高磷通过SET8调控p53/Bcl-2/Caspase信号通路促进血管平滑肌细胞钙化  

High phosphorus promotes calcification of vascular smooth muscle cells via SET8 regulating p53/Bcl-2/Caspase signaling pathway

作　　者：张东雪 路静[1] ZHANG Dong-xue;LU Jing(School of Basic Medical Science, Zhengzhou University, Zhengzhou 450001, China;Departments of Nephrology,the Fourth Hospital of Hebei Medical University)

机构地区：[1]郑州大学基础医学院,450001 [2]河北医科大学第四医院肾内科

出　　处：《天津医药》2019年第10期1030-1034,共5页Tianjin Medical Journal

基　　金：国家自然科学基金面上资助项目(81572972);河北省医学科学研究重点课题(20180513,20190702)

摘　　要：目的探讨组蛋白赖氨酸甲基转移酶SET8在高磷诱导血管平滑肌细胞(VSMCs)钙化中的作用及机制。方法选取80~100g的清洁雄性SD大鼠6只,取其胸主动脉,分离VSMCs后进行原代培养。VSMCs传至第3~ 4代,铺6孔板,并给予相应刺激。将VSMCs随机分为正常组和高磷组(10mmol/Lβ-甘油磷酸),培养4d后茜素红染色观察钙结节形成情况,甲基麝香草酚蓝比色法钙含量,流式细胞数检测细胞凋亡,RT-PCR和Westernblot检测SET8、 p53、 Bcl-2、 Bax、Caspase3的mRNA和蛋白的表达水平。之后用脂质体将SET8-shRNA质粒与NS-shRNA转染VSMCs作为SET8-shRNA组和空质粒组,未转染组作为正常对照组,RT-PCR和Westernblot检测p53、 Bcl-2、 Bax、Caspase3的mRNA和蛋白的表达水平。结果(1)高磷组钙盐沉积较正常组明显增加(P<0.05)。( 2)流式细胞仪检测结果显示,与正常组相比,高磷组VSMCs的细胞凋亡率明显增加(P<0.05)。( 3)与正常组相比,高磷组Bcl-2、 SET8的mRNA和蛋白表达水平下降;p53、 Bax、Caspase3的mRNA和蛋白表达水平升高(P<0 .05)。( 4)干扰SET8基因表达后钙盐沉积增加(P<0.05), Bcl-2的mRNA和蛋白表达水平下降;p53、 Bax、Caspase3的mRNA和蛋白表达水平升高(P<0.05)。结论高磷通过SET8调控p53/Bcl-2/Caspase信号通路,下调抗凋亡蛋白Bcl-2表达,上调促凋亡蛋白Bax和Caspase3的表达,进而参与调控VSMCs的钙化。Objective To explore the role and mechanism of histone lysine methyltransferase SET8 in the calcification of vascular smooth muscle cells (VSMCs) induced by high phosphorus. Methods The clean male SD rats weighting 80 g to 100 g were selected, and their thoracic aortae were taken for primary culture. VSMCs were passed to the 3rd to 4th generation, and the cells were plated into six-well plates, and then VSMCs were given corresponding stimulation. VSMCs were randomly divided into normal group and high phosphorus group (10 mmol/L β-glycerophosphate). The formation of calcium nodules was observed after four-day culture. Flow cytometry was used to detect the apoptosis of cells. The expression levels of mRNA and protein of SET8, p53, Bcl-2, Bax and Caspase3 were detected by RT-PCR and Western blot assay. Then SET8-shRNA plasmid was transfected into VSMCs that was used as SET8-shRNA group. The un-transfected group was used as normal control group. The effects of SET8 on mRNA and protein expression levels of p53, Bcl-2 Bax and Caspase3 were detected by RT-PCR and Western blot assay. Results (1) The calcium deposition was significantly increased in the high phosphorus group compared with that of normal group (P<0.05).(2) The result of flow cytometry showed that compared with the normal group, the apoptosis rate of VSMCs was significantly increased in the high phosphorus group (P<0.05).(3) Compared with the normal group, the expression levels of Bcl-2 and SET8 mRNA and protein were significantly decreased, and the expression levels of p53, Bax and Caspase3 increased in the high phosphorus group (P<0.05).(4) Calcium salt deposition increased after interference with SET8 (P<0.05). The expression level of Bcl-2 decreased and the expression levels of p53, Bax and Caspase3 increased after interference with SET8 gene expression (P<0.05). Conclusion High phosphorylation regulates p53/Bcl-2/Caspase signaling pathway through SET8, down-regulates the expression of anti-apoptotic protein Bcl-2, up-regulates the expressions of pro-a

关 键 词：肌 平滑 血管 基因 p53 RNA干扰 甘油磷酸类 

分 类 号：R543.3[医药卫生—心血管疾病]