| ObjectiveTo investigate the effect of the proliferation and angiogenesis of ectopic and eutopic endometrial stromal cells from women with endometriosis induced by Leuprolide acetate salt(LA)in vitro.MethodsThe ectopic and eutopic endometrial stromal cells were isolated and cultured in vitro.Then they were identified by vimentin immunocytochemistry staining.Both the ectopic and eutopic endometrial stromal cells were exposed to LA in different concentration(0,0.01,0.1,1,10ug/ml)for 0～96h.The proliferation and morphological changes were observed by inverted microscope.The growth and inhibition of ectopic and eutopic stromal cells were measured by MTT assay.The apoptosis and cell cycle distribution induced by different concentration of LA on ectopic and eutopic stromal cells were assayed by flow cytometric analysis.The expression of VEGF induced by LA was analyzed by immunocytochemistry.ResultsTwelve cases were successfully cultured in all 14 cases of ectopic endometrial cells. The successful rate was 85.7%.All the 16 cases of eutopic endometrial cells were cultured successfully.The serial subcultivation of ectopic and eutopic endometrial cells were successful.The positive rate of vimentin was above 95%in ectopic and eutopic endometrial cells identified by immunocytochemistry staining.The morphous of ectopic and eutopic cells had no obviously difference,the cells became fusiform growing after 24 hours.They became longer followed the time,and arranged to braid appearance.The cells had no clear boundary and contact inhibition.Both ectopic and eutopic endometrial stromal cells had no morphological changes in 10 serial subcultivations.The growth and proliferation depressed in ectopic and eutopic cells after the action of LA.The cells morphological changes were slightly when exposed to LA at a concentration of 0.01ug/ml for 48 hours.There were only individual cells floated in the culture fluid.The number of adherent cell was decreased and the morphological change was obviously at a concentration of 10ug/ml for 48 hours. These changes were more significant accompany to the time spread.MTT assay demonstrated that LA could inhibit the growth and proliferation of ectopic and eutopic endometrial stromal cells in a time-and-dose dependent fashion.The cell inhibition rates of ectopic cells induced by LA at a concentration of 0.1 ug/ml for 24h,48h,72h,96h were 33.74%,52.23%,62.04%,68.72%,and those ofeutopic cells were 13.13 %,30.29%,44.26%,58.14%.The inhibition rates of ectopic cells at a concentration of 1ug/ml were 51.61%,60.64%,70.00%,74.31%,and those of eutopic cells were 24.97%,36.70%,52.68%,67.35%.The inhibition of cell growth induced by LA in ectopic cells was stronger than in eutopic cells in a time-and-dose dependent fashion (P＜0.05)Annexin V—FITC and PI double staining showed that LA could increased the apoptotic rate of ectopic and eutopic stromal cells in a dose depend fashion(P＜0.05).These were more obvious in ectopic cells than in eutopic cells (P＜0.05).LA blocked the cell cycle of the two groups.The number of ectopic cells and eutopic cells were increased in G1 stage and decreased in S stage.It was more manifest in the higher concentration(P＜0.05).The percentage of G1 stage of ectopic cells was increased from 19.45±3.47 to 34.92±4.14(0.1ug/ml)and 51.47±5.07 (1ug/ml),and that of eutopic cells increased from 58.50±8.96 to 74.80±6.96 (0.1ug/ml)and 84.35±5.92(1ug/ml).The expression of VEGF in ectopic cells and eutopic cells was significantly decreased after the action of LA(P＜0.01).The positive rate accumulated points of VEGF in ectopic cells was decreased from 120.80 ±19.46 to 70.60±5.32(0.1ug/ml)and 50.80±9.09(1ug/ml),and that in eutopic cells was decreased from 83.00±9.35 to 54.40±5.32(0.1ug/ml)and 40.60±4.88 (1ug/ml).The expression of VEGF was decreased more obvious in ectopic cells than in eutopic cells(P＜0.05).Conclusion1,LA could significantly inhibit the proliferation and enhance the apoptosis of ectopic and eutopic endometfial stromal cells from women with endometfiosis in a time-and-dose dependent fashion.2,LA could obviously decrease the expression of VEGF.3,The actions above were more stronger in ectopic endometrial stromal cells than in eutopic endometrial stromal cells. |
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