hTERT-逆转录病毒载体构建及其介导的脐血间质干细胞基因转移研究

Construction of recombinant retrovirus vector carrying hTERT and transfected to MSCs in human cord blood

目的:构建hTERT-逆转录病毒载体,向脐血间质干细胞(MSCs)中导入hTERT基因,观察该基因对细胞端粒酶活性的影响,能否延长细胞的寿命以及转基因细胞的分化能力。方法:PCR法扩增出hTERT全部片段,定向克隆入pLNCX2载体。将病毒质粒导入包装细胞内,筛选阳性产毒细胞,测定病毒滴度。取病毒上清感染脐血MSCs,筛选转基因细胞。检测转染前后hTERT在mRNA水平的表达,端粒酶活性的变化,hTERT转入后对细胞增殖的影响。用5-氮杂胞苷将转基因细胞向心肌方向诱导,检测心肌特异抗体的表达。结果:用BglⅡ和NotⅠ双酶切构建质粒,证明PLNCX2-hTERT构建成功,转基因细胞的hTERT基因在mRNA水平得到表达,细胞端粒酶阳性。生长曲线显示转基因细胞增殖速度快于未转染细胞。向心肌细胞诱导后,心肌特异性抗体α-sarcom ericactin和connexin-43均有表达。结论:在逆转录病毒介导下,外源性hTERT基因转入脐血MSCs后得到表达,激活细胞端粒酶的活性,有效延长了细胞的寿命,不影响其分化功能。

AIM： To construct a recombinant retrovirus vector carrying hTERT for establishing UCBMSCs with hTERT （hTERT - MSCs） to overcome their limited life span and detecting whether telomerized UCBMSCs line maintained long -term self- renewal and differentiation capacity. METHODS： The whole cDNA was generated by PCR amplifications from the plasmid pEGFP - hTERT - C1. The hTERT segments were subcloned into pLNCX2. The target cells were infected with these retroviral particles. The stably transfected cells were selected by neomycin and expanded life span which were designated hTERT - MSCs was observed. The expression of hTERT in mRNA level was detected by RT - PCR and the telomerase activity was measured by TRAP （PCR） -ELISA assay. The hTERT- MSCs were induced with 5 -azacytidine to cardiac muscle cells and the specific marker of myocardiocyte was detected. RESULTS ： The constructed plasmids were digested with restriction endonucleases （Bgl Ⅱ and Not Ⅰ ）. Two characteristic segments including 6. 1 kb and 3.6 kb were obtained. The hTERT - MSCs expressed hTERT in mRNA level. The telomerase activity of hTERT - MSCs was positive. The growth kinetics of hTERT - MSCs was higher than those in UCBMSCs. The hTERT - MSCs were induced to myocardiocyte. CONCLUSION： The hTERT recombinant retrovirus vector has been successfully constructed. The hTERT gene activates the telomerase and prolongs the life - span of cells. No effect of hTERT gene on some type of differentiation potential of MSCs is present.