miR-29b通过抑制N2a细胞p53凋亡通路减轻氧糖剥夺/再灌注损伤  

The potential mechanism of mi R-29b in reducing oxygen and glucose deprivation/reperfusion-induced injury through inhibiting the apoptotic pathway of N2a p53 cells

作　　者：陈立 邹伟婕 张宇 张帅 王黎洲 周石 CHEN Li, ZOU Weijie, ZHANG Yu, ZHANG Shuai, WANG Lizhou, ZHOU Shi.(Department of lnterventional Radiology, Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou Province 550004, Chin)

机构地区：[1]贵州医科大学附属医院介入科,贵阳550004

出　　处：《介入放射学杂志》2018年第5期451-457,共7页Journal of Interventional Radiology

基　　金：国家自然科学基金(81460276)

摘　　要：目的探讨微小核糖核酸(miR)-29b过表达是否抑制脑缺血损伤,进一步探讨其潜在机制。方法体外培养小鼠成神经细胞瘤N2a细胞,构建氧糖剥夺/再灌注(OGD/R)模型,模拟体外脑缺血损伤。N2a细胞随机分为空白对照组、OGD/R组、OGD/R+转染miR-29b激动剂组、OGD/R+转染miR-29b抑制剂组、转染miR-29b激动剂阴性对照组、转染miR-29b抑制剂阴性对照组。实时定量聚合酶链反应(RT-q PCR)检测各组miR-29b表达,溴化噻唑蓝四氮唑(MTT)比色法、乳酸脱氢酶(LDH)法检测miR-29b激动剂和抑制剂对OGD/R诱导细胞活性和凋亡的影响,Hoechst 33258染色法观察N2a细胞形态学特征及半胱氨酸天冬氨酸特异性蛋白酶(caspase)-3活性,免疫印迹法定量分析促凋亡蛋白Bax、Bcl-2及p53表达。结果经OGD/R处理的N2a细胞中miR-29b水平明显降低。miR-29b激动剂显著增加细胞活力,减少LDH漏出率,改善细胞核在凋亡过程中形态学变化,增强OGD/R条件下caspase-3活性;miR-29b抑制剂加剧OGD/R诱导的细胞毒性和细胞凋亡。miR-29b激动剂阻断OGD/R诱导的Bax和p53蛋白表达增加,降低Bcl-2蛋白表达;miR-29b抑制剂加剧OGD/诱导的这些凋亡相关蛋白改变。p53基因敲除的p53 si RNA降低细胞活力,增加LDH漏出率,逆转miR-29b激动剂对细胞损伤的改善作用。结论 miR-29b通过负调控p53依赖性凋亡途径减轻脑缺血性损伤,可能为缺血性脑卒中提供一潜在的治疗靶点。Objective To clarify whether the over-expression of micro RNA-29 b（miR-29 b） will inhibit cerebral ischemia injury, and to make a further discussion on its potential mechanism. Methods Mouse-derived neuroblastoma cells（N2 a cells） were cultured in vitro, the oxygen and glucose deprivation/reperfusion（OGD/R） model was established to make simulation of cerebral ischemic injury in vitro. The N2 a cells were randomly divided into six groups： blank control group（control）, OGD/R group, OGD/R ＋transfected with miR-29 b mimics group（OGD/R＋miR-29 b M）, OGD/R＋transfected with miR-29 b inhibitor group（OGD/R＋miR-29 b I）, transfected miR-29 b mimics negative control group（miR-29 b M） and transfected miR-29 b inhibitor negative control group（miR-29 b I）. Real-time quantitative polymerase chain reaction（RTq PCR） was used to test the expression of miR-29 b in each group. MTT colorimetric assay and lactate dehydrogenase（LDH） method were employed to detect the effect of miR-29 b mimics and inhibitors on the activity and apoptosis of OGD/R-induced cells. Hoechst 33258 staining was adopted to observe the morphological characteristics of N2 a cells and the activity of caspase specific protease-3（caspase-3）. The expressions of proapoptotic proteins, including Bax, Bcl-2 and p53, were quantitatively analyzed by immunoblotting. Results In OGD/R-treated N2 a neuroblastoma cells, the level of miR-29 b decreased obviously. The miR-29 b mimics could significantly increase cell＇s viability and decrease LDH leakage, thereby improving the morphological changes of nuclei during apoptosis and enhancing caspase-3 activity under OGD/R conditions. The inhibitors of miR-29 b could exacerbate OGD/R-induced cytotoxicity and apoptosis. The mimics of miR-29 b could block OGD/R-induced increase in the expressions of Bax and p53 proteins, and could decrease the expression of Bcl-2 protein in N2 a cells. The inhibitors of miR-29 b could exacerbate OGD/R-induced changes in these apoptosis-associated pr

关 键 词：miR-29b 脑缺血 P53基因 细胞凋亡 成神经细胞瘤N2a细胞 

分 类 号：R743.3[医药卫生—神经病学与精神病学]