端粒酶逆转录酶基因修饰对人骨髓间质干细胞端粒酶活性的影响

Ectopic hTERT gene expression on the telomerase activity in human bone marrow mesenchymal stem cell

目的：观察外源性端粒酶逆转录酶（hTERT）基因的异位表达对人骨髓间质干细胞（MSCs）端粒酶活性的影响。方法：无菌条件下，抽取健康志愿者骨髓2mL，经离心、洗涤及传代培养后备用。取第5代人MSCs接种至6孔培养板中，待其融合达90％～95％时，用脂质体法对其进行转染，转染共分4组：正常对照组、脂质体组、pEG-FP-C1质粒组、pEGFP-hTERT质粒组。选用G418进行抗性克隆的筛选。30d后，正常对照组、脂质体组细胞全部死亡，而pEGFP-C1质粒组、pEGFP-hTERT质粒组则获得抗性克隆；将获得的抗性克隆进一步扩增后，选用pEGFP-C1质粒组、pEGFP-hTERT质粒组和未转染的人骨髓MSCs分别进行RT-PCR，检测转染前后hTERT mRNA的表达情况，并通过PCR-ELISA检测上述细胞的端粒酶活性。结果：未转染组和pEGFP-C1质粒组的人骨髓MSCs hTERT mRNA表达阴性，且端粒酶呈阴性；pEGFP-hTERT质粒组的人骨髓MSCs hTERT mRNA表达阳性，且端粒酶呈阳性。结论：外源性hTERT基因可以在人骨髓MSCs中获得异位表达，并能诱导人骨髓MSCs的端粒酶活性。

Aim ： To investigate the introduction of hTERT gene into human mesenchymal stem cells（MSCs） on the telomerase activity. Methods：Human bone marrow was obtained from healthy donator. The MSCs were separated and cultured. The 5th generation of MSCs were planted and transfected with lipofectamine, pEGFP-C1 plasmid, and pEGFP- hTERT plasmid,respectively. The untransfected MSCs were used as normal control. Drug selection（ including G418 200 mg/L） was started 24 h after transfection and continued for 14 days. After 30 days, cells in normal control and lipo- fectamine group did not survive, but clones of the pEGFP-C1 and the pEGFP-hTERT group were observed. Then the hTERT mRNA expression in MSCs was detected by RT-PCR. Telomerase activity of the transfected MSCs was detected by PCR-ELISA. Results： hTERT fragment was identified in the hTERT-transfected cells but not in the untransfected human bone marrow MSCs. The untransfected human MSCs remained telomerase-negative but the hTERT-transfected cells showed telomerase activity. Conclusion ： Ectopic expression of the hTERT gene in human MSCs can reconstitute telomerase activity.