HO-1通过Adipophilin途径对脂肪干细胞在低氧无血清条件下的保护机制探讨

Discussion on the Protective Mechanism of Heme Oxygenase-1 to Adipose-deprived Stem Cells by the Way of Adipophilin under the Circumstance with Serum Free and Oxygen Deprivation

目的探讨血红素加氧酶-1(HO-1)对脂肪干细胞(ADSCs)在低氧无血清的条件下的保护机制。方法分离培养SD大鼠ADSCs,取第三代ADSCs分为四组:对照组(Control,Con);低氧无血清(serum—free and oxygen deprivation,SOD)组;慢病毒介导的HO-1组;HO-1+Znpp组。蛋白印迹(Western-blot)法检测四组细胞外信号调节蛋白激酶1/2(ERK1/2)、脂肪分化相关蛋白(Adipophilin,Adi)的表达量;用ERK1/2抑制剂PD98059孵育四组细胞,检测各组细胞中ERK1/2、Adipophilin的变化情况。ELISA法测定四组细胞培养上清液中TNF-a,hs-CRP含量。结果(1)对照组ERK1/2、Adipophilin的表达量极低;与对照组相比,SOD组ERK1/2、Adipophilin的表达量显著升高(P<0.01),HO-1组与SOD组相比ERK1/2、Adipophilin的表达量显著降低(P<0.05);HO-1+Znpp组与HO-1组相比ERK1/2、Adipophilin的表达量显著升高(P<0.05)(2)用ERK1/2抑制剂PD98059孵育四组细胞,较孵育前:SOD组ERK1/2、Adipophilin的表达量显著降低(P<0.01);HO-1组、HO-1+Znpp组ERK1/2、Adipophilin的表达量降低,但差异无统计学意义(P>0.05);(3)SOD组TNF-a,hs-CRP浓度较对照组显著升高(P<0.01);HO-1可显著降低TNF-a,hs-CRP分泌,而这种保护效应可被HO-1抑制剂锌原卟啉(Znpp)所逆转;(4)随着Adipophilin的表达减少,TNF-a,hs-CRP浓度相应降低。结论HO-1降低ADSCs在低氧无血清条件下的凋亡,其机制与抑制ERK1/2激活,降低Adipophilin表达,从而减少炎症因子TNF-a,hs-CRP分泌有关。

Objective To explore the protective effects of Heme oxygenase-1 （HO-1） to Adipose-deprived stem cells（ADSCs） under the circumstance with serum free and oxygen deprivation. Methods Methods Fractional cultivate the ADSCs of SD rats ,divided the third generation of ADSCs into four groups ： control group, serum free and oxygen deprivation group （SOD group）, viral mediated HO-1 group and HO-l＋Znpp group. Western blot was used to identify the expressions of ERK1/2 and Adipophilin ineach group; and the changes of ERKI/2 and Adipophilin and were detected after using PD98059. The concentrations of serum TNF-aand hs-CRP were determined by using ELISA. Results （1） The expression of ERK1/2 and Adipophilinin is extremely low in the control group; Compared with the control group, the expression of ERKI/2 and Adipophilin increased significantly in SOD group （P〈0.01）; The expression of ERK1/2 and Adipophilin was significantly lower in HO-1 group than that of SOD group （P〈0.05） ; When compared with HO-1 group, the expression of ERK1/2 and Adipophilin is increased significantly in HO-l＋Znpp group（P〈0.05）. （2） The expression of ERK1/2 and Adipophilin decreased significantly in SOD group with the ERK1/2 inhibitor PD98059（P〈0.01）; But the difference is not statistically significant in HO-1 group and HO-l＋Znpp group （P〉0.05）; （3）In SOD group, TNF-a, hs-CRP were significantly higher than that of control group （P〈0.01）; HO-1 can significantly reduce TNF-a, hs-CRP secretion, and this effect can be reversed by the HO-1 inhibitor zinc protoporphyrin （Znpp）; （4） With the decrease in the expression of Adipophilin, TNF-aand hs-CRP concentration decreased. Conclusions HO-1 reduced apoptosis of ADSCs under serum- free and oxygen deprivation conditions, the mechanism is that HO-lcan inhibit ERK1/2 activation, reduce the expression of Adipophilin, and thereby reducing the inflammatory cytokines TNF-a, hs-CRP secretion.