自然流产胚胎组织中人类纺锤体有丝分裂俘获缺陷基因的表达及意义
자연유산배태조직중인류방추체유사분렬부획결함기인적표체급의의
Expression of human spindle mitosis arrest deficiency gene in spontaneous abortion embryo tissues
  • 万方数据
    中华妇产科杂志 중화부산과잡지
    CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
    2008年 5期 346-351 ,共6页

    蔡燕%王箭%袁泰先%施琼%翁亚光%王应雄%蒋洪彦%刘子杰

    채연%왕전%원태선%시경%옹아광%왕응웅%장홍언%류자걸

    流产,自然%胚胎%钙结合蛋白质类%阻遏蛋白质类%细胞周期蛋白质类%RNA,小分子干扰%细胞增殖 流產,自然%胚胎%鈣結閤蛋白質類%阻遏蛋白質類%細胞週期蛋白質類%RNA,小分子榦擾%細胞增殖
    유산,자연%배태%개결합단백질류%조알단백질류%세포주기단백질류%RNA,소분자간우%세포증식
    Abortion,spontaneous%Embryo%Calcium-binding proteins%Repressor proteins%Cell cycle proteins%RNA,small interfering%Cell Proliferation
    目的 探讨自然流产胚胎组织中人类纺锤体有丝分裂俘获缺陷(hsMAD)2基因的表达,以及hsMAD2基因表达降低与染色体数目异常的相关性.方法 采集2006年3月至2007年3月重庆医科大学附属第一、第二医院妇产科自然流产患者的胚胎组织标本33份,其中流产1次者23份,流产2次及以上者10份;同时采集人工流产患者的胚胎组织标本35份.采用FQ-PCR和蛋白印迹法检测自然流产和人工流产胚胎组织中hsMAD2基因的mRNA和蛋白表达水平;原代培养人工流产胚胎组织并经染色体分析筛选出5例具有正常核型的胚胎细胞,构建hsMAD2基因的短发夹RNA(shRNA)表达载体,转染筛选出来的胚胎细胞以抑制其内源性hsMAD2基因的表达,将细胞分为实验1组(转染重组干扰质粒pshRNA-hsMAD2-1)、实验2组(转染pshRNA-hsMAD2-2)、实验3组(转染pshRNA-hsMAD2-3)、对照1组(未做任何处理)、对照2组(转染pTZU6+1干扰质粒空载体)、无关对照组(转染pshRNA-N1).用蛋白印迹法和定量PCR技术评价shRNA的干扰效果,四甲基偶氮唑蓝比色法测定胚胎细胞增殖抑制率,流式细胞技术检测胚胎细胞周期的分布,并计算染色体数目的 变化.结果 (1)自然流产1次者、自然流产2次及以上者、人工流产者的胚胎组织中hsMAD2基因mRNA的表达量分别为0.00879±0.00035、0.00901±0.00033、0.00941±0.00026,3者分别比较,差异均无统计学意义(P＞0.05);hsMAD2蛋白的表达量分别为0.2791±0.0311、0.0431±0.0020、0.5790±0.0331,3者分别比较,差异均有统计学意义(P＜0.05).(2)shRNA的重组质粒表达载体能明显抑制胚胎细胞中hsMAD2基因的表达,转染有效干扰质粒后,实验1组胚胎细胞的增殖抑制率为54%,分别与对照1组(4%)、对照2组(3%)比较,差异均有统计学意义(P＜0.05);G2/M期细胞比例实验1组为17.9%,与对照1组(8.2%)、对照2组(8.0%)比较均明显升高,差异均有统计学意义(P＜0.05);实验1组染色体异常率平均值为30.0%,与对照1组(4.8%)比较,差异有统计学意义(P＜0.01).结论 hsMAD2基因的表达下调可能是导致患者染色体数目异常、胚胎发育异常乃至自然流产发生的重要原因之一.
    목적 탐토자연유산배태조직중인류방추체유사분렬부획결함(hsMAD)2기인적표체,이급hsMAD2기인표체강저여염색체수목이상적상관성.방법 채집2006년3월지2007년3월중경의과대학부속제일、제이의원부산과자연유산환자적배태조직표본33빈,기중유산1차자23빈,유산2차급이상자10빈;동시채집인공유산환자적배태조직표본35빈.채용FQ-PCR화단백인적법검측자연유산화인공유산배태조직중hsMAD2기인적mRNA화단백표체수평;원대배양인공유산배태조직병경염색체분석사선출5례구유정상핵형적배태세포,구건hsMAD2기인적단발협RNA(shRNA)표체재체,전염사선출래적배태세포이억제기내원성hsMAD2기인적표체,장세포분위실험1조(전염중조간우질립pshRNA-hsMAD2-1)、실험2조(전염pshRNA-hsMAD2-2)、실험3조(전염pshRNA-hsMAD2-3)、대조1조(미주임하처리)、대조2조(전염pTZU6+1간우질립공재체)、무관대조조(전염pshRNA-N1).용단백인적법화정량PCR기술평개shRNA적간우효과,사갑기우담서람비색법측정배태세포증식억제솔,류식세포기술검측배태세포주기적분포,병계산염색체수목적 변화.결과 (1)자연유산1차자、자연유산2차급이상자、인공유산자적배태조직중hsMAD2기인mRNA적표체량분별위0.00879±0.00035、0.00901±0.00033、0.00941±0.00026,3자분별비교,차이균무통계학의의(P＞0.05);hsMAD2단백적표체량분별위0.2791±0.0311、0.0431±0.0020、0.5790±0.0331,3자분별비교,차이균유통계학의의(P＜0.05).(2)shRNA적중조질립표체재체능명현억제배태세포중hsMAD2기인적표체,전염유효간우질립후,실험1조배태세포적증식억제솔위54%,분별여대조1조(4%)、대조2조(3%)비교,차이균유통계학의의(P＜0.05);G2/M기세포비례실험1조위17.9%,여대조1조(8.2%)、대조2조(8.0%)비교균명현승고,차이균유통계학의의(P＜0.05);실험1조염색체이상솔평균치위30.0%,여대조1조(4.8%)비교,차이유통계학의의(P＜0.01).결론 hsMAD2기인적표체하조가능시도치환자염색체수목이상、배태발육이상내지자연유산발생적중요원인지일.
