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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 investigate the protective mechanism of heme oxygenase-1 (HO-1) on adipose-derived stem cells (ADSCs) under hypoxic and serum-free conditions. Methods The ADSCs of SD rats were isolated and cultured. The third generation ADSCs were divided into four groups: control group (Con), serum-free and oxygen deprivation (SOD) group, lentivirus-mediated HO- Group; HO-1 + Znpp group. The expression of extracellular signal-regulated protein kinase 1/2 (ERK1 / 2) and Adipophilin (Adi) in the four groups was detected by Western-blot. Four groups of cells were incubated with ERK1 / 2 inhibitor PD98059 The changes of ERK1 / 2 and Adipophilin in each group were detected. The contents of TNF-a and hs-CRP in the four cell culture supernatants were determined by ELISA. Results Compared with the control group, the expression of ERK1 / 2 and Adipophilin in the control group was significantly increased (P <0.01). The expression of ERK1 / 2 and Adipophilin in the control group was significantly lower than that in the control group The expression of Adipophilin in HO-1 + Znpp group was significantly higher than that in HO-1 group (P <0.05). (2) ERK1 / 2 inhibitor PD98059 incubated four groups of cells, compared with pre-incubation: SOD group ERK1 / 2, Adipophilin expression was significantly reduced (P <0.01); HO-1 group, HO-1 + Znpp ERK1 / 2, Adipophilin (P> 0.05); (3) The concentration of TNF-a and hs-CRP in SOD group was significantly higher than that in control group (P <0.01); HO-1 significantly decreased the expression of TNF- , hs-CRP secretion, and this protective effect can be reversed by HO-1 inhibitor zinc protoporphyrin (Znpp); (4) With the decrease of Adipophilin expression, the concentration of TNF-a and hs-CRP is correspondingly decreased. Conclusions HO-1 can reduce the apoptosis of ADSCs under the condition of hypoxia and serum-free, the mechanism is related to the inhibition of ERK1 / 2 activation, the decrease of Adipophilin expression and the decrease of the secretion of TNF-a and hs-CRP.