| Objective:Endometriosis(EMs) which is defined as endometriotic tissues growing outside uterine cavity is one of the most common gynecological disorders of reproductive aged women. Although it is benign,its behavior is alignant.The incidence reaches 10%-15%,and the tendency has being evaluated in recent years.This disease can bring chonic pelvic pain and infertility for women.Although there are various etiogenic theories,the etiology and pathogenesis of endometriosis is remain obscure. The stduy has been to emphasize on the treatment by triterpenoid saponin and the initial establishment of cultural method of endometrim cells in vitro.To investigate the effects of triterpenoid saponin on growth and apoptosis,as well as the secretion of VEGF by eutopic endometrial stromal cells cultured in vitro.It has provided experimental model in order to study effect of Triterpenoid Saponin on endometriosis,and to approach its mechanism of action.Methods:1.Primary culture of endometrial cells was performed .the eutopic endometrium cells were obtained from the digestation of trypsinase and collagenase typeⅣ. Morphology of endometrial cells of each group was observed under phase-contras microscope.SP immunocytochemistry to identify the cells.The endometriotic cell types were observed and differentiated under optical and electron microscopes .2.Eutopic endometrium cells were cultured and exposed to Triterpenoid Saponin of different doses of 200μg/ml,100μg/ml,50μg/ml,25μg/ml respectively.The inhibition of the cell during 24 hours was determined by methylthiazolyl tetrazolium(MTT)assay,and the cell growth curve was made.Triterpenoid Saponin was administered to the cells and at 24 hours emorphological changes were observed by transmission electron microscopy ,the apoptosis rate and cell cycle were monitored by flow cytometry(FCM)at the same time .3.Eutopic stromal cells cultured in vitro were treated with different doses of Triterpenoid Saponinand and the control group was not treated .the VEGFprotein in the medium of the ahove groups was measured by Enzyme—linked immunosorbent assay(ELISA).Results:1.Primary culture of endometrial cells was performed .we observed two types of endometrial cells.One is the stromal cell with polygon or fusiform shape.But the shape is not clear enough .The cells grew in parallel .The nuclear was in center, with round or oval-shaped.The other is the glandular cell with the shape of Tadpole.They grew in group,the shape is clear.Cytoplasm is full ang the nuclear is large and round.We found there were only a little of glandular cells(30%or so)and the stromal cells were a lot (70% or so)after SP immunocytochemistry under optical and electron microscopes.2.The dose of 200μg/ml Triterpenoid Saponin can inhibit the proliferation of eutopic endometrim cells(P<0.05) .Although there is no significant inhibition of the other doses(P> 0.05), but its average value were lower than the control group.inhibitory trend is existed, and each dose dosenot inhibit the proliferation of the nomal eutopic endometrial cells (P> 0.05).3.Triterpenoid saponin may lead to changes of cell cycle of the EMs eutopic endometrim cells.The G1 phase cells increased, and the S phase cells decreased. The cell growth arrest at the G 1 period, but it did not induce significant apoptosis.4.The dose of 200μg/ml Triterpenoid Saponin can inhibit the secretion of VEGF (P<0.05).Although there is no significant inhibition of the other doses(P> 0.05), but its average value were lower than the control group.inhibitory trend is existed.Conclusion:1.Enzyme digestion method to Isolate and culture the endometrial cells is scientific and effective, and the cells have the typical characteristics of endometrial cell biology.2.Triterpenoid Saponin can inhibit the proliferation of eutopic endometrim cells and dosenot inhibit the proliferation of the nomal eutopic endometrial cells .3.Triterpenoid saponin may lead to changes of cell cycle of the EMs eutopic endometrim cells but it did not induce significant apoptosis.4.Triterpenoid Saponin can inhibit the secretion of VEGF . |
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