细胞因子信号转导抑制因子-3对子痫前期滋养细胞增殖和迁移能力的影响

Suppressor of cytokine signaling-3 improves proliferation and migration of human trophoblast cells during pre-eclampsia

目的 探讨细胞因子信号转导抑制因子-3（suppressor of cytokine signaling-3,SOCS-3）基因在人子痫前期胎盘组织中的表达情况,及其对HTR-8/SVneo细胞增殖与迁移能力的影响. 方法 （1）选择2010年10月至2011年3月在南京医科大学第一附属医院住院分娩的重度子痫前期孕妇15例为子痫前期组,1 5例正常孕妇作为正常妊娠组.（2）体外培养的HTR-8/SVneo细胞分别转染SOCS-3小分子干扰RNA（实验组）和无意义的阴性对照小分子干扰RNA（阴性对照组）.采用实时荧光定量逆转录 聚合酶链反应和Western印迹技术分别检测胎盘组织及体外培养细胞中SOCS-3 mRNA和蛋白表达水平;采用3-（4,5）-二甲基噻唑-（2,5）-二苯基溴化四氮唑蓝法检测细胞增殖能力;应用流式细胞仪检测细胞周期;采用Transwell小室实验检测细胞迁移能力.2组数据间比较采用两独立样本f检验. 结果 （1）子痫前期组胎盘组织SOCS-3 mRNA和蛋白表达水平分别为0.25±0.03和0.21±0.05,均低于正常妊娠组（0.71±0.08和0.75±0.12）（f值分别为15.94和14.29,P值均＜0.05）.（2）小分子干扰RNA转染24 h后,实验组SOCS-3 mRNA水平低于阴性对照组（0.39±0.02与1.00±0.04,t=27.58,P＜0.05）,蛋白水平也较低（0.003 7±0.001 4与1.514 9±0.035 7,t=73.35,P＜0.05）.转染后,实验组细胞增殖能力降低,转染后48、72和96 h增殖能力分别为0.23±0.01、0.32±0.02和0.37±0.02,均低于阴性对照组（分别为0.39±0.02、0.55±0.04和0.86±0.04）（f值分别为2.60、6.64和42.44,P值均＜0.05）.转染10d时实验组细胞克隆形成个数也低于阴性对照组[（116±15）个与（312±24）个,t=9.96,P＜0.05].转染48 h后,实验组G1/G0期细胞比例为（55.75±2.21）％,高于阴性对照组[（47.88±1.87）％]（t=45.43,P＜0.05）;S期细胞比例为（31.59±0.83）％,低于阴性对照组[（37.38±1.34）％]（t=20.06,P＜0.05）.转染48 h后,实验组穿膜细胞数为（93±11）个,低于阴性对照组[（167±17）个]（t=21.36,P＜0.05）. 结论 SOCS-3表达水平下降可能通过抑制滋养细胞的增殖和迁移能力,从而参与子痫前期的发生发展.

Objective To investigate the expression of suppressor of cytokine signaling-3 （SOCS-3） gene in placenta,its role in the pathogenesis of pre-eclampsia and its effect on proliferation and migration of HTR-8/SVneo cells.Methods Fifteen women with severe pre-eclampsia hospitalized in the First Affiliated Hospital of Nanjing Medical University from October 2010 to March 2011 and t 5 normal pregnant women during the same time period were investigated.Cultured HTR-8/SVneo cells were transfected with SOCS-3 specific small interfering RNA （siRNA） or negative siRNA as the controls.The expression of SOCS-3 mRNA and protein in placenta and these cells was detected by real-time quantitative reverse transcription-polymerase chain reaction and Western blot.Cell proliferation was detected by methyl thiazolyl tetrazolium,cell cycle by flow cytometry and migration by the Transwell test.Two independent t tests were used for statistical analysis.Results The SOCS-3 mRNA and protein levels in the severe pre-eclampsia group were lower than those in the normal group （0.25±0.03 vs 0.71±0.08 and 0.21±0.05 vs 0.75±0.12,t=15.94 and 14.29,respectively,both P＜0.05）.SOCS-3 mRNA and protein levels in the transfection group at 24 hours were lower than those in the negative control group （0.39±0.02 vs 1.00±0.04 and 0.003 7±0.001 4 vs 1.514 9±0.035 7,t=27.58 and 73.35,respectively,both P＜0.05）.The integral absorbance values of cell proliferation in the transfection group at 48,72 and 96 hours after transfection were 0.23 ± 0.01,0.32±0.02 and 0.37± 0.02,respectively,which were lower than those in the negative control group （0.39± 0.02,0.55 ± 0.04 and 0.86± 0.04,t=2.60,6.64 and 42.44,respectively,all P＜0.05）.The cell clonal formation was lower in the transfection group compared with the negative group （116± 15 vs 312±24,t=9.96,P＜0.05）.The ratios of G1/G0 and S phase cells in the transfection group were （55.75±2.21） ％ and （31.59±0.83） ％,respectively,and were significantly different from those in the negative control group [（47.88± 1.87） ％ and （37.38± 1.34） ％,t=45.43 and 20.06,respectively,P＜0.05].After 48 hours,cell migration in the transfection group was lower than that in the negative control group （93 ± 11 vs 167± 17,t=21.36,P＜O.05）.Conclusion SOCS-3 expression is probably involved in the pathogenesis of pre-eclampsia by being down-regulated and therefore impeding proliferation and migration of the trophoblast.