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

Expression of human spindle mitosis arrest deficiency gene in spontaneous abortion embryo tissues

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。

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 Affiliated 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 hsMAD2 genes in embryonic ceils which have normal karyotypes ; the groups were defined as the first experimental group （ transfected 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; ceil proliferation was measured by methyl thiazolyl tetrazolium （MTr） assay; ceil-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 induced abortion tissue were 0. 00879±0. 00035, 0. 00901±0. 00033 and 0. 00941±0. 00026 respectively, and there was no significant difference（ P 〉 0. 05 ）compared with each other; however, the protein levels of hsMAIY2 in three groups were 0. 2791±0. 0311, 0. 0431±0. 0020 and 0. 5790±0. 0331 respectively, and there were significant differences（P 〈 0. 05） compared with each other. （2） Recombinant shRNA plasmids could significantly and specifically inhibit hsMAD2 gene expression in embryonic ceils. Compared with the first control group （4%）and the second control group （3%）, the recombinant shRNA could inhibit embryonic ceil 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 ceils 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.