缺氧对滋养细胞VEGF分泌与基因表达调控作用的研究

目的:研究原代培养人早孕绒毛滋养细胞VEGF分泌、基因表达的特点,以及缺氧的调控作用。方法:原代培养早孕绒毛滋养细胞,研究正常和缺氧(氯化钴化学诱导)条件下细胞培养上清液中VEGF含量(ELISA法)以及滋养细胞VEGF mRNA表达(RT-PCR方法)特点。结果:(1)免疫组化技术证实培养细胞角蛋白阳性,波形蛋白阴性,纯度达95%以上;(2)正常培养的滋养细胞在培养12h后分泌产生的VEGF明显增加,至培养结束时的72h达到最高点;缺氧诱导滋养细胞大量分泌产生VEGF的时间提前至缺氧培养后6h,持续至72h无下降,VEGF水平明显高于正常,最高达到2200ng/ml,较正常上升47%;(3)滋养细胞VEGF mRNA表达于培养12h时开始明显上升,至48h时达峰值;缺氧则使VEGF mRNA表达显著上升的时间提前至6h,约12h达峰值。结论:早孕绒毛滋养细胞中存在VEGF基因转录并分泌至细胞外;缺氧诱导VEGF转录增强、分泌增加。

The expression and regulation of vascular endothelial growth factor in trophoblast cells

Objective：To characterize the trophoblast cell＇s expression of vascular endothehal growth factor, key regulators of conventional vasculogenesis and angiogenesis, and how the hypoxia regulates these expressions. Methods： Cytotrophoblasts were isolated from the chorionic villi of 6 to 12 week gestation by trypsin/DNase digestion and purifcafion through changing culture plate. The results of immunohistochemistry confirm that a human trophoblast system was estabhshed. The cells were cultured respectively in normal and hypoxia circumstance which indued by CoCl2. The cell extracts were tested for VEGF by enzyme linked immunosorbent assay and the RNA of the cultured cells were isolated and tested for VEGF mRNA by real-time RTPCR. Results： （ 1 ） The cultured cells were positive for cytokeratin and negative for vimentin, indicating the absence of contaminating elements and the cell viability evaluated by trypan blue exclusion was greater than 95% ; （2）The levels of VEGF in the cell extracts and the VEGF mRNA of the cultured cells all increased gradually as the culturing time continuted during the 72hours＇ cultured time. Hypoxia could up-regulate the expression of VEGF. Conclusion：VEGF is expressed by the cytotrophoblast and the expression is up-regulated by hypoxia.