NO水平下降通过氧化应激引起人胎盘滋养细胞凋亡  

The levels of NO decrease induced apoptosis in human placental trophoblast cells through oxidative stress

作　　者：王艳华[1] 张慧萍[2] 田珏[3] 周龙霞[3] 陈久凯 马文斌[1] 孔繁琪[3] 赵丽[1] 刘现梅 韩学波[1] 杨晓玲[3] 姜怡邓[3] 

机构地区：[1]宁夏医科大学检验学院,宁夏银川750004 [2]宁夏医科大学总医院,宁夏银川750004 [3]宁夏医科大学基础医学院,宁夏银川750004

出　　处：《中国药理学通报》2014年第9期1287-1292,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81260105);宁夏高等学校科学研究项目(No NGY2013054)

摘　　要：目的探讨一氧化氮(NO)水平下降诱导人胎盘滋养细胞凋亡的可能机制。方法体外培养人胎盘滋养细胞株(HTR-8),分别加入10、100、500、1 000μmol·L-1L-NAME,并设置对照组(0μmol·L-1L-NAME),孵育48 h;四甲基偶氮唑蓝法(MTT)检测L-NAME对细胞存活率的影响;采用硝酸还原酶法测定细胞中NO的含量;利用透射电镜、流式细胞术和Annexin-V FITC染色检测L-NAME对HTR-8细胞凋亡的影响;Fe3+还原比色法检测总抗氧化能力(T-AOC)、黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性及硫代巴比妥酸比色法测定MDA含量。结果与对照组比较,100、500、1 000μmol·L-1L-NAME组细胞存活率及NO水平明显降低(P<0.05,P<0.01);Annexin-V FITC染色和流式分析均显示L-NAME能诱导细胞凋亡,并且呈剂量依赖性;NO水平与细胞凋亡数呈负相关(r=-0.5210);透射电镜结果显示:与对照组比较,实验组细胞核形态不规则,核膜固缩呈不规则状,染色质凝集或边集,线粒体肿胀或浓缩、嵴断裂或溶解,甚至消失,形成空泡,尤其在100μmol·L-1L-NAME组可见凋亡小体;同时,HTR-8细胞内T-AOC、SOD水平明显降低(P<0.05),MDA的含量明显升高(P<0.05);细胞凋亡数与T-AOC(r=-0.3212)、SOD(r=-0.2779)水平均呈负相关,与MDA(r=0.2807)含量呈正相关。结论氧化应激在NO水平降低引起的人胎盘滋养细胞凋亡中起重要作用。Aim To investigate the possible mecha-nisms of the levels of NO decrease induced apoptosis in human placental trophoblast cells. Methods Human placental trophoblast cells （ HTR-8 ） were cultured in 5 ml DMEM-F12 culture medium with 37℃ 5% CO2 . Then, the old culture medium was discarded and re-placed with 10,100,500,1 000 μmol·L-1 L-NAME, and the group without L-NAME was set as the control group, cultured for 48h. The effects of L-NAME on the survival of cells were detected by methylthiazolyldiphe-nyl tetrazolium bromide （ MTT）; the content of NO in cells was tested by nitrate reductive enzymatic;trans-mission electron microscopy, flow cytometry analysis and Annexin-V FITC dyeing were used to test the effects of L-NAME on apoptosis in HTR-8 cells;restore Fe3＋ colorimetric assay was applied for detection of to-tal antioxidant capacity （ T-AOC ） , xanthine oxidase for detection of superoxide dismutase （ SOD） activity, and thiobarbituric acid colorimetry for determination of content of MDA. Results Compared with the control group, the survival rate of HTR-8 cells and the levels of NO in 100,500,1 000 μmol·L-1 L-NAME group were significantly reduced（P〈0.05,P〈0.01）. Flow analysis and Annexin-V FITC staining showed that L-NAME could induce cell apoptosis in a dose-dependent manner. The number of cell apoptosis was negatively correlated with the content of NO （ r = -0.5210 ） in HTR-8 cells. Transmission electron microscopy results showed that compared with the control group, the ex-perimental group＇s cell nucleus shape was irregular, nuclear pyknosis in irregular shape, the chromatin ag-glutination or side the collection, mitochondrial swell-ing or enrichment, crest fracture or dissolved, even vanished, forming the vacuole, especially in 100 μmol ·L-1 L-NAME group, the apoptotic bodies obviously appeared. At the same time, T-AOC, SOD levels in HTR-8 cells decreased （ P 〈0.05 ） , and the MDA content increased （ P〈0.05 ） . The number of cell ap-optosis was negatively cor

关 键 词：一氧化氮 N-硝基-L-精氨酸甲酯 人胎盘滋养细胞 凋亡 氧化应激 妊娠期高血压疾病 

分 类 号：R321.4[医药卫生—人体解剖和组织胚胎学] R329.25[医药卫生—基础医学]