Objective: To evaluate the effect of endoplasmic reticulum stress in trophocytes, in patients with intrahepatic cholestasis of pregnancy (ICP). Methods: Sixty-one pregnant women who were hospitalized in Women's Hospital, School of Medicine, Zhejiang University from January to December 2015 were recruited. Thirty-one women who were diagnosed as ICP were defined as the ICP group and 30 healthy pregnant women were defined as the control group. The localization and expression intensity of glucose regulated protein 78 (GRP-78) in placental tissues were detected by immunohistochemistry technique. Electronic microscope was used to observe ultra-microstructure change of the endoplasmic reticulum in trophocytes and cell line Swan71. Reverse transcription (RT)-PCR and western blot were used to investigate the expression of GRP-78 mRNA and protein in Swan 71 cell. Results: (1) GRP-78 protein was mainly expressed in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts. The protein expression of GRP-78 in placentas of the ICP group (13.2±2.4) was significantly higher than that in the control group (7.8±1.3, P<0.01). (2) The volume of endoplasmie reticulum did not increase and the microvilli developed well, with no swelling and no expansion of endoplasmic reticulum in the control group.In the ICP group, microvilli injury, endoplasmic reticulum edema were found; the volume of endoplasmic reticulum increased, with dilation, vacuolation and significant degranulation. After treated with 100 μmol/L cholyglycine for 24 hours, universal dilatation of the endoplasmic reticulum were seen in the Swan71 cells. (3) In Swan71 cells, cholylglycine displayed a concentration-dependent up-regulation on the expression of GRP-78. The expressions of GRP-78 mRNA in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0.17, 2.17±0.16, 5.47±0.36, 5.65±0.82, respectively. The expression of GRP-78 protein in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0.04, 1.17±0.15, 1.33±0.13, 1.73±0.13, respectively. The expression of GRP-78 mRNA and protein in 100 and 50 μmol/L cholylglycine experimental group were significantly higher than 0 μmol/L (all P<0.01). Conclusion: The obvious expansion of endoplasmic reticulum and the increased expression of GRP-78 in trophocytes indicated that endoplasmic reticulum stress of trophocytes may be involved in the pathogenesis of ICP.
目的： 探讨滋养细胞内质网应激在妊娠期肝内胆汁淤积症（ICP）孕妇胎盘组织的作用。 方法： 收集2015年1月至12月在浙江大学医学院附属妇产科医院单活胎、初产分娩的孕妇61例，其中ICP孕妇31例（ICP组），健康孕妇30例（对照组）。免疫组化法检测两组孕妇胎盘组织滋养细胞中葡萄糖调节蛋白78（GRP-78）的表达部位和表达水平；透射电镜观察两组孕妇胎盘组织滋养细胞、未处理及100 μmol/L甘胆酸处理24 h后的滋养细胞系Swan71细胞中内质网超微结构的变化；逆转录（RT）-PCR技术和蛋白印迹法检测不同浓度（分别为0、25、50、100 μmol/L）的甘胆酸处理24 h后的Swan71细胞中GRP-78 mRNA和蛋白的表达水平。 结果： （1）免疫组化法：两组孕妇胎盘组织的滋养细胞中均可检测到GRP-78蛋白的表达，GRP-78蛋白主要表达于滋养细胞的胞质。与对照组孕妇（7.8±1.3）比较，ICP组孕妇绒毛细胞中GRP-78蛋白的表达水平（13.2±2.4）升高，两组比较，差异有统计学意义（P<0.01）。（2）透射电镜：①两组孕妇的绒毛细胞：对照组孕妇的胎盘组织中滋养细胞内可见发育良好的微绒毛与内质网，内质网体积无明显增大，无扩张、肿胀变化；ICP组孕妇的胎盘组织中滋养细胞的微绒毛消失，内质网腔明显扩张，内质网水肿、体积增大、液泡化，且内质网脱颗粒现象明显。②Swan71细胞：未处理的Swan71细胞内可见发育良好的微绒毛与大量的内质网，内质网无扩张、肿胀变化；100 μmol/L甘胆酸处理24 h后的Swan71细胞内质网病变弥漫，显著扩张，细胞质呈筛状外观。（3）RT-PCR技术与蛋白印迹法：①RT-PCR技术：用0（生理盐水）、25、50、100 μmol/L的甘胆酸处理24 h后的4组Swan71细胞中GRP-78 mRNA的表达水平分别为1.01±0.17、2.17±0.16、5.47±0.36、5.65±0.82，50、100 μmol/L甘胆酸处理细胞分别与生理盐水处理细胞比较，差异均有统计学意义（P<0.01）。②蛋白印迹法：4组细胞中GRP-78蛋白的表达水平分别为1.01±0.04、1.17±0.15、1.33±0.13、1.73±0.13，50、100 μmol/L甘胆酸处理细胞分别与生理盐水处理细胞比较，差异均有统计学意义（P<0.05）。 结论： ICP孕妇胎盘组织滋养细胞中内质网较正常孕妇有明显的扩张及肿胀性改变；ICP孕妇的绒毛细胞中GRP-78蛋白的表达水平较对照组孕妇明显升高。甘胆酸可引起滋养细胞内质网肿胀，致GRP-78 mRNA和蛋白的表达水平升高。滋养细胞的内质网应激是ICP胎盘的重要病理生理表现。.
Keywords: Cholestasis, intrahepatic; Endoplasmic reticulum stress; Heat-shock proteins; Pregnancy complications; Trophoblasts.