染色体数目异常自然流产胚胎有丝分裂关卡蛋白基因的研究  

THE STUDY ON hsMAD2 GENE IN SPONTANEOUS ABORTION EMBRYOS WITH CHROMOSOMAL NUMERICAL ABNORMALITY

作　　者：施琼[1] 王箭[1] 袁泰先[1] 翁亚光[1] 王应雄[1] 徐远久[1] 蔡燕[1] 蒋洪彦[1] 

机构地区：[1]重庆医科大学临床检验诊断学省部共建教育部实验室,重庆市重点实验室,重庆400016

出　　处：《解剖学报》2008年第2期197-201,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(30371485)

摘　　要：目的探讨染色体数目异常自然流产胚胎中有丝分裂关卡蛋白(hsMAD2)基因的功能。方法分别用定量PCR和Western blotting在mRNA和蛋白水平检测染色体数目异常(实验组)和正常(对照组)的自然流产胚胎组织中目的基因的表达;构建针对hsMAD2基因的shRNA表达载体,抑制胚胎细胞内源性hsMAD2基因的表达,用Western blotting和定量PCR方法评价shRNA的干扰效果;用MTT法测定细胞增殖率;流式细胞术检测细胞周期。结果hsMAD2蛋白在实验组中的表达显著低于对照组。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。胚胎细胞转染有效干扰质粒48 h后,细胞增殖抑制率达58%。有效干扰质粒引起G0/G1期和S期细胞的明显降低,G2/M期细胞增多。结论hsMAD2基因表达下调可能在染色体数目异常的自然流产胚胎发生中起着很重要的作用,为寻找可靠的临床检测指标奠定了基础。Objective To investigate the function of hsMAD2 gene in spontaneous abortion embryos with chromosomal numerical abnormality.Methods The results of quantitative real-time RT-PCR and Western blotting method were used to display the mRNA and protein level of hsMAD2 gene both in spontaneous abortion embryos with chromosomal numerical abnormality（experimental group）and with chromosomal numerical normality（control group）.Recombinant shRNA plasmids was constructed targeting hsMAD2 gene to inhibit the expression of endogenous hsMAD2 genes in embryonic cells;interference efficiency was demonstrated by FQ-PCR and Western blotting; cell proliferation was measured by MTT assay;cell-cycle was assessed by FCM.Results Western blotting analysis showed that compared with the control group,the protein levels of hsMAD2 in the experimental group decreased significantly.Recombinant shRNA plasmids could significantly and specially inhibit hsMAD2 gene expression in embryonic cells and could inhibit up to 58% of embryonic cells proliferation 48 hours after the transfection.Efficient shRNA can decrease G_0/G_1 and S phase cells,and increase G_2/M phase cells.Conclusion The down-expression of hsMAD2 gene probably plays an important role in the development of spontaneous abortion embryos with chromosomal numerical abnormality.The study provides preliminary data for further establishment of diagnostic or therapeutic criteria on spontaneous abortion.

关 键 词：有丝分裂关卡基因 RNA干扰 细胞增殖 染色体数目异常 免疫印迹法 

分 类 号：R714.21[医药卫生—妇产科学] Q343.244[医药卫生—临床医学]