| Objective:1. This study was designed to set up the method of separating mesenchymal stem cells from umbilical cord, and the biological properties and phenotypic character-istics in vitro.2. Simulation of the chemotherapy-induced granulosa cells damage, and then co-culture with UC-MSCs to study if UC-MSCs could repair the chemotherapy-ind-uced damage of ovaries.Methods:1. In sterile condition, obtain the umbilical cord samples from healthy full-term fetus and apply double enzymatic method, adherent method to separate and purify umbilical cord mesenchymal stem cells. Make a contrast during these two methods. UC-MSCs are cultivated with 10% fetal bovine serum (FBS) in DME-M/F12 medium.2. Patients accept IVF-ET from reproduction center of the first affiliated hospital of Sun Yat-sen, ageing 27-25, having evidences of ovulation and normal reproductive hormone in basic state because of the uterine tube diseases and spouses' abnormality. The ovarian granulosa cells from the follicular fluid were separated and cultured in vitro when collected ovarian follicle through type-B ultrasonic. Add various concentrations of chlormethine(0.5mg/l,2.5mg/l,5mg/l) for 24h to induce apoptosis, then chose the optimal concentration by apoptotic detection. Two groups were divided, one group was that the granulosa cells were co-cultured with UC-MSCs for 48h at different ratio of GCs/UC-MSCs. The other was that the granulose cells were co-cultured with UC-MSCs for 6h,then optimal concentration of chlormethine was added. The apoptosis rates were detected by Hoechst33342-PI staining, DNA-ladder. The expressions of Bcl-2 and Caspase-3 in ovarian gramulosa cells were tested by Western-blot method. Results:1. HUC-MSCs cultured in vitro were morphologically resembled fibroblast.97 percent of the third passage of HUC-MSCs expressed CD44,CD29,CD59,CD105,but negative for markers of the hematopoietic and endodermis lineages including CD28,CD31,CD34,CD45, and the immunologic rejection fos-related antigen including CD40,CD86,HLA-DR, which were indentified as MSCs.2. Most of the HUC-MSCs were in still and DNA presynthetic phase(G0/G1 phase),and only few were in mitotic phase and DNA synthetic phase.3. At the Chlormethine concentration of 5mg/l, the proliferation of ovarian granulosa cells was inhibited obviously, and apoptotic ratio elevated, and typical DNA ladder was displayed on agarose gel eletrophoresis.4. The effect of UC-MSCs co-culture in vitro to repair the chemotherapy-induced ovarian damage was not so ideal, but the effect of prevention had statistical difference with control group. the granulosa cells were co-cultured with UC-MSCs for 6h then optimal concentration of Chlormenthine was added, and kept the station of co-culture with UC-MSCs for 72h, the apoptotic ratio was lower than negative control group(co-culture:early apoptotic ratio was 22.43±0.45%,late apoptotic ratio was 13.57±1.58%;negative control group:early apoptotic ratio was 25.23±1.07%, late apoptotic ratio was 29.57±2.74%). There was statistic difference between these two groups(P=0.000).5. The results of Western-blot displayed that the GCs co-cultured with MSCs expressed higher level of bcl-2, and lower level of caspase-3.Conclusions:HUC-MSCs could effectively prevent the chemotherapy-induced ovarian damage. The probable potential mechanism is up-regulation the expression of bcl-2 and down-regulation the expressed of caspade-3, and then inhibit the apoptosis.
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