摘要
目的探讨p53凋亡刺激蛋白2(apoptosis stimulating protein 2 of p53,ASPP2)调控葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)在N-硝基-L-精氨酸甲酯(L-nitroarginine methyl ester,L-NAME)诱导胎盘滋养细胞凋亡中的作用,为临床研究妊娠期高血压疾病提供理论依据。方法体外培养HTR-8/SVneo人胎盘滋养细胞,分为对照组(0μmol·L^(-1)L-NAME)和L-NAME组(100μmol·L^(-1)LNAME),干预48 h后,分别采用Annexin V-FITC/PI双标记流式细胞术和吖啶橙/溴化乙锭染色,检测胎盘滋养细胞凋亡水平的变化;运用Western blot检测caspase-12、GRP78和ASPP2的蛋白变化; ASPP2干扰腺病毒感染人胎盘滋养细胞后,采用实时荧光定量PCR(qRT-PCR)检测ASPP2的mRNA表达水平,Western blot检测GRP78的蛋白表达水平;给予100μmol·L^(-1) L-NAME干预48 h后,Western blot和免疫荧光分别检测caspase-12和GRP78的蛋白表达。结果与对照组相比,L-NAME组胎盘滋养细胞凋亡水平明显增加(P <0. 05);吖啶橙染色显示,与对照组相比,L-NAME组细胞多数呈现鲜亮的橙色,晚期凋亡细胞数明显增加;同时caspase-12、GRP78和ASPP2蛋白的表达明显增加(P <0. 05,P <0. 01);干扰ASPP2后,caspase-12和GRP78蛋白表达明显降低(P <0. 05)。结论下调ASPP2可降低GRP78的表达,进而抑制L-NAME诱导的胎盘滋养细胞凋亡。
Aim To explore the role of apoptosis stimulating protein 2 of p53 (ASPP2) on L-NAME induced apoptosis of placental trophoblast cells by regulating glucose-regulated protein78 (GRP78),and provide a theoretical basis for the study of clinical pregnancy-induced hypertension. Methods The HTR-8/SVneo human placental trophoblast cells were cultured in vitro,and in the absence (control group) or presence of 100 μmol·L -1 L-NAME (L-NAME group) for 48 h. The effects of L-NAME on placental trophoblast cell apoptosis were tested using flow cytometry and AO/EB assay. The expressions of caspase-12,GRP78 and ASPP2 were detected by Western blot. The ASPP2 interference with adenovirus was used to transfect the cells,and the mRNA expression level of ASPP2 and the protein expression level of GRP78 were detected by qRT-PCR and Western blot,respectively. After treated with 100 μmol·L -1 L-NAME for 48 h,the protein expression of caspase-12 and GRP78 was detected by Western blot and immunofluorescence. Results Compared with control group,the placental trophoblast cell apoptosis significantly increased in L-NAME group ( P <0.05). AO/EB staining showed that compared with control group,the majority of cells in L-NAME group showed bright orange and the number of late apoptotic cells increased significantly. At the same time,caspase-12,GRP78 and ASPP2 protein expression increased ( P <0.05, P <0.01). After interfering with ASPP2,caspase-12 and GRP78 protein expressions decreased ( P <0.05). Conclusions Down-regulation of ASPP2 could decrease GRP78 expression and inhibit L-NAME-induced apoptosis in placental trophoblast cells.
作者
谢琳
张辉
丁宁
王艳华
吴凯
曹军
王青青
刘昆
张慧萍
姜怡邓
XIE Lin;ZHANG Hui;DING Ning;WANG Yan-hua;WU Kai;CAO Jun;WANG Qing-qing;LIU Kun;ZHANG Hui-ping;JIANG Yi-deng(School of Basic Medical SciencesNingxia Medical University,Yinchuan 750004,China;College of Clinical MedicineNingxia Medical University,Yinchuan 750004,China;General Hospital, Ningxia Medical University,Yinchuan 750004,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2019年第6期765-770,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81760270)
宁夏回族自治区重点研发计划一般项目(No 2018BEG03026)
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)资助项目(No NXYLXK2017B07)
