法检测外源基因的整合效率，将其分别置于亚低温（33℃）和正常体温（37℃）两种温度下培养，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，流式细胞术检测细胞周期，Western Blot法检测细胞周期调控蛋白——周期素D1（cyclin D1）、CyclinE、周期素依赖性蛋白激酶-2（CDK2）、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；SPSS13.0软件进行统计学分析。结果：PCR结果显示tsSV40LT抗原的基因片段被成功整合入干细胞，该细胞系在33℃条件下增殖生长旺盛，端粒酶活性高，Cyclin D1、CyclinE、CDK2、CDK4、CDK6均高表达，P16和P21低表达，同时高表达nestin，不表达NSE和GFAP；在37℃条件下细胞增殖缓慢甚至停滞，端粒酶活性低，Cyclin D1、CyclinE、CDK2、CDK4、CDK6的表达强度明显低于33℃条件，P16和P21则相反，与此同时nestin表达强度明显下降，NSE和GFAP表达水平上升。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，为在亚低温条件下实现干细胞移植救治颅脑创伤提供了实验支持。

关键词: 干细胞, 颅脑创伤, 低温疗法, 分化

Abstract: Objective:To study the mechanism of temperature control proliferation of the stem cells modified by tsSV40LT gene for establishing a basement on stem cells transplantation into injured brain area during hypothermia treatment. Methods: After transfecting plasmid containing temperature-sensitive simian virus 40 large T-antigen (tsSV40LT） into umbilical cord mesenchymal stem cells (UCMSCs), PCR method was used to detect gene integration. Then we cultured these UCMSCs modified by tsSV40LT in 33℃ and 37℃ incubators respectively. Immunofluorescence was used to detect proliferating cell nuclear antigen (PCNA) activity, telomerase activation analysis by PCR-ELISA telomerase detect kit, and cell cycle by flow cytometry. Then cell cycle regulating proteins, including Cyclin D1, CyclinE, CDK2, CDK4, CDK6, P16 and P21, were detected by western blot. The expression of nestin, neurone specific enolase (NSE) and glial fibrillary acidic protein(GFAP) were detected by immunofluorescence method. Results:The tsSV40LT gene was integrated into UCMSCs successfully. When cultured at 33℃ incubator, the new cell line displayed high proliferation activity, as well as its telomerase activation, highly expressed Cyclin D1, CyclinE, CDK2, CDK4 and CDK6, and lowly expressed P16 and P21, meanwhile, highly nestin, lowly NSE and GFAP. But when cultured at 37℃ incubator, the cell line showed a completely converse profile. Conclusion:The proliferation of stem cells can be regulated by temperature through effective tsSV40LT gene transfection, and that supports the clinical application of stem cells transplantation into injured brain area during hypothermia treatment.

Key words: Stem Cell, Traumatic Brain Injury, Hypothermia Treatment, Differentiation