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目的建立彗星分析小鼠卵母细胞凋亡的模型,研究靶向酸性鞘磷酯酶1(SMPD1)的siRNA对卵子自发性凋亡的保护作用,为建立新的生殖保护方法奠定基础。方法通过同源性分析并结合siRNA设计软件设计并化学合成靶向SMPD1的3条siRNA,用超排卵技术对雌性BALB/c小鼠进行超排卵并杀鼠取卵,利用显微注射的方法将siRNA分别导入超排获得的小鼠卵子,分别于48h和72h观察卵子形态学变化,通过彗星分析检测卵细胞DNA破坏程度来分析卵子自发性凋亡情况。结果彗星分析法观察到小鼠卵母细胞在体外培养24h可见少量DNA泳出,48h可见大量DNA泳出;通过显微注射导入siRNA48h后的彗星分析结果显示,其中1对siRNA注射组,即siRNA003组的卵母细胞泳出量与其它2个siRNA注射组及对照组相比显著降低(P<0.01),其它2对siRNA注射组与对照组无明显差异。结论靶向SMPD1的siRNA能够有效保护卵子的自发性凋亡,有望成为新的生殖保护方法。
OBJECTIVE: To establish a model of mouse oocyte apoptosis induced by comet assay and to study the protective effect of SMPD1 targeting siRNA against spontaneous apoptosis of ovum in order to establish a new method of reproductive protection. Methods Three siRNAs targeted to SMPD1 were designed and synthesized by homology analysis and siRNA design software. Ovulation and ovulation were performed on female BALB / c mice by superovulation. Microinjection siRNA were respectively introduced into the mouse ovum obtained by superovulation, and morphological changes of the ovum were observed at 48h and 72h, respectively. The degree of DNA damage was analyzed by comet assay to analyze the spontaneous apoptosis of the ovum. Results The comet assay showed that a small amount of DNA could be seen in the mouse oocytes after culturing in vitro for 24 hours and a large amount of DNA could be eluted after 48 hours. The comet assay results after siRNA 48 h administration by microinjection showed that 1 pair of siRNA injection groups, siRNA003 Compared with the other two siRNA injection groups and the control group, the amount of the outflow of the oocytes in the two groups was significantly decreased (P <0.01), while the other two pairs of siRNA injection group and the control group had no significant difference. Conclusion siRNA targeting SMPD1 can effectively protect the ovum from spontaneous apoptosis and is expected to become a new reproductive protection method.