脂多糖对胚胎附植期Toll样受体4及相关免疫因子的影响

试验旨在探讨脂多糖（LPS）对绵羊胚胎附植期Toll样受体4（TLR4）及相关免疫因子表达的影响。以构建好的pcDNA3.1-TLR4过表达载体转染绵羊子宫内膜基质细胞，运用Western blotting技术鉴定细胞转染效果，然后用1 μg/L LPS刺激转染后的子宫内膜基质细胞，建立内膜基质细胞炎症模型。将经LPS处理的细胞分别培养12、24、48、72 h，采用实时荧光定量PCR技术和Western blotting法检测内膜基质细胞中TLR4 mRNA及蛋白表达量，以及免疫因子IL-1β和IL-6的mRNA表达水平，并以未经LPS处理的细胞作为对照。结果显示，与对照组相比，LPS促进了免疫因子IL-1β、IL-6的释放量，但随LPS作用时间的延长，细胞中IL-6的表达量逐渐下降，而IL-1β的表达量逐渐升高，使得Th1/Th2偏向不利于妊娠的Th1方向表达；TLR4 mRNA相对表达量在12、24、72 h均显著高于对照组（P<0.05），48 h时极显著高于对照组（P<0.01），且LPS处理后的细胞TLR4蛋白表达量也始终高于对照组。综上所述，pcDNA3.1-TLR4过表达载体成功转入绵羊子宫内膜基质细胞；LPS有效激活了子宫内膜细胞中TLR4信号通路，并促进了下游因子的表达；子宫蜕膜组织中TLR4受体蛋白对胚胎附植早期妊娠微环境的平衡维持也起到了重要作用。

Effects of LPS on TLR4 and Related Immune Factors During Embryo Implantation Stage

The aim of this study was to investigate the effect of LPS on the expression of TLR4 and related immune factors in embryo implantation stage of sheep.The constructed pcDNA3.1-TLR4 overexpression vector was used to transfect sheep endometrial stromal cells,and Western blotting was used to identify the transfection effect.Then the endometrial stromal cells were stimulated with 1 μg/L LPS,and the inflammatory model of endometrial stromal cells was established.The TLR4 and related immune factors IL-1β and IL-6 of cell treated with LPS were detected after cultured for 12,24,48 and 72 h by Real-time quantitative PCR and Western blotting methods with untreated cell as the control.The results showed that compared with the control group,LPS promoted the release of IL-1β and IL-6,but the expression of IL-6 in the cells gradually decreased with time,and the expression of IL-1β increased gradually,which made Th1/Th2 biased to the Th1 direction.The expressions of TLR4 mRNA at 12,24 and 72 h were all significantly higher than that in control group (P<0.05),and the expression at 48 h was extremely significantly higher than that in control group (P<0.01),and the expression of TLR4 protein after LPS treatment was always higher than that of the control group.In conclusion,the pcDNA3.1-TLR4 overexpression vector was successfully transferred into the endometrial stromal cells of sheep.LPS effectively activated the TLR4 signaling pathway in endometrial cells and promoted the expression of downstream factors.The TLR4 protein in the decidua tissue also played an important role in maintaining the balance of early pregnancy microenvironment.