Hsp70在小鼠体外早期胚胎发育中的表达

The Expression of Hsp70 in Early Embryonic Development of Mice in vitro

为探究热休克蛋白Hsp70在小鼠体外早期胚胎发育中的表达情况以及Hsp70在胚胎发育中的作用机理,本试验采集8~12周龄SPF小白鼠的卵巢,在体式显微镜下采集卵母细胞并培养成熟,用0. 1%透明质酸酶消化卵丘卵母细胞复合体后,放入SrCl2+CB液中进行5 h(37℃)的孤雌激活,然后移入G1培养液进行培养。取培养至2细胞期、4~8细胞期、9~16细胞期、桑椹期和囊胚期的小鼠胚胎提取总RNA,通过实时荧光定量PCR检测Hsp70在各个时期的表达情况,免疫荧光染色检测其在各个时期小鼠胚胎的表达定位。实时荧光定量PCR结果表明,Hsp70在小鼠体外早期胚胎的2细胞期、4~8细胞期、9~16细胞期、桑椹期、囊胚期都有表达,但在2细胞期胚胎和4~8细胞期胚胎的相对表达水平较高,在9~16细胞期胚胎、桑椹期胚胎和囊胚期胚胎的相对表达水平较低。免疫荧光染色结果显示,Hsp70定位于各个时期胚胎的细胞核和细胞质中,但9~16细胞期以后Hsp70主要定位于细胞核中。综上所述,Hsp70对小鼠体外早期胚胎的发育具有潜在的调控作用,这为进一步明确热休克蛋白Hsp70在胚胎抗热应激中的作用及机理研究提供了理论依据。

To research the expression of heat shock protein Hsp70 in early embryonic development of mice in vitro,and to explore the effect and mechanism of Hsp70 in embryonic development.The ovaries of SPF mice aged 8-12 weeks were collected in this study,then oocytes were collected under the microscope and cultivated to maturity,we placed COCs into the SrCl2+CB solution for parthenogenetic activation for 5 hours(37℃)after digesting it with 0.1%hyaluronidase,then we transferred it into G1 culture fluid for cultivating.We extracted the total RNA from the mouse embryos that had been cultivated to 2 cell embryo,4-8 cell embryo,9-16 cell embryo,morula and blastocyst,then we detected the expression of Hsp70 in each period by real-time Q-PCR,and the expression and location of Hsp70 at various stages of mouse embryos by immunofluorescence staining.The result of the real-time Q-PCR shows that Hsp70 expressed in the 2 cell embryo,4-8 cell embryo,9-16 cell embryo,morula and blastocyst in early extracorporeal embryo of mice,but the expression of Hsp70 is higher in the 2 cell embryo and 4-8 cell embryo,and lower in the 9-16 cell embryo,morula and blastocyst.The result of immunofluorescence staining shows that Hsp70 locates in the nucleus and cytoplasm of mouse embryos at various stages,but Hsp70 mainly locates in the nucleus after the 9-16 cell embryo.In summary,Hsp70 has the potential regulatory effects in early embryonic development of mice in vitro,and this study provides a theoretical basis for further clarifying the role and mechanism of heat shock protein Hsp70 in embryo heat stress resistance.