体外培养对小鼠囊胚的发育潜能及DNA损伤的影响研究

Effects of in vitro culture on developmental potential and DNA damage of mouse blastocysts

目的研究体外培养对小鼠囊胚的发育潜能及DNA损伤的影响及作用机制。方法将实验小鼠随机分为两组,自然妊娠组(NC组)和胚胎体外培养组(IVC组),每组各6例雌鼠,收集两组囊胚期胚胎各60枚。荧光实时定量聚合酶链反应检测两组胚胎早期细胞定向分化因子以及DNA损伤相关基因的表达水平,免疫荧光法检测两组囊胚中DNA损伤因子γH2AX的表达水平。结果实时定量PCR检测结果显示,相比于NC组,IVC组的多能因子Pou5f1、Nanog基因表达显著下降(P<0.05),滋养层细胞分化基因Tead4表达显著上调(P<0.05),滋养层细胞分化基因Cdx2、Eomes和Elf5的表达均显著下降(P<0.05),调控原始外胚层分化的基因Gata6、Sox17、Fgfr2以及Fgfr4表达显著下降(P<0.05),Lif基因表达也在IVC组显著下降(P<0.05)。免疫荧光检测结果显示,IVC组囊胚DNA损伤因子γH2AX的相对荧光密度显著大于NC组(P<0.05)。同时,与NC组相比,IVC组的DNA损伤相关基因Fas、Gadd45a和Ddit3的表达均显著上升(P<0.05)。结论体外培养可导致小鼠囊胚多能因子及早期胚胎命运决定基因表达水平发生改变,影响小鼠囊胚发育潜能。这可能与IVC组小鼠囊胚DNA损伤增加、DNA损伤相关基因的表达异常具有潜在联系。

Objective:To study the effects of in vitro culture on the developmental potential and DNA damage of mouse blastocysts.Methods:The experiment mice were divided into two groups:natural conception(NC)group and in vitro culture(IVC)group,6 female mice in each group.60 blastocysts were collected for each group.Fluorescence quantitative polymerase chain reaction(RT-PCR)was used to detect the expression of early embryonic cell fate determinants and DNA damage-related genes in blastocysts of both groups.The DNA damage factorγH2AX of the blastocysts from the two groups was detected by immunofluorescence assay.Results:The results of RT-PCR showed that the expression of multiple potential factor Pou5f1 and Nanog in IVC group were decreased compared with NC group(P<0.05).The trophoblast cell differentiation gene Tead4 was significantly up-regulated in IVC group(P<0.05).The expressions of trophoblast cell differentiation genes Cdx2,Eomes and Elf5 in IVC group were significantly decreased(P<0.05).The expressions of Gata6,Sox17,Fgfr2 and Fgfr4,which regulate primary ectodermal differentiation,were decreased in IVC group(P<0.05).Compared with NC group,Lif gene was also significantly decreased in IVC group(P<0.05).Moreover,the fluorescence density of DNA damage factorγH2AX in IVC group was significantly higher than that in NC group(P<0.05).Meanwhile,compared with the NC group,DNA damage-related genes Fas,Gadd45a and Ddit3 were significantly increased in IVC group(P<0.05).Conclusions:The expression level of pluripotency factor and early embryonic cell fate-determining gene were affected when blastocysts cultured in vitro.This may be associated with increased DNA damage and abnormal expression of DNA damage-related genes in mouse blastocysts.