木尼孜其血清对大鼠卵巢早衰颗粒细胞增殖的影响及其机制

Effect of Muniziqi serum on proliferation of ovarian granulosa cells in rats with premature ovarian failure and its mechanism

目的观察木尼孜其血清对大鼠卵巢早衰(POF)颗粒细胞增殖的影响,并探讨其可能作用机制。方法(1)木尼孜其血清制备50只雌性SD大鼠随机分为5组,每组10只。空白对照每天灌胃生理盐水2 mL/次;阳性对照组(戊酸雌二醇组)每天灌服戊酸雌二醇0.15 mg/kg;木尼孜其低、中及高剂量组分别用0.9、1.8、3.6 mL的木尼孜其合剂灌胃,2次/天,连续灌胃4 d,第5天早灌入2次剂量,灌胃1 h取各组大鼠,股动脉采血,分离血清。(2)大鼠卵巢早衰(POF)颗粒细胞制备取幼年SD大鼠,腹腔皮下注射孕马血清促性腺激素(40 U/只)48 h,处死大鼠分离卵巢,培养大鼠卵巢颗粒细胞(HE染色和免疫细胞化学法鉴定为大鼠卵巢颗粒细胞),将细胞随机分为正常组、顺铂模型组、阳性对照组、低剂量组、中剂量组及高剂量组。除正常组外其余5组加入终浓度为5μg/mL的顺铂制备POF。(3)木尼孜其合剂血清给予方法及其对增殖的影响观察正常组加入无血清DMEM/F-12培养基,阳性对照组加入含有戊酸雌二醇血清,低、中、高剂量组分别加入0.9、1.8、3.6 mL的木尼孜其合血清。分别于培养24、48、72 h,取各组细胞MTT法观察细胞增殖情况,取细胞培养液ELISA法检测抗苗勒氏激素(AMH)、雌二醇(E2)及孕酮(P)。结果与正常组比较,培养24、48、72 h时模型组细胞OD值降低(P均<0.05);与模型组比较,培养48 h高剂量组细胞OD值降低(P<0.05);与培养24 h比较,模型组、阳性对照组、低剂量组、中剂量组及高剂量组细胞OD值降低(P均<0.05)。与正常组比较,模型组培养液AMH、E2、P水平降低(P均<0.05);与模型组相比较,阳性对照组、低剂量组、中及高剂量组培养液AMH水平升高,高剂量组培养液E2水平升高,中、高剂量组培养液P水平升高(P均<0.05)。结论木尼孜其血清可促进大鼠POF颗粒细胞的增殖,其机制可能为木尼孜其可促进POF卵巢颗粒细胞AMH、E2、P分泌。

Objective with premature ovarian failure(POF),and to explore its possible mechanism.Methods rum Fifty female SD rats were randomly divided into five groups with 10 rats in each group.The rats in the blank control group were intragastrically given normal saline 2 mL/time every day,the rats in the positive control group(estradiol valerate group)were given estradiol valerate 0.15 mg/kg daily,the rats in the low-dose,medium-dose and high-dose groups were intragastrically administrated 0.9,1.8 and 3.6 mL Muniziqi mixture,twice a day,for 4 days.On the fifth day,two doses were intragastrically administrated for 1 h,the blood was collected from the femoral artery,and serum was separated.(2)Preparation of POF granulosa cells in rats The young SD rats were intraperitoneally injected with pregnant mare serum gonadotropin(PMSG)(40 U/rat)for 48 h.Rats were sacrificed and their ovaries were separated.Rat ovarian granulocytes were cultured and identified as granulocytes by HE staining and immunocytochemistry,the cells were randomly divided into the normal group,cisplatin model group,positive control group,low-dose group,medium-dose group,and highdose group.Except the normal group,the rats in the other five groups were added with cisplatin at a final concentration of 5μg/mL to prepare POF.(3)Observation on the administration method of Muniziqi mixture serum and its effect on proliferation The normal group was added with DMEM/F-12 medium without fetal bovine serum,the normal group with DMEM/F-12 medium without fetal bovine serum,and the low-dose group,medium-dose group and high-dose group with 0.9,1.8 and 3.6 mL Muniziqi serum.At 24,48,and 72 h,the cell proliferation of each group was observed by MTT colorimetry,and anti-Mullerian hormone(AMH),estradiol(E2)and progesterone(P)in cell culture medium were detected by ELISA.Results after culture(all P<0.05).Compared with the model group,OD value of cells in the high-dose group decreased at 48 h after culture(P<0.05).The OD values of the model group,positive control group,low-dose group,medium-dose group and high-dose group decreased as compared with those at 24 h after culture(all P<0.05).Compared with the normal group,the levels of AMH,E2,and P in culture medium of the model group decreased(all P<0.05).Compared with the model group,the level of AMH in the culture medium increased in the positive control group,low-dose group,medium-dose group,and high-dose group,the level of E2 increased in the high-dose group,and the P level of the medium-dose and high-dose groups increased(all P<0.05).Conclusion mechanism may be that Muniziqi can promote the secretion of AMH,E2 and P in POF ovarian granulosa cells.