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目的探讨慢病毒介导的血红素加氧酶-1(Heme oxygenase-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分为三组:对照组(Control);低氧无血清(serum—free and oxygen deprivation,SOD)组;慢病毒介导的HO-1组。采用DAPI染色检测细胞凋亡率;蛋白印迹(Western-blot)法检测三组HO-1的表达量;ELISA法测定三组细胞培养上清液中TNF-а,IL-1β,IL-10含量。结果 (1)携带HO-1基因的慢病毒转染ADSCs可明显增加ADSCs中HO-1的表达;(2)对照组凋亡率对照组(4.21±2.56)%;SOD组(26.69±1.29)%;HO-1组(12.32±1.28)%。SOD组与对照组相比,凋亡率有显著升高(P<0.01),HO-1组与SOD组相比凋亡率显著降低(P<0.05);(3)与对照组比较,SOD组TNF-а,IL-1β浓度显著升高(P<0.01);IL-10浓度显著降低(P<0.01)。与SOD组比较,HO-1组TNF-а,IL-1β浓度显著降低(P<0.05);IL-10浓度显著升高(P<0.05)。结论提高ADSCs中HO-1的表达可以显著降低ADSCs在低氧无血清条件下的凋亡,其机制与降低炎症因子TNF-а,IL-1β分泌,升高抗炎因子IL-10表达有关。
Objective To investigate the protective effect of lentivirus-mediated Heme oxygenase-1 (HO-1) on adipose-derived stem cells (ADSCs) under hypoxic and serum-free conditions. Methods ADSCs were isolated and cultured in SD rats. The third generation ADSCs were divided into three groups: Control group, serum-free and oxygen deprivation (SOD) group and lentivirus-mediated HO-1 group. The apoptosis rate was detected by DAPI staining. The expression of HO-1 in three groups was detected by Western-blot. The levels of TNF-а, IL-1β and IL-10 in the three cell culture supernatants were determined by ELISA . Results (1) Transfection of ADSCs with lentivirus carrying HO-1 gene could obviously increase the expression of HO-1 in ADSCs. (2) The apoptotic rate of control group (4.21 ± 2.56)%, SOD group (26.69 ± 1.29) %; HO-1 group (12.32 ± 1.28)%. Compared with the control group, the apoptosis rate in SOD group was significantly increased (P <0.01), while the apoptosis rate in HO-1 group was significantly lower than that in SOD group (P <0.05); (3) Compared with control group, The levels of TNF-а and IL-1β were significantly increased (P <0.01), and the concentrations of IL-10 were significantly decreased (P <0.01). Compared with SOD group, the concentrations of TNF-а and IL-1β in HO-1 group were significantly decreased (P <0.05); the concentration of IL-10 in HO-1 group was significantly increased (P <0.05). CONCLUSION: Increasing the expression of HO-1 in ADSCs can significantly reduce the apoptosis of ADSCs under hypoxia and serum-free conditions. The mechanism is related to decreasing the secretion of inflammatory cytokines TNF-а and IL-1β and increasing the expression of anti-inflammatory cytokine IL-10.