瘦素对绵羊子宫内膜上皮细胞增殖、迁移和凋亡的影响

Effect of leptin on endometrial epithelial cell proliferation,migration and apoptosis in sheep

试验旨在明确瘦素在妊娠和非妊娠绵羊子宫内膜组织中的定位特征,探究其对绵羊子宫内膜上皮细胞中胚胎附植的影响。使用免疫组化法检测瘦素及其受体在妊娠和未妊娠绵羊的子宫内膜上皮组织的表达及定位;利用组织块分离培养原代绵羊子宫内膜上皮细胞,CCK8法确定瘦素最适浓度,RT-qPCR和Western blot检测瘦素对绵羊子宫内膜上皮细胞凋亡的作用,细胞划痕试验检测瘦素对绵羊子宫内膜上皮细胞迁移的作用,RT-qPCR检测瘦素对胚胎附植相关因子血管内皮生长因子(VEGF)、白血病抑制因子(LIF)、白细胞介素-6(IL-6)、子宫内膜附植位点黏蛋白(Muc1)、基质金属蛋白9(MMP9)、白细胞介素-1β(IL-1β)、骨桥蛋白(OPN)、泛素样修饰因子(ISG15)等基因mRNA的表达情况。结果显示,瘦素蛋白主要表达在未妊娠绵羊子宫内膜的基质和上皮组织中,主要在妊娠绵羊子宫内膜上皮组织中表达,瘦素受体表达无明显变化;组织块法可成功获得具有典型上皮样细胞特征并表达角蛋白CK18的原代绵羊子宫内膜上皮细胞。250 mg/L瘦素促进绵羊子宫内膜上皮细胞增殖和迁移,并显著上调Bcl-2,下调BAX、Casepase-3的转录和BAX及Casepase-3蛋白的表达。研究表明,瘦素可能通过抑制绵羊子宫内膜上皮细胞凋亡,促进细胞增殖、迁移,影响调控绵羊胚胎附植过程。

The study was to identify the localization characteristics of leptin in pregnant and non-pregnant sheep endometrial tissues and to investigate its effect on embryo attachment in sheep endometrial epithelial cells.The expression and localization of leptin and its receptor in endometrial epithelial tissues of pregnant and non-pregnant adult healthy sheep were detected by immunohistochemistry.Primary sheep endometrial epithelial cells were isolated and cultured using tissue blocks,the optimal concentration of leptin was determined by CCK8 method,the effect of leptin on apoptosis of sheep endometrial epithelial cells was detected by RT-qPCR and Western blot,and the effect of leptin on migration of sheep endometrial epithelial cells was detected by cell scratch test.RT-qPCR was used to detect the mRNA expression of VEGF,LIF,IL-6,Muc1,MMP9,IL-1β,OPN,ISG15 and other genes related to embryo implantation.The results showed that leptin protein was mainly expressed in the matrix and epithelial tissues of the endometrium of non-pregnant sheep,while the expression of leptin receptor was mainly expressed in the endometrium epithelial tissues of pregnant sheep,and the expression of leptin receptor was not significantly changed.Primary sheep endometrial epithelial cells with typical epithelioid cell characteristics and keratin CK18 expression were successfully obtained by tissue block method.250 mg/L leptin promoted the proliferation and migration of sheep endometrial epithelial cells,significantly upregulated Bcl-2,down-regulated the transcription of BAX and Casepase-3 genes and the expression of BAX and Casepase-3 proteins.The study indicates that leptin may inhibit the apoptosis of sheep endometrial epithelial cells,promote cell proliferation and migration,and affect the regulation of sheep embryo attachment process.