Effect Of EGCG On The Uterine Leiomyoma Cells In Vitro Based On The Wnt/?-catenin Signal Transduction Pathway

Objective:1.To improve the technique of cultivating human uterine leiomyoma cells in vitro culture?Improving the rate of adherenting human uterine leiomyoma cells and shorten the time of passage for the first time.2.To investigate the suppressive effect of EGCG on the proliferation and apoptosis of human uterine leiomyoma cells in vitro,and the expression of Wnt5b??-catenin?EGFR based on the Wnt/?-catenin signal transduction pathway.Our purpose was to clarify the mechanism of action of EGCG treatment of uterine leiomyoma cells,enrich the diet of TCM clinical applicability.Methods:1.Removed fibroids group into 4? Hanks fluid after surgery,to prolong cells digestion time,digestion and tissue pieces of secondary modified method to get the original generation of uterine flesh tumour cells.After the action of EGCG with different concentration(OuM?50uM?100uM?200uM),CCK-8 was used to examine the proliferative activity of UL cells,the rate of apoptosis was determined by flow cytometry.Application of inverted Microscope on the cell morphology and growth characteristics of observation analysis;After interventing 48 hours,immunphistochemical method was applied to detect the UL cell Wntb??-catenin,expression,western blot assay was used to detect on the UL cell Wntb??-catenin?EGFR elements meanwhile.Results:1.UL cells were successfully culturedCompared with the normal group,the improved group was better:he amount of cells(2.33±0.48 vs 1.16±0.29)×108/L;the adherenting rate of cells(41.33±7.43 vs 22.07±6.82)%.To extend the time for the first time(10.27±1.67 vs 14.67±1.18)days;P<0.05;Cells under a microscope show typical "peak-valley"shape growth,SM alpha actin)expressed in the cytoplasm and cell purity of more than 98%.2.All groups of UL cells grow well,under inverted Microscope on the cells in the control group showed fusiform or polygon adherent growth,number density,the outline clear,compact connection between cells.The CCK-8 shows EGCG could inhibit the proliferation of uterine leiomyoma cells in vitro as direct relation of concentrations and times(P<0.05).After 24h the proliferative activity of cells were inhibited(P<0.05).3.FCM results showed that the human uterine leiomyoma cells apoptosis rate increased as the concentration of EGCG.4.Immunohistochemical method,Western-blot test results showed that EGCG can reduce Wnt5b??-catenin protein in human uterine leiomyoma cells with a concentration-dependent;Conclusion:1.Modified cell culture technique can acquire the original generation of uterine flesh tumour cells both in speed and quantity.2.Based on Wnt/?-catenin signal transduction pathway,EGCG could down-regulate the expression of Wnt5b??-catenin?EGFR,which lead to inhibite growth of UL cells.