    Objective To investigate the expression of human spindle mitosis arrest deficiency gene (hsMAD2)in spontaneous abortion embryos and the relationship between low expression of hsMAD2 and numerical chromosomal aberration. Methods Spontaneous abortion embryo tissues were collected,including 23 cases of once spontaneous abortion tissue and 10 cases of twice or more spontaneous abortion tissue and induced abortion embryos(35 cases)from the Department of Gynaecology and Obstetrics of the Affilisted Hospitals of Chongqing University of Medical Science during the period of March 2006 to March 2007.FQ-PCR and western blot were used to evaluate the endogenous expression level of hsMAD2 mRNA and hsMAD2 protein;primary culturing of cells from the induced abortion embryos was conducted and 5 embryonic cells were selected by chromosomes karyotype analysis.Recombinant shRNA plasmids targeting hsMAD2 gene were constructed to inhibit the expression of endogenous hsMAIY2 genes in embryonic cells which have normal karyotypes;the groups were defined as the first experimental group(transfeeted with pshRNA-hsMAD2-1),the second experimental group(transfected with pshRNA-hsMAD2-2),the third experimental group(transfected with pshRNA-hsMAD2-3),the first control group(transfected with nothing),the second control group(transfected with pTZU6+1)and the independent group(transfected with pshRNA-N1).Interference efficiency was demonstrated by FQ-PCR and western blot:cell prolireration was meagured by methyl thiazolyl tetrazolium(MTT)assay;cell-cycle was assessed by flow cytometry (FCM):the chromosome numbers were calculated to analyze the variation of chromosomes.Results(1) The mRNA levels of hsMAD2 in the once spontaneous abortion tissue,twice or more spontaneous abortion tissue and indueed abortion tissue were 0.00879±0.00035.0.00901±0.00033 and 0.00941±0.00026 respectively,and there Wag no significant ditierence(P＞0.05)compared with each other;however,the protein levels of hsMAD2 in three groups were 0.2791±0.0311.0.0431±0.0020 and 0.5790±0.0331 respectively,and there were significant difierences(P＜0.05)compared with each other.(2)Recombinant shRNA plagmids could significantly and specifically inhibit hsMAD2 gene expression in embryonic cells.Compared with the first control group(4%)and the second control group(3%),the recombinant shRNA could inhibit embryonic cell proliferation to 54% at 48 h after transfection(P＜0.05):compared with the first control group(8.2%)and the second control group(8.0%),the ratios of G2/M phase cells in the experimental group(17.9%)was significantly increased(P＜0.05);compared with the first control group (4.8%),the ratios of abnormal chromosomes in the experimental group was increased to 30.0%(P＜0.05).Conclusions Down-expression of hsMAD2 gene may be one of the mechanisms inducing numerical chromosome aberration,abnormal embryo development and the occurrence of spontaneous abortion.
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