OGD/R星形胶质细胞DNMT3a、tPA表达与凋亡变化及腺苷甲硫氨酸的干预作用

Changes of DNMT3a,tPA expression and apoptosis in OGD/R astrocytes and the intervention of S-adenosyl methionine

目的观察糖氧剥夺/复氧复糖(OGD/R)大鼠星形胶质细胞中DNA甲基转移酶(DNMT)3a、组织型纤溶酶原激活物(tPA)表达与细胞凋亡变化,以及S-腺苷甲硫氨酸(SAM)的干预作用。方法培养并鉴定大鼠皮质星形胶质细胞,将细胞分为对照组、OGD/R组、OGD/R+SAM组。对照组常规培养。OGD/R组、OGD/R+SAM组以缺糖缺氧3 h后再复氧复糖24 h建立OGD/R细胞模型,OGD/R+SAM组在OGD/R过程中细胞培养基始终保持有50μmol/L的SAM。使用DNA甲基化试剂盒检测各组DNMT活性,采用RT-PCR法检测各组细胞中的DNMT3a、tPA mRNA,采用Western blotting法检测DNMT3a、tPA、Bcl-2、Bax、Cleaved Caspase-3蛋白。结果OGD/R组DNMT活性、DNMT3a mRNA和蛋白表达低于对照组,tPA mRNA和蛋白表达高于对照组(P均<0.05);OGD/R+SAM组DNMT活性、DNMT3a mRNA表达高于OGD/R组,tPA mRNA和蛋白表达低于OGD/R组(P均<0.05)。OGD/R组Bcl-2蛋白表达及Bcl-2/Bax低于对照组,Bax、Cleaved Caspase-3蛋白表达高于对照组(P均<0.05);OGD/R+SAM组Bcl-2蛋白表达及Bcl-2/Bax高于OGD/R组,Bax、Cleaved Caspase-3蛋白表达低于OGD/R组(P均<0.05)。结论OGD/R的星形胶质细胞DNA甲基化水平降低,tPA表达上调,促进细胞凋亡;SAM干预可逆转OGD/R导致的上述改变。

Objective To observe the expression changes of DNA methyltransferase 3a(DNMT3a),tissue plasminogen activator(tPA)and apoptosis induced by oxygen glucose deprivation and reoxygenation(OGD/R)in rat cortical astrocytes,as well as the intervention of S-adenosyl methionine(SAM).Methods Primary cultured cortical astrocytes were isolated and identified,and then were divided into the control group,OGD/R group,and OGD/R+SAM group.Cells in the control group were routinely cultured.In the OGD/R group and OGD/R+SAM group,the OGD/R cell injury models were established(oxygen glucose deprivation 3 h,reoxygenation 24 h),and the cells in the OGD/R+SAM group were treated with 50μmol/L SAM during the modeling process.DNMT activity was measured by DNA methylation assay;DNMT3a,tPA,Bcl-2,Bax,and Cleaved Caspase-3 protein expression levels were determined by Western blotting;and RT-PCR was used to detect the expression of DNMT3a and tPA mRNA expression level after OGD 3 h and reoxygenation and glucose 24 h.Results DNMT activity and DNMT3a mRNA and protein expression in the OGD/R group were lower than those in the control group,but tPA mRNA and protein expression levels were higher than those in the control group(all P<0.05);DNMT activity and DNMT3a mRNA expression in the OGD/R+SAM group were higher than those in the OGD/R group,and the expression levels of tPA mRNA and protein were lower than those in the OGD/R group(all P<0.05).The Bcl-2 protein expression and the Bcl-2/Bax ratio of the OGD/R group were lower than those of the control group,and the Bax and Cleaved Caspase-3 protein expression levels were higher than those of the control group(all P<0.05);the Bcl-2 protein expression and Bcl-2/Bax ratio in the OGD/R+SAM group were higher than those in the OGD/R group,and the Bax and Cleaved Caspase-3 protein expression levels were lower than those in the OGD/R group(all P<0.05).Conclusion OGD/R decreases DNA methylation,increases tPA expression,and induces apoptosis of astrocytes;SAM can reverse these changes.