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目的探讨重组慢病毒(lentivirus,LVs)介导Akt1基因转染大鼠BMSCs能否提高细胞缺氧耐受能力,为提高干细胞移植治疗心肌梗死效果提供理论依据。方法以LVs作为转染载体,增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)为标记,构建携带Akt1基因的p LVX-EGFP-3FLAG病毒载体。取第3代3~5周龄SD大鼠BMSCs分别转染p LVX-EGFP空白病毒液(B组)和p LVX-EGFP-3FLAG病毒液(C组),以未转染病毒的BMSCs为空白对照组(A组)。转染后2~3 d荧光显微镜观察细胞绿色荧光表达情况,并于48 h时采用Western blot法检测B、C组Akt1蛋白表达情况。将B、C组培养的BMSCs分别置于94%N2、1%O2和5%CO2缺氧箱进行缺氧干预(分别为B1、C1组),取B1、C1组缺氧干预0、3、6、9、12 h的细胞以膜联蛋白V-FITC/碘化丙啶双染法行流式细胞仪分析细胞凋亡率和死亡率、MTT法分析细胞增殖情况、Western blot法检测凋亡相关基因半胱氨酸天冬氨酸蛋白酶3(Caspase-3)表达情况。结果转染后A组荧光显微镜下未见绿色荧光表达,B、C组可见明显绿色荧光,转染效率约60%;Western blot可检测到B、C组Akt1表达,且C组Akt1表达明显高于B组(t=17.525,P=0.013)。B1组缺氧干预后各时间点细胞凋亡率和死亡率均逐渐增高,差异有统计学意义(P<0.05);C1组细胞凋亡率和死亡率在缺氧干预后3 h短暂下降,之后逐渐增高,差异有统计学意义(P<0.05)。除0 h外,缺氧干预各时间点C1组细胞凋亡率和死亡率均显著低于B1组,差异有统计学意义(P<0.05)。MTT法检测示,常氧条件下B、C组各时间点吸光度(A)值均显著高于缺氧条件下B1、C1组(P<0.05),C组各时间点A值均显著高于B组(P<0.05),缺氧干预后6、9、12 h B1组A值显著低于C1组(P<0.05)。Western blot法检测示,与B1组比较,C1组缺氧干预各时间点Caspase-3表达均显著下调(P<0.05)。结论通过重组LVs介导Akt1基因转染可通过抑制凋亡显著提高BMSCs的缺氧耐受能力,从而为改善干细胞移植治疗心肌梗死效果提供新思路。
Objective To investigate whether the transfection of Akt1 gene into rat BMSCs by recombinant lentivirus (LVs) can improve cell hypoxia tolerance and provide a theoretical basis for improving stem cell transplantation in treating myocardial infarction. Methods LVs was used as transfection vector and enhanced green fluorescent protein (EGFP) was used as a marker to construct p LVX-EGFP-3FLAG vector carrying Akt1 gene. The third generation of BMSCs from 3 to 5 weeks old SD rats were transfected with the pVXX-EGFP blank virus solution (group B) and the p LVX-EGFP-3FLAG virus solution (group C), respectively. The untransfected BMSCs were blank Control group (group A). The expression of green fluorescence was observed by fluorescence microscopy 2 ~ 3 days after transfection. At 48 h, the expression of Akt1 protein in B and C groups was detected by Western blot. BMSCs cultured in B and C groups were exposed to 94% N2, 1% O2 and 5% CO2 hypoxia for hypoxia (B1 and C1, respectively) The cell apoptosis rate and mortality were analyzed by flow cytometry with annexin V-FITC / propidium iodide double staining method at 6, 9 and 12 h. Cell proliferation was analyzed by MTT assay and apoptosis was detected by Western blot Related gene caspase-3 expression. Results After transfection, no fluorescence was observed in group A under fluorescence microscope, and obvious green fluorescence was observed in group B and C, and the transfection efficiency was about 60%. Akt1 expression in group B and C was detected by Western blot and Akt1 expression was significantly higher in group C In group B (t = 17.525, P = 0.013). The apoptosis rate and mortality of B1 group were significantly increased at each time point after hypoxia (P <0.05). The apoptosis rate and mortality of C1 group decreased temporarily 3 hours after hypoxia intervention, Then gradually increased, the difference was statistically significant (P <0.05). Except for 0 h, the apoptosis rate and mortality of C1 group were significantly lower than those of B1 group at each time point of hypoxia intervention (P <0.05). MTT assay showed that the absorbance (A) of B and C groups at each time point under normoxia was significantly higher than that of B1 and C1 groups under hypoxia (P <0.05), and the A value at each time point in C group was significantly higher than In group B (P <0.05), the value of A in group B1 at 6, 9 and 12 h after hypoxia intervention was significantly lower than that in group C1 (P <0.05). Western blot showed that compared with B1 group, the expression of Caspase-3 in C1 group was significantly down-regulated at each time point (P <0.05). Conclusion Transfection of Akt1 gene by recombinant LVs can significantly enhance the hypoxia tolerance of BMSCs by inhibiting apoptosis, and provide new ideas for improving the effect of stem cell transplantation in treating myocardial infarction.