hTERT介导脐带间充质干细胞实现永生化的研究

Establishment of immortalized umbilical cord mesenchymal stem cells mediated by hTERT

目的通过转染人端粒酶逆转录酶催化亚单位（hTERT）建立永生化脐带间充质干细胞（UCMSCs），用以获得足够多的UCMSCs满足体外研究与临床需要。方法原代分离培养人UCMSCs。取第4代UCMSCs行流式细胞术检测UCMSCs表面特异标志物表达：利用脂质体介导pLXSN—hTERT正义表达质粒转染UCMSCs，建立永生化UCMSCs; RT-PCR检测转染前后hTERTmRNA表达水平，细胞免疫荧光方法检测hTERT蛋白表达，流式细胞术观察hTERT转染后细胞周期改变。结果流式细胞术结果显示UCMSCs强表达CD29、CD44、CD105，极低表达CD31、CD34、CD45，检测结果符合人UCMSCs特征；RT-PCR检测单纯UCMSCs低表达hTERTmRNA，pLXSN-hTERT正义表达质粒转染后hTERTmRNA表达明显增高；细胞免疫荧光显示hTERT全部表达于细胞核．转染后胞核荧光强度明显增强：细胞周期检测结果显示转染后处于S期UCMSCs数目明显增多，细胞可获得长期稳定传代（〉35代）。结论以hTERT转染UCMSCs在体外可获得长期稳定传代培养细胞，可基本满足体外研究与临床需要。

Objective To establish the immortalized umbilical cord mesenchymal stem cells （UCMSCs） mediated by human telomerase reverse transcriptase （hTERT） so as to offer enough UCMSCs for in vitro or clinical researches. Methods Human UCMSCs were isolated and cultured to passage 4, and then identified by flow cytometry analysis. The pLXSN-hTERT plasmid was transfected into UCMSCs by liposome to establish the immortalized UCMSCs. Expressions of hTERT mRNA and protein were respectively tested by RT-PCR and immunocytochemistry. The cell cycle kinetics of the passage of hTERT-UCMSCs and UCMSCs were detected with flow cytometry. Results Flow cytometry analysis showed that UCMSCs expressed CD29, CD44 and CD105 strongly, but CD31, CD34 and CD45 slightly; these were right the features of human UCMSCs. RT-PCR showed that hTERT mRNA was expressed low in UCMSCs, and high in pLXSN-hTERT transfected UCMSCs. Immunocytochemistry revealed that fluorescence intensity of cell nuclei was increased significantly after transfection and that hTERT was expressed all in cell nuclei. Flow cytometry analysis suggested that the number of hTERT-UCMSCs at phase S was increased significantly, and the cells would be able to passage stably （more than 35 passages）. Conclusions The immortalized UCMSCs can be established by hTERT transfection, and the number of immortalized UCMSCs can meet the need of in vitro or clinical researches.