Effects Of EP₂ And FP Receptor Agonist On Expression Of TGFβ₁ And VEGF In Bovine Endometrial Epithelial Cell

Purpose:Aim to reveal the impact of an anti-inflammatory drug------indomethacin on gene expression of transforming growth factor β1（TGFβ₁） and vascular endothelial growth factor （VEGF） in cow’s endometrial epithelial cells. And then aim to reveal regulatorymechanism of expression of EP₂ （Butaprost） and FP （Fluprostenol） to TGFβ₁ and VEGF in cow’s endometrial epithelial cells after treatment of indomethacin, which provide a theoretical guidance for clinical treatment of endometrial inflammation and restoration and protection of endometrium in treatment process of inflammation so as to increase cure rate of diseases of cow’s reproductive system.Method:1) Performed pronase digestion and mechanical method to isolate cow’s endometrial epithelial cells, and using immunofluorescence assay to identify these primary isolated cells.After amplified these cells to four passage, using MTTassay to identify cellsgrowth curve; 2) Performed fluorescence quantitative PCR （RT-PCR） to detect impact of 10-5mol/L indomethacin treatment group and 10-6mol/L Butaprost and 10-6mol/L Fluprostenol treatment groups processed by indomethacin on gene expression of TGFβ₁ and VEGF in cow’s endometrial epithelial cells at different time （Oh,2h,4h,8h,16h,24h and 48h）; 3) Performed RT-PCR to detect impact of Butaprost （10-7-10-5mol/L） and Fluprostenol （10-7～10-5mol/L） with different concentrations on gene expression of TGFβ₁ and VEGF in cow’s endometrial epithelial cells after treatment of 10-5mol/L indomethacin.4) Using Western Blot assay to detect impact of 10-5mol/L indomethacin treatment group and 10-6mol/L Butaprost. and10-6mol/L Fluprostenol treatment groups processed by indomethacin on synthesis and secretion of protein TGFβ₁ and VEGF in cow’s endometrial epithelial cells.Result:First, gained cow’s endometrial epithelial cells bypronasedigestion and mechanical method successfully. The cell had typical epithelial cell morphology with high proliferative capacity, and it could highly express the labeled proteins in cow’s endometrial epithelial cell: keratin. We selected cells of the passage four during subculture for subsequent tests. Second, the results of fluorescence quantitative PCR showed that through process of 10-5mol/L indomethacin, gene expression of TGFβ₁ and VEGF in cow’s endometrial epithelial cells declined in different degrees, in which the gene expression of TGFβ₁ reduced significantly in 4h,8h,16h and 24h after treatment, and gene expression of VEGF reduced significantly in the process of 4h,8h andl6h; while added 10-6mol/L Butaprost and 10-6mol/L Fluprostenol after processed by 10-5mol/L indomethacin, the gene expression of TGFβ₁ and VEGF in cow’s endometrial epithelial cells increased in different degrees. Compared to Butaprost, the impact of Fluprostenol on gene expression of TGFβ₁ is greater. Third, the concentration of Butaprost and Fluprostenol had little impact on gene expression of TGFβ₁, while Butaprost and Fluprostenol are concentration-dependent on gene expression of VEGF. If the concentration is higher, the expression of VEGF is higher. Forth, test results of Western Blot showed that added 10-6mol/L Butaprost and 10-6mol/L Fluprostenol after treatment of 10-5mol/L indomethacin, protein expression level of TGFβ₁ and VEGF in treatment group was similar as results tested by fluorescence quantitative PCR technology.Conclusions:1. Indomethacin can cause reduction of gene expression of TGFβ₁ and VEGF in endometrial epithelial cells.2. Butaprost and Fluprostenol are capable of promotinggene expression of TGFPi and VEGF in endometrial epithelial cells, respectively.3. Both Butaprost and Fluprostenol are dependent on gene expression of TGFβ₁ and VEGF, respectively.