滋养细胞自噬在子痫前期胎盘中的形成及调控机制

Induction and molecular mechanism of placental trophoblast cell autophagy in preeclampsia

目的探讨自噬在子痫前期患者胎盘滋养细胞中的形成及调控机制。方法选择2016年8月至2016年11月在南京医科大学附属常州妇幼保健院产科剖宫产的20例重度子痫前期患者为子痫前期组，随机选取同期剖宫产的血压正常、无蛋白尿的20例正常孕妇为对照组。同时收集2组孕妇胎盘组织，采用透射电镜观察胎盘滋养细胞的超微结构及自噬体形成情况，实时定量聚合酶链反应和Western印迹法检测胎盘组织中微管相关蛋白1轻链3B（microtubule associated protein 1 light chain 3B，MAP1LC3B，亦称LC3B）和Beclin 1的表达情况，以及Western印迹法检测胎盘组织中蛋白激酶B（protein kinase B，PKB，亦称Akt）/哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）通路的活性。采用两独立样本t检验或Mann-Whitney U检验对数据进行统计学分析。 结果透射电镜可观察到重度子痫前期患者胎盘滋养细胞胞膜微绒毛稀少、排列紊乱，胞质中可见典型的自噬体形成增加。子痫前期组孕妇胎盘组织中LC3B mRNA和蛋白的表达水平及LC3-Ⅱ/ LC3-Ⅰ比值均高于对照组[分别为3.37（2.37~6.11）与0.62（0.25~4.15）、1.40±0.17与1.00±0.13及1.57±0.25与1.00±0.31，Z或t值分别为－4.440、3.274及3.113，P值均〈0.05]。而2组孕妇胎盘组织中Beclin 1 mRNA和蛋白的表达差异无统计学意义（P值均〉0.05）。与对照组相比，子痫前期组孕妇胎盘组织中Akt和mTOR蛋白的磷酸化修饰水平明显下降（分别为1.00±0.29与0.64±0.21、1.00±0.32与0.60±0.22，t值分别为－3.672及－2.895，P值均〈0.05）；但2组胎盘组织中Akt及mTOR蛋白表达差异均无统计学意义（P值均〉0.05）。结论重度子痫前期患者胎盘中Akt/mTOR通路活性下降，滋养细胞自噬过度激活，提示Akt/mTOR通路活性下降致使滋养细胞自噬过度激活可能是子痫前期发病的重要机制。

ObjectiveTo investigate the induction and regulatory mechanism of placental trophoblast cell autophagy in women with preeclampsia （PE）.MethodsTwenty gravidas with severe PE who underwent cesarean section in the Department of Obstetrics and Gynecology of Changzhou Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University from August 2016 to November 2016 were enrolled in PE group. An equal number of normotensive gravidas without proteinuria who also underwent cesarean section during the same period were randomly selected as control group. Placental tissue samples were collected from all gravidas. Ultrastructure of placental trophoblast cells and changes in autophagosome formation were observed by transmission electron microscope. Expressions of microtubule associated protein 1 light chain 3B （MAP1LC3B, or LC3B） and Beclin 1 in placental tissue samples were detected by quantitative real-time polymerase chain reaction （PCR） and Western blot. Activities of protein kinase B （PKB, also known as Akt）/mammalian target of rapamycin （mTOR） pathway in placental tissue samples were detected by Western blot. Two independent samples t-test or Mann-Whitney U test was used for statistical analysis.ResultsSparse and disordered villi and many typical autophagosomes were observed in placental trophoblast cells from patients with severe PE. Significantly enhanced expression of LC3B at mRNA and protein levels and increased ratio of LC3-II/LC3-I were observed in the PE group as compared with the control group [3.37 （2.37-6.11） vs 0.62 （0.25-4.15）, 1.40±0.17 vs 1.00±0.13, 1.57±0.25 vs 1.00±0.31, Z or t=-4.440, 3.274 and 3.113, all P〈0.05]. No significant difference in the expression of Beclin 1 at mRNA or protein level in placental tissues was found between the two groups （both P〉0.05）. Furthermore, Akt and mTOR phosphorylation in the PE group was significantly suppressed as compared with that in the control group （1.00±0.29 vs 0.64±0.21, 1.00±0.32 vs 0.60±0.22, t=-3.672 and -2.895, both P〈0.05）. However, the two groups showed no significant difference in the expression of Akt or mTOR protein （both P〉0.05）.ConclusionsSuppressed activity of Akt/mTOR pathway and enhanced induction of trophoblast cell autophagy are detected in placental tissues of patients with severe PE, indicating that excessive trophoblast cell autophagy, induced by decreased activity of Akt/mTOR pathway, may be the pathogenesis for PE.