MB-MSCs May Protect The Epirubicin-induced Granulose Cells In Vitro

Objective: GCs were isolated from donor and to explore the mechanisms of epirubicin-induced damage to human ovarian granulosa cells(GCs); MB-MSCs isolate from human menstrual blood, and amplification and identification in vitro; To explore MB-MSCs effects on epirubicin-induced damage to human ovarian granulosa cells(GCs), MB-MSCs co-culture with epirubicin-induced apoptosis of GCs, and provide evidence for further study.Methods:GCs and MB-MSCs were isolated from donor using density centrifugation method; FSHR expression was examined by immunohistochemical staining method; cell immunological phenotype was examined by flow cytometry analysis; the adipogenic, osteogenic, and chondrogenic differentiation of MSCs were examined by Oil-Red-O staining, ALP staining, and Aldrich staining; and the secretion of cytokines, including vascular endothelial growth factor(VEGF), hepatocyte growth factor(HGF) and insulin-like growth factor-1(IGF-1), was detected using enzyme-linked immunosorbent assay. To discuss their effects on epirubicin-induced damage to human ovarian granulosa cells(GCs); GCs were cultured alone(Control group) or co-cultured with 0.025 mg/ml of epirubicin(G+E group) or 0.025 mg/ml of epirubicin and MB-MSCs(G+E+M group) in a transwell that allowed the passage of cytokines but impeded the passage of MB-MSCs. The morphology and growth of GCs were observed under an inverted microscope. The apoptosis of GCs was determined using Annexin V and 7-AAD double staining, and the levels of estradiol(E2), progestin(P), anti-Mullerian hormone(AMH), inhibin A, and inhibin B were determined using enzyme-linked immunosorbent assay(ELISA). To detect MB-MSCs may secrete a number of factors affect the function of GCs, the levels of vascular endothelial growth factor(VEGF), insulin like growth factor-1(IGF-1), and hepatocyte growth factor(HGF) were determined using ELISA kits.Results: GCs and MB-MSCs were isolated and cultured by density gradient centrifugation successful; GCs were expression FSHR; MB-MSCs were positive for CD73, CD105, CD29 and CD44, but negative for CD31 and CD45. The differentiated cells MB-MSCs were positive for ALP staining, Oil-Red-O staining, and Aldrich staining. MB-MSCs can secreted VEGF(4.927 ±0.890 ng/ml), HGF(122.953± 4.377 pg/ml), and IGF-1(1.77 ± 0.027ng/ml); Epirubicin-induced damage to ovarian GCs apoptosis. Compared with the control group, the apoptosis of GCs increased from 4.58%(control group) to 36.14%(G+E group), when co-cultured with 0.025 mg/L of epirubicin and MB-MSCs, However, the apoptosis of GCs was increased to 27.24%, and cell viability was increased to71.7%. Early apoptosis of GCs were(32.40±7.21%)、Late apoptosis of GCs were(16.61±8.11), showed statistically significant differences compared with the control group of early apoptotic rate(2.18±2.042%)late apoptotic rate(16.61±8.11%)(p< 0.05); ELISA analysis showed that GCs secrete hormones E2, P, AMH, inhibin A, inhibition of B was significantly reduced compared with the control group, And after co-cultured with MB-MSCs, GCs secrete the hormone levels were significantly increased above.(P <0.05).Conclusions: MB-MSCs can partially inhibit the apoptosis of GCs induced by epirubicin, probably by the secretion of anti-apoptosis cytokines such as VEGF, HGF, and IGF-1.