【作者】 石鑫玮；

【导师】 乔福元；

【作者基本信息】 华中科技大学 ， 妇产科学， 2012， 博士

【摘要】 目的检测正常孕妇及子痫前期患者胎盘中maspin基因的表达情况，探讨maspin基因在子痫前期发病机制中的作用。方法采用免疫组织化学法检测正常孕妇和子痫前期患者胎盘中maspin基因的蛋白表达情况，并采用RT-PCR法检测maspin基因的mRNA表达水平。结果1. maspin蛋白在子痫前期患者及正常孕妇胎盘组织中均有表达，主要位于合体滋养细胞、细胞滋养细胞及内皮细胞胞浆中。2.子痫前期组胎盘中maspin mRNA表达水平与正常组相比明显增高，差异有统计学意义（P＜0.05）。两组孕妇年龄、身高、孕龄、胎儿Apgar评分和体重均无统计学差异。结论胎盘组织中maspin基因主要表达在滋养细胞，子痫前期患者胎盘maspin表达明显升高。我们推测maspin基因表达水平的异常可能参与了子痫前期的发生。目的体外实验研究缺氧状态对绒毛外滋养细胞maspin表达及生物学行为（增殖、迁移、凋亡）的影响，探讨maspin基因参与子痫前期发病的机制。方法利用化学物质二氯化钴建立细胞缺氧模型，采用细胞免疫组化、实时荧光定量PCR、CCK8细胞增殖实验、Transwell细胞迁移实验和细胞凋亡检测研究绒毛外滋养细胞maspin蛋白的表达变化及对绒毛外滋养细胞生物学行为（增殖、迁移和凋亡）的影响。结果1. maspin蛋白在绒毛外滋养细胞株TEV-1中明显表达，主要位于细胞浆2.与正常对照组相比，缺氧组中绒毛外滋养细胞的maspin的mRNA表达明显增加；增殖能力和迁移能力明显下降，凋亡显著增加。结论缺氧状态下滋养细胞maspin表达增加，其浸润性行为明显受到抑制，这与子痫前期胎盘中maspin表达情况及滋养细胞侵袭能力下降的现象一致，因此推测maspin基因表达异常与滋养细胞的浸润性能力改变相关，参与子痫前期的发病机制。目的利用脱甲基化药物（5-Aza-dC）研究绒毛外滋养细胞生物学行为（增殖、迁移和凋亡）的变化，探讨maspin基因的表观遗传学调控在子痫前期发病机制中的作用。方法采用实时荧光定量PCR、CCK8细胞增殖实验、Transwell细胞迁移实验和Annexin/PI凋亡检测等方法检测脱甲基化药物对绒毛外滋养细胞的maspin mRNA表达及增殖、迁移和凋亡的影响。结果1.与PBS对照组相比，5-Aza-dC组中绒毛外滋养细胞的maspin mRNA表达显著升高，迁移能力明显下降，而增殖和凋亡率没有明显改变。2.与缺氧组相比，缺氧+5-Aza-dC组中绒毛外滋养细胞的maspin mRNA表达明显降低，早期凋亡率有一定程度的降低，但增殖能力没有明显改变结论脱甲基化药物使绒毛外滋养细胞的maspin表达明显增加，并抑制其迁移能力；同时抑制了缺氧导致的绒毛外滋养细胞凋亡的增加，提示了表观遗传学调控在子痫前期胎盘浅着床发生机制中的作用，并为子痫前期的早期诊断和治疗提供了新的潜在的研究方向。更多还原

【Abstract】 ObjectiveThe study detected the expression of maspin in placenta from normal and preeclampticpregnancies.MethodsBy immunohistochemical assay and real-time PCR, the protein expression and themRNA expression level of maspin in placenta from normal and preeclamptic pregnancieswere detected.Results1. The protein of maspin was expressed in placenta of normal and preeclampticpregnancies. The protein of maspin was strongly expressed in syncytiotrophoblast,cytotrophoblast and endothelial cell, and was mainly located in the cytoplasm.2. By contrast with normal group, the mRNA expression of maspin in preeclampsiagroup was significantly increased(P＜0.05). Between the two groups, there was nosignificantly difference in maternal age, maternal height, gestational age, fetal Apgar scoreand fetal weight.ConclusionIn placenta, the maspin protein was mainly expressed in trophoblast. The mRNAexpression level of maspin in preeclamptic placenta was significantly increased. So wespeculate that abnormal expression of maspin may contribute to the pathogenesis of preeclampsia. ObjectiveTo investigate the effect of hypoxia on the expression of maspin and biologicalbehavior (include proliferation, migration and apoptosis) in extravillous trophoblast invitro.MethodsThe study used chemical reagent (CoCl2) to establish the model of hypoxic cell. Byimmunocytochemistry assay, RT-PCR method, CCK8cellular proliferation assay,Transwell cellular migration assay and cellular apoptosis assay, the effect of hypoxia on theexpression of maspin and biological behavior (include proliferation, migration andapoptosis) in extravillous trophoblast was detected.Results1. Maspin protein was expressed in extravillous trophoblast cell line (TEV-1), wasmainly located in the cytoplasm.2. In hypoxic group, the mRNA expression of maspin in extravillous trophoblast wassignificantly up-regulated; the ability of proliferation and migration was obviouslydecreased; the apoptosis rate was significantly increased.Conclusion:The expression of maspin was increased in hypoxic trophoblast, and the aggressive behavior was obviously suppressed. The results in vitro were in accord with thephenomenon that expression of maspin in preeclamptic placenta was increased and theinvasion ability of trophoblast was decreased. Therefore, we speculate that abnormalexpression of maspin is related to the change of aggressive behavior of trophoblast, andplay an important role in the pathogenesis of preeclampsia. ObjectiveTo investigate the effect of demethylating reagent (5-Aza-dC) on the expression ofmaspin and biological behavior in extravillous trophoblast in vitro.MethodsBy immunocytochemistry assay, RT-PCR method, CCK8cellular proliferation assay,Transwell cellular migration assay and cellular apoptosis assay, the effect of demethylatingreagent (5-Aza-dC) on the expression of maspin and biological behavior (includeproliferation, migration and apoptosis) in extravillous trophoblast was detected.Results1. By contrast with PBS group, the mRNA expression of maspin in extravilloustrophoblast in5-Aza-dC group was significantly increased; the ability of migration wasobviously inhibited; the ability of proliferation and the rate of apoptosis had no obviouschange.2. By contrast with hypoxic group, the mRNA expression of maspin in extravilloustrophoblast in hypoxia+5-Aza-dC group was significantly down-regulated; the apoptosis rate in early stage was decreased; the ability of proliferation had no significant change.Conclusion:Demethylating reagent up-regulated the expression of maspin in trophoblast, andinhibited the ability of migration; demethylating reagent inhibited the effect of hypoxia onexpression of maspin and apoptosis in trophoblast. The results revealed that epigeneticmodification contribute to inadequate invasion of trophoblast, and provide us a potentialnovel direction in the study of preeclampsia.更多还原