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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 study the dynamic changes and regulation of perivascular cells in the process of corpus luteum angiogenesis and maturation in mice and to clarify the changes of the structure of corpus luteum. Methods Eighteen day old mice were injected intraperitoneally with gonadotropin (PMSG, HCG) to induce ovulation. The corpus luteum was induced by intraperitoneal injection of HCG for 20 h (early corpus luteum, H20), 48 h (corpus luteum development, H48) (HCD), H72, 96h (late corpus luteum, H96) were collected ovarian tissue, the use of laser confocal technology to detect vascular density (vascular endothelial cell marker CD31), maturation (PMG marker Desmin); qPCR and immunofluorescence detection of angiogenesis The expression and distribution of regulatory factors (VEGF, HIF1-α, ANGPT-1 and ANGPT-2). Results At the H20 stage, vascular endothelial cells and pericytes entered the granulosa cell layer and some of the pericytes were at the anterior end of endothelial cells. The expression of VEGF began to rise, the expression of HIF1-α was up-regulated, and the ratio of ANGPT-1 / ANGPT-2 was increased. At the time of H48, the vessel density reached the maximum with pericyte coverage, the highest VEGF and HIF1-α levels, and the highest ANGPT-1 / ANGPT-2 ratio (all P <0.0001). At H72, blood vessels began to degenerate, with reduction of vascular endothelial cells preceded by reduction of pericytes; HIF1-α and VEGF expression decreased, and the ratio of ANGPT-1 / ANGPT-2 decreased and reached the lowest at H96. Conclusion In the early stage of corpus luteum, pericytes accompany with vascular endothelial cells crawling into the corpus luteum and guiding angiogenesis. In the developmental stage, pericytes promote the stability of vascular maturation. In the middle and late stages, pericytes and endothelial cells degenerate together. The changes of the structure of corpus luteum are affected by HIF1 -α-VEGF pathway and the synergistic regulation of ANGPT1 / ANGPT2.