子宫内膜巨噬细胞通过调控血管内皮生长因子A的表达影响胚胎着床

摘要

目的：研究小鼠子宫内膜巨噬细胞（macrophages, Mφ）对血管内皮生长因子A（VEGFA）表达的影响，探讨其在胚胎着床过程中的作用。方法取第3.5（雌鼠交配后次日晨阴道见栓为第0.5）孕鼠30只，随机分为3组，实验组（E）、对照组（C）和空白组（B）。E组向左侧宫腔内注射消除巨噬细胞的药物：氯膦酸二钠脂质体（clodronate liposomes, CL），右侧注射磷酸缓冲盐脂质体（PBS liposomes, PL）；C组向双侧宫腔内注射PL；B组向双侧宫腔内注射等体积灭菌PBS溶液。注射后48 h，获取第5.5小鼠子宫和卵巢，统计各单侧子宫的胚胎着床数。采用免疫组化技术检测子宫内膜、卵巢内Mφ数量，并观察着床位点和非着床位点VEGFA的表达变化。流式细胞学方法检测子宫F4/80+CD11b+Mφ相对百分比，观察注射CL对子宫Mφ的选择性抑制作用。结果流式细胞学和免疫组织化学方法均显示，E组左侧子宫注射氯膦酸二钠脂质体后，与E组右侧和C、B组比较，局部巨噬细胞被显著抑制（P<0.05），抑制率达74%。各组卵巢Mφ数量间则未见明显差异。E组左侧子宫的胚胎着床部位宫腔未闭合，平均胚胎着床数为2.20±1.81个，显著低于E组右侧5.10±1.91个（P<0.05）。在着床位点，伴随子宫Mφ受到抑制，子宫内膜的VEGFA蛋白表达明显降低（P<0.05）。结论子宫内膜中的Mφ为胚胎着床所必需，其机制可能是通过调控VEGFA的表达，影响宫内膜容受性及胚胎着床。%Objective To investigate the role of endometrial macrophages in embryo implantation and in regulating the expression of vascular endothelial growth factor A (VEGFA) in mouse endometrium during the peri-implantation period. Method At D3.5 (D0.5 defined as the morning when a vaginal plug was observed), pregnant mice were divided randomly into experimental group, control group and blank group. In the experimental group, the mice were subjected to intrauterine injection of clodronate liposomes on the left side of uterus to eliminate the macrophages, and PBS liposomes on the right side. PBS liposomes and PBS were administered in the control and blank groups, respectively. The uterine tissues were collected on D5.5 and stained with trypan blue to show the implantation sites. Flow cytometry was performed to examine the percentage of F4/80+ CD11b+ macrophages macrophages in the uterus. F4/80+ macrophage population within the endometrium and ovary and changes in VEGFA expression at the implantation and non-implantation sites were examined using immunohistochemistry. Results Endometrial F4/80 + CD11b + macrophages macrophages were significantly reduced by 74%following intrauterine injection of clodronate liposomes (P<0.05). The number of macrophages in the ovaries showed no significant difference among the 3 groups. In the experimental group, the left side of the uterine showed imcomplete cavity closure with a lower number of implantation site than the right side (2.20±1.81 vs 5.10±1.91, P<0.05). VEGFA expression at the implantation site were significantly decreased in the endometrium on the left side with macrophage suppression as compared with that on the right side (P<0.05). Conclusion Endometrial macrophages appear to modulate uterine receptivity by regulating the expression of VEGFA to affect embryo implantation, suggesting the important role of macrophages in embryo implantation.