HCG对人子宫内膜细胞VEGF表达调控的初步研究

The preliminary study on the regulation of VEGF by HCG in human endometrium in vitro

目的:探讨人子宫内膜细胞体外培养模型中,HCG对VEGF表达的调控作用和机制。方法:采用机械分离加梯度离心法分别获得腺上皮与基质细胞进行体外原代细胞混合培养;实验设对照组、HCG组(对照组使用常规DF/12培养液提取总蛋白;HCG组在DF/12培养液中加终浓度为20U/ml HCG)。采用Q-PCR测定子宫内膜细胞中VEGF mRNA表达水平。免疫组化或荧光法进行LH-R和VEGF蛋白质定位表达,Western blot法测定蛋白半定量表达。结果:子宫内膜腺上皮与基质细胞均存在LH/HCG-R和VEGF蛋白表达。HCG对子宫内膜细胞VEGF蛋白的峰值作用时间点为24h。与对照组相比,20IU/ml HCG体外干预24h后,子宫内膜细胞VEGF mRNA和蛋白质水平均显著上调(Q-PCR:1 vs 2.0132±0.6024;WB:0.4069±0.1897 vs 0.7781±0.1651)(P均<0.05)。结论:成功建立了人子宫内膜细的体外培养模型,并完成HCG的干预实验。HCG与HCG/LH-R结合后上调子宫内膜细胞VEGF的基因和蛋白质水平表达。

Objective：To investigate the mechanism and effection of HCG on the expression of VEGF on human endometrial cells in vitro culture model. Method： Endometrial cells were isolated by mechanical dissociation and centrifugation from endometrial tissue for the pri- mary cells culture, which divided into two groups：Control group and HCG group. The mRNA was analyzed by RT-PCR semi-quantitative. The location of protein was identified by immuno- histochemistry and immunofluorescence. Quantity of proteins was performed by Western blot. Results：The LH/hCG-R and VEGF were detected in cytomembrane and cytoplasm of endometrium cells by immunohistochemistry respectivity. After HCG intervention we detected VEGF mRNA and protein expression levels at different times and cansure that the optimal stimulation time of HCG acting on VEGF in endometrial cells was 24 hours. Both the mRNA and protein expressions of VEGF were significantly up-regulated in HCG group compared with that in controlgroup （P〈0.05） （Q-PCR：I vs 2. 0132±0.6024;WB：0.4069±0. 1897 vs 0.7781±0. 1651 ）. Conclusion：Successfully established the in vitro culture model of human endometrial cells and completed intervention trials of HCG;HCG up-regulated the gene and protein expression levels of VEGF after combining with HCG/LH-R.