慢病毒介导的血红素加氧酶-1对脂肪干细胞在低氧无血清条件下的保护作用

Lentiviral mediated HO-1 gene transfer can enhance the survival of transfected ADSCs in vitro under ischemic environmen

目的探讨慢病毒介导的血红素加氧酶-1(HO-1)对脂肪干细胞(ADSCs)在低氧无血清的条件下的保护作用。方法分离培养SD大鼠ADSCs,取第三代ADSCs分为五组:对照组;单纯低氧无血清组;空载体病毒组;HO-1组;HO-1+ZnPP组。采用DAPI染色检测各组细胞凋亡率;Western blot法检测各组HO-1、Cleaved-Caspase-3的表达量。结果携带HO-1基因的慢病毒转染ADSCs(HO-1组)可明显增加ADSCs中HO-1的表达;ADSCs在缺氧状态下发生大量凋亡,其凋亡率为(27.29±1.18)%,转空载体的细胞凋亡率达(27.47±1.35)%,而转HO-1基因的ADSCs的凋亡率仅为(12.00±1.18)%,HO-1+ZnPP组:凋亡率(25.52±2.98)%。与对照组相比,低氧无血清组与空载体病毒组ADSCs的Cleaved-Caspase-3的表达有显著升高(P<0.05);HO-1组Cleaved-Caspase-3的表达与低氧无血清组与空载体病毒组相比有明显下降(P<0.05);而HO-1+ZnPP组Cleaved-Caspase-3的表达又明显高于HO-1组(P<0.05)。结论提高ADSCs中HO-1蛋白的表达可以通过降低凋亡效应因子Caspase-3的活性显著降低ADSCs在低氧无血清条件下的凋亡率。

Objective To identify wether Lentiviral mediated HO-1 gene transfer can enhance the survival of transfected Adipose-derived mesenchymal stem cells （ADSCs） in vitro under ischemic environment which are principal components of myocardial ischemia. Methods ADSCs were isolated from Sprague-Dawley （SD） rats adipose tissue and purified by centrifuge. The proliferation and growth characteristics were observed in primary and passage culture. ADSCs at passage 3 were equally randomized into five groups： group sham control, group serum and oxygen deprivation, group blank transferred ADSCs, group HO-1-ADSCs and groups HO-1-ADSCs＋ZnPP. Cell apoptosis and death of ADSCs in each group through SOD method in the incubator for 6 hours were detected by DAPI staining.Western blot was used to identify protein expression of Cleaved-caspase-3 and HO-1. Results Compared to other groups, HO-1-ADSCs can significantly increased the expression of HO-1 of stem cells, which suppressed Cleaved-caspase-3 expression （P〈0.05）. Primary cells in hypoxia and lack of nutritional status undergoing a lot of apoptosis, while the apoptosis rate was 27.29 percent compared to the HO-1 transferred ADSCs of （12.00±1.18）%. A large number of deaths occurred in the blank transferred cells, while the mortality rate was （27.47±1.35）%. Compared to the sham control group, the expression of Cleaved-Caspase-3 protein in serum and oxygen deprivation group and blank transferred ADSCs group＆amp;nbsp;were significantly increased （P〈0.05）; Compared to the serum and oxygen deprivation group and blank transferred ADSCs group, the expression of Cleaved-Caspase-3 protein in HO-1 group were significantly decreased （P〈0.05）;The expression of Cleaved-Caspase-3 protein in HO-1＋ZnPP group was significantly higher than HO-1 group （P〈0.05）. Conclusions The HO-1 transferred ADSCs in vitro showed more tolerance to apoptosis induced by lack of nutrition and hypoxia than simple ADSCs. The reason maybe was that the stable expression of HO-1 inhibit apoptosis by restraining the expression of Cleaved-caspase-3.