DNA甲基转移酶在胚胎停育绒毛组织中的表达差异及临床意义

Different Expression and Clinical Significance of DNA Methyltransferases in the Chorionic Villi of Early Embryo Growth Arrest

目的:探讨胚胎停育绒毛组织中DNA甲基转移酶(DNMTs)4种亚型DNMT1、DNMT3A、DNMT3B与DNMT3L的mRNA及蛋白表达差异,并探讨其临床意义。方法:随机选取2013年1月-2014年6月在青岛市立医院妇产科门诊就诊的经B型超声(B超)证实为胚胎停育而行清宫术的40例患者为观察组,并选取同期正常早孕要求人工流产的40例患者为对照组。1采用实时荧光定量聚合酶链反应(qRT-PCR)方法检测2组患者绒毛组织中DNMT1、DNMT3A、DNMT3B与DNMT3L mRNA的表达量;2用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶连结法(SP法)及蛋白质印迹法(Western blot)检测DNMT1、DNMT3A、DNMT3B与DNMT3L蛋白在观察组和对照组患者绒毛组织中的表达部位及定量差异。结果:1qRT-PCR结果显示,2组患者绒毛中DNMT1、DNMT3A、DNMT3B和DNMT3L mRNA的表达量比较差异无统计学意义(P>0.05);2免疫组化结果显示,DNMT1、DNMT3A、DNMT3B与DNMT3L在2组绒毛滋养细胞的细胞核或细胞质呈不同程度的阳性染色,同时,半定量分析结果显示观察组的DNMT3A蛋白表达量低于对照组,差异有统计学意义(P<0.05);3Western blot分析结果显示,观察组患者绒毛中DNMT3A蛋白的相对表达量低于对照组,差异有统计学意义(P<0.05);4胚胎停育绒毛组织中DNMT3A蛋白的表达量与DNMT1、DNMT3B和DNMT3L蛋白表达量之间无明显关联(均P>0.05)。结论:胚胎停育绒毛组织中,DNMT3A蛋白水平的低表达可能参与了胚胎停育的发病机制。

Objective：To study the expression of DNMT1, DNMT3A, DNMT3B and DNMT3L mRNA and protein in the chorionic villi of early embryo growth arrest and explore its clinical significance. Methods：We randomly selected 40 women as observational group in which were diagnosed with early embryo growth arrest by B ultrasound and accepted complete curettage of uterine cavity after visiting the Obstetrical Department of the Qingdao Municipal Hospital between January 2013 and June 2014,during the same period, select another 40 women who performed induced abortion with normal early pregnancy as control group.① Real-time quantitative PCR（qRT-PCR） was used on the normal groups（40 cases） and the early embryo growth arrest group（40 cases） to quantitatively determine DNMT1, DNMT3A, DNMT3B and DNMT3L mRNA expression in the chorionic villi tissues. ② Streptavidin-Perosidase（SP） immunochemistry and Western blot were performed on the two groups to detect the expression and distribution of DNMT1, DNMT3A, DNMT3B and DNMT3L. Results： ① qRT-PCR showed there were no statistically significant difference of DNMT1, DNMT3A, DNMT3B and DNMT3L expression in two groups（P〉0.05）. ② Immunochemistry showed DNMTs were predominately distributed on the villous trophoblasts which the cytoplasm and nuclear had varying degrees of positive staining. And semi-quantitative analysis indicated that the expression of DNMT3A of early embryo growth arrest was significantly lower, compared with that of the normal group（P〈0.05）. ③ Western blot also showed expression of DNMT3A of early embryo growth arrest was lower, differences between the two groups were statistical significance（ P〈0.05）.④ There was no significant correlation between DNMT3A and DNMT1, DNMT3B, DNMT3L in the chorionic villi of early embryo growth arrest（all P〉0.05）. Conclusions：The lower expression of the DNMT3A protein might be involved in the pathogenesis of early embryo growth arrest.