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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。
Objective To investigate the possible mechanism of the apoptosis of human placental trophoblast cells induced by the decrease of nitric oxide (NO) level. Methods Human placental trophoblast cell line (HTR-8) was cultured in vitro. The cells were incubated with 0, 100, 500 and 1 000 μmol·L-1 L-NAME for 48 h. The effect of L-NAME on cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. The content of NO was determined by nitrate reductase method. The content of NO was determined by transmission electron microscopy, flow cytometry and Annexin-V FITC staining. NAME on the apoptosis of HTR-8 cells. The total antioxidant capacity (T-AOC) was measured by Fe3 + reduction colorimetric assay. The activity of superoxide dismutase (SOD) and thiobarbituric acid ratio Colorimetric determination of MDA content. Results Compared with the control group, the cell viability and the level of NO in 100, 500 and 1 000 μmol·L-1 L-NAME groups were significantly decreased (P <0.05, P <0.01). Annexin-V FITC staining and flow cytometry showed L- NAME could induce apoptosis in a dose-dependent manner; the level of NO was negatively correlated with the number of apoptotic cells (r = -0.5210); the results of transmission electron microscopy showed that compared with the control group, the nuclear morphology of the experimental group was irregular, Especially in 100μmol·L-1L-NAME group. At the same time, apoptotic bodies were observed in HTR- The levels of T-AOC and SOD were significantly decreased (P <0.05) and the contents of MDA were significantly increased (P <0.05) ) Levels were negatively correlated with MDA (r = 0.2807) content was positively correlated. Conclusion Oxidative stress plays an important role in the apoptosis of human placental trophoblast cells caused by the decrease of NO level.