构建人端粒酶催化亚单位基因慢病毒表达载体转染人骨髓基质细胞

背景：骨髓基质细胞不表达端粒酶，因而在体外传代扩增过程中端粒长度逐渐缩短，导致细胞衰老，这是限制其用于细胞治疗应用的一个重要因素。 目的：构建人端粒酶催化亚单位基因慢病毒表达载体，探讨以慢病毒介导的人端粒酶催化亚单位基因修饰人骨髓基质细胞的可行性。 方法：以pReceiver-M02-hTERT质粒为模板PCR扩增获得目的基因。用BP重组系统将目的片段重组到载体pDONR221上。然后使用LR重组系统将目的序列重组到载体pLenti6.3/V5-DEST上。将重组载体与包装质粒充分混合，利用脂质体共转染293FT细胞获得慢病毒颗粒。 结果与结论：成功构建人端粒酶催化亚单位慢病毒表达载体，病毒的平均物理滴度为1.07×10¹² LP/L。以之转染人骨髓基质细胞，目的基因表达水平有显著提升，细胞正确表达人端粒酶反转录酶蛋白。说明以慢病毒介导的人端粒酶催化亚单位基因修饰人骨髓基质细胞能强化细胞表达人端粒酶催化亚单位。

Construction of human telomerase catalytic subunit gene lentiviral expression vector and preliminary study on transfection of human bone marrow stromal cells

BACKGROUND:Bone marrow stromal cells (BMSCs) lack telomerase activity. Therefore, cell telomere length gradually shortens while proliferation in vitro, resulting in cellular senescence. This is an important factor that limits cell therapy application of BMSCs. OBJECTIVE:To construct human telomerase catalytic subunit (hTERT) gene lentiviral expression vector, and to investigate the feasibility of transfection of human BMSCs with lentivirus-mediated hTERT gene. METHODS:hTERT cDNA was obtained by PCR amplification from pReceiver-M02-hTERT. The coding sequence of hTERT was transferred into pDONR221 using BP recombination system, and then was transferred from pDONR221 into pLenti6.3/V5-DEST using LR clonase. The recombinant plasmid vector and packaging mixture were transfected into 293FT using Lipofectamine 2000 reagent, and the lentiviral particles were collected. ELISA was employed to detect the lentiviral titers. Q-PCR, Western blot and fluorescence immunocytochemistry were carried out to detect gene expression in human BMSCs after lentiviral transfecton. RESULTS AND CONCLUSION:The hTERT gene lentiviral expression vector was constructed successfully. The average virus physical titer is 1.07×10¹² LP/L. Q-PCR confirmed that the hTERT gene expression level was significantly improved in modified human BMSCs. Western blot and immunocytochemistry confirmed the correct expression of hTERT protein in cells. The results suggest that transfection of human BMSCs with lentivirus-mediated hTERT gene can enhance hTERT expression.