小鼠黄体血管中周细胞的动态变化及调控因素研究

摘要

目的 研究血管周细胞在小鼠黄体血管新生及成熟过程中的动态变化及其调控因素,阐明黄体血管结构的变化规律.方法 对21日龄小鼠腹腔注射促性腺激素(PMSG,HCG)促排卵,建立黄体生成模型,分别于注射HCG后20h(黄体早期,H20)、48h(黄体发展期,H48)、72h(黄体中期,H72)、96h(黄体晚期,H96)收取卵巢组织,利用激光共聚焦技术检测血管密度(血管内皮细胞marker CD31)、成熟度(周细胞marker Desmin);通过qPCR及免疫荧光检测血管新生关键调控因子(VEGF、HIF1-α、ANGPT-1及ANGPT-2)的表达和分布.结果 在H20阶段,血管内皮细胞和周细胞进入颗粒细胞层,部分周细胞在内皮细胞前端;VEGF表达开始上升,HIF1-α表达上调,ANGPT-1/ANGPT-2的比值增加.在H48时,血管密度达最大,有周细胞覆盖,VEGF和HIF1-α达最高,ANGPT-1/ANGPT-2的比值达最高,与其他时间组比较差异有统计学意义(P＜0.0001).在H72时,血管开始退化,血管内皮细胞的减少先于周细胞的减少;HIF1-α和VEGF表达下降,ANGPT-1/ANGPT-2的比值下降,并在H96时降至最低.结论 黄体早期,周细胞伴随血管内皮细胞爬行进入黄体,引导血管新生;发展期,周细胞促进血管成熟稳定;中、晚期,周细胞与血管内皮细胞一起退化;且黄体血管结构组成的变化受HIF1-α-VEGF通路和ANGPT1/ANGPT2的协同调控.%Objective To investigate pericyte's distribution and regulatory factors in mouse corpus luteum angiogenesis and to clarify luteal vascular structure variation during corpus luteum angiogenesis.Methods The model of corpus luteum was established by injecting exogenous gonadotropin(PMSG,HCG)into 21days mice intraperitoneally.Mice ovaries were collected after injected HCG for 20 hours (early luteal phase),48hours (Luteal phase of development),72hours (middle luteal phase) and 96hours (late luteal phase) respectively.Vascular density(CD31) and maturation(Desmin) were measured by immunofluorescence by using laser confocal microscopy.HIF1-α,VEGF,ANGPT-1 and ANGPT-2 in each group were detected by immunofluorescence and.Results In the early luteal phase,the expression of VEGF in luteal began to increase while the expression of HIF1-α was upregulated and the ratio of ANGPT-1/ANGPT-2 was increased.At the same time,the endothelial cells and pericytes were activated and some pericytes preceded the corpus luteum.In the Luteal development phase,the expression of VEGF and HIF1-o in corpus luteum reached the highest level and the ratio of ANGPT-1 / ANGPT-2 became the highest level while the vascular density reached the maximum and the mature vascular network was formed.In the middle luteal phase2,the expression of HIF1-α,VEGF and the ratio of ANGPT-1 / ANGPT-2 decreased while the blood vessels began to degenerate and vascular endothelial cells turned to apoptosis before pericyte's degeneration.In the late luteal phase,almost all of the endothelial cells disappeared,but part of the peripheral cell remnants.Conclusion Pericytes existed in the early luteal phase,and some of them are sprouted before endothelial cells.In middle phase,luteal pericytes promote vascular maturation.In the late stage,pericytes degenerate with endothelial cells,which is mainly affected by HIF1-α-VEGF and ANGPT1 / ANGPT2 synergistic regulation.