Rap1A DNA甲基化在Hcy促进巨噬细胞增殖中的作用  

Effect of DNA methylation of Rap1A on macrophages proliferation induced by Hcy

作　　者：张玲 李思睿 赵迅霞[4] 田荣 袁茵 孙卓成 马生贤 郝银菊[5] 杨晓玲 ZHANG Ling;LI Sirui;ZHAO Xunxia;TIAN Rong;YUAN Yin;SUN Zhuocheng;MA Shengxian;HAO Yinju;YANG Xiaoling(Ningxia Key Laboratory of Vascular Injury and Repair;School of Basic Medicine,Ningxia Medical University;School of Public Health and Management,Ningxia Medical University;Experimental Center,School of Basic Medicine,Ningxia Medical University;School of Pharmacy,Ningxia Medical University,Yinchuan,Ningxia 750004,China)

机构地区：[1]宁夏血管损伤与修复研究重点实验室 [2]宁夏医科大学基础医学院,宁夏银川市750004 [3]宁夏医科大学公共卫生与管理学院,宁夏银川市750004 [4]宁夏医科大学基础学院实验中心,宁夏银川市750004 [5]宁夏医科大学药学院,宁夏银川市750004

出　　处：《中国动脉硬化杂志》2020年第6期483-489,共7页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金项目(81760095);宁夏高等教育科学研究项目(NGY2017122);宁夏高等学校一流学科建设(宁夏医科大学西部一流建设学科基础医学)(NXYLXK2017B07)。

摘　　要：目的探讨Rap1A DNA甲基化在同型半胱氨酸(Hcy)促进小鼠单核巨噬细胞RAW264.7增殖中的作用。方法将处于对数生长期的RAW264.7细胞分为对照组(Hcy 0μmol/L)和不同浓度的Hcy(20,40,60,80,100μmol/L)干预组,24 h后用XTT检测细胞活力;Edu检测细胞增殖情况;qRT-PCR和Western blot检测Rap1A的mRNA和蛋白的表达;甲基化特异性PCR法(MSP)检测Rap1A启动子区甲基化改变;激光共聚焦显微镜观察Rap1A的变化;将Rap1A干扰腺病毒转染RAW264.7细胞并用Hcy刺激,检测Rap1A的mRNA和蛋白水平的变化及细胞的增殖情况。结果与对照组相比,不同浓度的Hcy干预细胞后,细胞活力增强,其中100μmol/L Hcy浓度最为明显(P<0.01),且细胞增殖水平明显增加(P<0.01);经Hcy刺激后,细胞Rap1A的mRNA和蛋白表达量显著增加(P<0.01),启动子区甲基化水平降低(P<0.01);干扰Rap1A的表达后能够部分逆转Hcy所致的细胞增殖(P<0.01)。结论 Rap1A启动子区低甲基化介导了Hcy导致的RAW264.7细胞增殖。Aim To investigate the role of DNA methylation of Rap1 A in Hcy-induced RAW264.7 cell proliferation. Methods RAW264.7 cells in logarithmic growth phase were divided into control group(Control) and Hcy treated groups with different concentrations(20, 40, 60, 80, 100 μmol/L Hcy). Cell viability was detected by XTT to screen optimal intervention time and concentration of Hcy after cells were treated for 24 hours. Edu test was used to analyze cell proliferation. The expression levels of Rap1 A mRNA and protein were measured by qRT-PCR and Western blot respectively. Methylation-specific PCR(MSP) was used to detect the change of Rap1 A promoter region. The changes of Rap1 A were examined by immunofluorescence staining. Rap1 A interference adenovirus was transfected into RAW264.7 cells and stimulated with Hcy to detect the changes of mRNA and protein levels of Rap1 A and cell proliferation. Results Compared with control group, the cell viability was enhanced after cells were treated with different concentration of Hcy, which was the most obvious when cells were treated by 100 μmol/L Hcy(P<0.01), as well as cell proliferation(P<0.01). But there was no time and concentration dependence. After Hcy stimulation, the expression of mRNA and protein of Rap1 A increased significantly(P<0.01). The result of MSP revealed that the methylation level of Rap1 A promoter region was decreased(P<0.01). Interfering with the expression of Rap1 A can partially reverse the proliferation of cells induced by Hcy(P<0.01). Conclusion DNA hypomethylation of Rap1 A promoter region may play an important role in macrophage proliferation induced by Hcy.

关 键 词：RAW264.7细胞 同型半胱氨酸 Ras相关蛋白1A DNA甲基化 

分 类 号：R5[医药卫生—内科学] R363[医药卫生—临床医学]