摘要
目的探讨有丝分裂关卡基因（hBUB1）在染色体数目异常自然流产胚胎发生中的可能机制。方法采用实时定量逆转录（RT）-PCR和免疫印迹法（WB）检测染色体数目异常组和正常对照组的自然流产胚胎组织中目的基因在mRNA和蛋白水平的表达；构建针对hBUB1基因的短发卡状（shRNA）表达载体，用细胞免疫化学、WB和实时定量RT-PCR方法评价其对胚胎细胞内源性hBUB1表达的干扰效果；用四甲基偶氮唑盐微量酶反应比色法（MTT）试验测定细胞增殖抑制率；用流式细胞术解析细胞周期分布。结果hBUB1蛋白在染色体数目异常组中的表达显著低于正常对照组（阳性表达率分别为8％和93．5％，P〈0．05）。shRNA的重组质粒表达载体能明显抑制胚胎细胞中hBUB1基因的表达（干扰前后mRNA水平分别为0．196±0．067和0．042±0．006，P〈0．05）。胚胎细胞转染有效干扰质粒48h后，染色体数目异常组细胞增殖抑制率达62％，显著高于正常对照组（4％，P〈0．05）。有效干扰质粒引起G2／M期细胞从对照组的40．2％和41．3％降低至21．3％。结论hBUB1基因表达下调可导致细胞增殖的抑制和周期的改变，在染色体数目异常自然流产胚胎发生中可能起很重要的作用，这将为寻找可靠的临床检测指标奠定基础。
Objective To explore the mechanism of hBUB1 gene in the developing of spontaneous abortion embryos with numerical chromosomal abnormality. Methods Quantitative real-time RT-PCR and Western blot were used to determine the mRNA and protein level of hsMAD2 gene both in spontaneous abortion embryos with numerical chromosomal abnormality （experimental group ） and with numerical chromosomal normality （control group ）. Recombinant shRNA plasmids targeting hBUB1 gene was constructed to inhibit the expression of endogenous hBUB1 genes in embryonic cells. Interference efficiency was demonstrated by fluorescent quantitative PCR and Western blot. The inhibitory rate of cell proliferation was measured by MTT assay and cells-cycle was assessed by flow cytometry. Results Western blot analysis showed that protein level of hBUB1 in the experimental group was decreased significantly （the rate of positivity and strong positivity were 8% and 93.5%, respectively, P 〈 0. 05 ） compared with the control group. The expression of hBUB1 gene in embryonic cells was significantly and specially inhibited by shRNA plasmids （the mRNA level before and after treatment with RNAi were 0. 196 ± 0. 067 and 0. 042 ± 0. 006, respectively,P 〈0. 05）. The inhibitory rate of cell proliferation was increased to 62% from 4% at 48 h after transfection. The rate of G2/M phase cells was decreased after transfection with efficient shRNA （control group： 40.2% and 41.3%, test group：21.3% ）. Conclusions Down-regulation of hBUB1 gene leads to the inhibition of cell proliferation and the arrest of cell-cycle. It also probably plays an important role in the development of spontaneous abortion embryos with numerical chromosomal abnormality. The clinical relevance warrant further investigation.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2008年第3期309-315,共7页
Chinese Journal of Laboratory Medicine
基金
国家自然科学基金资助项目（30371485）
国家教育委员会高等校博士学科点专项科研基金资助项目（[2007]226）
关键词
流产
自发性
胚胎
染色体畸变
蛋白质丝氨酸苏氨酸激酶
逆转录聚合酶链反应
免疫印迹法
Abortion, spontaneous
Embryo
Chromosome aberrations
Protein-serinethreonine kinases
Reverse transcriptase polymerase chain reaction
Immunoblotting
