The Study Of Leiomyoma Treatment With Mifepristone In Vitro

Objective: 1. To Observe the morphologic changes of the human leiomyoma cells cultured in the presence of mifepristone. (10-8~10-4mol/L). To find the optimal concentration of treating leiomyoma with mifepristone. 2. To investigate the machenisms of shrinking leiomyoma with mifeprtstone. 3. To investigate the relationship between apoptosis and treatment of leiomyoma with mifepristone.Methods: 1. The isolated and purified cells derived from human uterine leiomyoma were cultured in the different concentration of mifepristone for 3 days. We observed the morphologic changes of the cells under the microscope and took the pictures, then collected the cells and examine their ultrastructure with the electron microscope and took the pictures. The growth of the leiomyoma cells cultured for 3 days were detected by MTT assay. 2. The cells slides were stained with ER, PR, PCNA, VEGF, TGFB, Fas, FasL, bcl-2, caspase3 enzyme-labelled antibody methods. 3. PRL was detected by ELISAmethod. 4. The apoptosis was detected by FCM.Results: 1. 58.8% leiomyoma cells were cultured successfully.The myoma of different types need different digestion time. The separation cells having an initial viability of 90% by dye exclusion . The cells settled within 24 hours and commenced to multiply when plated in DMEM with 20% fetal bovine serum. About 5~7 days to confluence 70-80% of cells. 2. The cells morphologically appeared spindle-shaped extended over the bottom of the culture flask. The inhibitory effect of mifepristone on the cells was only expressed when the concentration of mifepristone is relatively high (10'6mol/L). The cell began to regression. Most of cells cultured in 10"4mol/L mifepristone became roundshape and died. a-SMA stain is positive. 3. Election microscocope shows: control group: The membranes of cells and nuclei were clear. The structure of membranes and cell organs were active. 10-6mol/L group: the cells were shrunk, membrane and chromosomes were resolved. 10-4mol/L group: the cells were edema and degenerated. The membrane of cells and nuclei were broken. Chromosomes became solid contraction and transformed. 4. MTT: the inhibitory effect of mifepristone on the cells was expressed when the concentration of mifepristone is 10-6mol/L .The inhibitory effect improved with the increase of concentration . 5.The expression ofER.PR.PCNA in the control group were higher than in the 10-6mol/L group and 10-4mol/L group. There was significant difference among 3 groups (P<0.05). The expression of VEGF. TGFB caspase3, bcl-2 and Fas in the control group and the 10-6mol/L group were lower than in 10-4mol/L group. There was significant difference among 3 groups (P<0.01). There was no expression of Fas and FasL in the control group and 10-6mol/L groups, There was weak expression in the 10-4mol/L group. The secretion of PRL in the control group was higher than in the 10-6mol/L group and 10-4mol/L group. There was significant difference among 3 groups (P<0.05). 6. There were no pominent apoptic peaks in 3 groups. Control group: there was no apoptic cell. G0/G1: 96.19%. S: 0.17%. G2/M: 3.64%; 10-6mol/L group:apoptic cells 3.18%, G0/G1: 94.61%. S: 2.19%. G2/M: 3.20%; 10-4mol/L group: apoptic cells 4.71%, G0/G1: 95.01%, S: 2.61%, G2/M: 2.38%Conclusion: 1.We can culture the human leiomyoma in vitro successfully if we use the optimal concentration of the collegenase and digestion time. 2. Mifepristione broke the struture of leiomyoma cells and interfere the membrane of cell and nuclei, organs of cell ,chromosome. 3. The inhibitory effect of mifepristone on human leiomyoma cells in vitro is concentration-dependent. 4. Mifepristonedown-regulated the expression of ER,PR,PCNA. The decrease of ER,PR in study group may be one of the important mechanisms of leiomyoma regression after treatment with mifepristione. 5. Mifepristone can inhibit the secretion of PRL of leiomyoma cells in vitro. Interfering the self-secretion may be another mechainism of leiomyoma regression after treatment with mifepristione. 6. Mifepristone up-regulated the expres...