hTERT正、反义表达载体转染大鼠骨髓间充质干细胞的实验研究

Transfection of expression plasmids of sense and antisense hTERT in rat bone marrow-derived mesenchymal stem cells

目的研究提高和阻断端粒酶活性对大鼠骨髓间充质干细胞(BMSCs)增殖及分化的影响。方法采用直接贴壁法分离、培养SD大鼠BMSCs,流式细胞仪检测BMSCs表面标记。阳离子脂质体介导hTERT正、反义表达载体转染BMSCs,RT-PCR和免疫组化方法检测TERT表达,并对转染前后BMSCs的生长增殖能力、神经细胞方向的分化能力和大鼠颅内移植存活情况进行研究。结果大鼠BMSCs表面CD29、CD44、CD90阳性,CD31、CD34、CD45阴性,转染正义hTERT后BMSCs增殖速度明显提高,而其向神经元和星形胶质细胞诱导分化及异体海马移植后的存活能力未受影响,转染反义hTERT后BMSCs增殖速度减慢,5￣6周内死亡。结论BMSCs存在端粒酶低表达,提高或阻断端粒酶活性对BMSCs的增殖分别起到促进或抑制作用。

Objective To study the influence of elevation or blockage of telomerase activity on proliferation and differentiation of rat bone marrow-derived mesenchymal stem cells （BMSCs）. Methods Rat BMSCs were obtained by complete marrow cell culture and certificated by flow cytometry analysis. Transfection of expression plasmids of sense and antisense human telomerase reverse transcriptase （hTERT） in BMSCs was mediated with cationic liposome and hTERT expression was examined with RT-PCR and immunocytochemical methods. Then the proliferation, differentiation and transplantation of BMSCs before and after transfection were studied. Results BMSCs expressed CD29, CD44 and CD90, but not CD31, CD34 or CD45. Sense hTERT transfection accelerated the proliferation of BMSCs without affecting differentiation potential into neurons or astrocytes or survival ability after transplantation into hippocampus of rats. Antisense hTERT transfection inhibited the proliferation of BMSCs and caused BMSCs death in 5-6 weeks. Conclusion Telomerase exists in BMSCs at a low level. Elevation or blockage of telomerase activity respectively accelerates or inhibits the proliferation of BMSCs.