促性腺激素释放激素类似物缓解环磷酰胺导致卵巢损伤的机制研究

The Mechanism of Gonadotropin-releasing Hormone Analogs to Relieve Ovarian Injury Induced by Cyclophosphamide

目的:通过动物实验和细胞实验初步探索促性腺激素释放激素类似物在环磷酰胺暴露下保护卵巢功能的机制。方法:构建乳腺癌荷瘤小鼠36只,随机分成Control组,CTX组和CG(CTX+GnRHa)组,药物干预后麻醉下剥离肿瘤和子宫卵巢并称重,计数卵巢原始卵泡和生长卵泡数目,提卵巢蛋白进行Western blot检测抗缪勒氏管激素(Anti-Müllerian hormone,AMH)蛋白表达量,提取卵巢RNA进行qRT-PCR实验检测mRNA水平,心尖取血检测血清AMH水平。卵巢颗粒细胞加药处理36小时后,收细胞蛋白进行Western blot检测AMH蛋白表达量,提细胞RNA进行qRT-PCR检测mRNA水平,ELISA法检测细胞培养基上清中AMH水平。结果:成瘤后第21天,CTX组和CG组小鼠肿瘤质量无统计学差异(P>0.05)且均小于Control组(P<0.05);CTX组子宫和卵巢的总重量均显著低于Control组和CG组(P<0.01),卵巢内原始卵泡数量均低于Control组和CG组(P<0.001),生长卵泡数量无统计学差异(P>0.05)。动物实验,Western blot结果提示,CTX组卵巢AMH蛋白含量显著高于Control组或CG组(P<0.05);由ELISA结果提示,CTX组血清AMH蛋白浓度显著低于CG组(P<0.01)。细胞实验,由Western blot可见,CTX组AMH蛋白含量显著高于Control组和CG组(P<0.05);由ELISA实验可见,CG 750组和CG1000组培养基上清的AMH蛋白浓度均高于对应CTX剂量组(P<0.01)。无论在动物实验还是细胞实验中,各组AMH的mRNA表达水平均无统计学差异(P>0.05)。结论:在CTX化疗的同时运用GnRHa,可以在不干扰化疗疗效的前提下,通过减少CTX所致细胞内AMH潴留程度,增加细胞外和血清中的AMH浓度从而发挥保护卵巢储备的功能。

Objective:To explore the mechanism of gonadotropin-releasing hormone analogs(GnRHa)on protecting ovarian function during cyclophosphamide(CTX)exposure via animal and cell experiments.Methods:Thirty six breast cancer tumor-bearing mice were randomly divided into the Control group,CTX group,and CG(CTX+GnRHa)group.After intervention,the tumor,uterine,and ovary were removed form mice and weighed under anesthesia.The number of primordial follicles and growing follicles in ovary were counted.The protein of the ovary was extracted to detect the protein expression of AMH by Western blot.The RNA of ovary was purified to detect the mRNA level of AMH by qRT-PCR assay.Then the blood of the heart apical was collected to detect the serum AMH level.Meanwhile,after 36 hours of treatment of ovarian granulosa cells,the cells were harvested for Western blot and qRT-PCR to detect AMH protein expression and mRNA level.ELISA assay was applied to detect AMH level in the cell culture supernatant.Results:On the 21st day after subcutaneous tumor formation,there was no significant difference in tumor mass from mice between the CTX group and the CG group(P>0.05),and both of them were smaller than the Control group(P<0.05).The weight of uterus and ovaries in the CTX group was significantly lower than Control group and CG group(P<0.01).Compared with the Control group and the CG group,the number of primordial follicles in the CTX group was significantly lower(P<0.001).There was no difference in the number of growing follicles between three group(P>0.05).In animal experiments,Western blot showed that the content of AMH protein in ovary of CTX group was significantly higher than that of Control group or CG group(P<0.05).The result of ELISA assay indicated that the concentration of AMH protein in serum of CTX group was significantly lower than that of CG group(P<0.01).In cell experiments,Western blot revealed that the content of AMH protein in CTX group was significantly higher than that in Control group and CG group(P<0.05).The result of ELISA assay indicated that the concentration of AMH protein in the medium supernatant of CG 750 and CG1000 groups was higher than that of CTX group(P<0.01).No matter in animal experiments or cell experiments,the mRNA expression levels of AMH in all the groups did not change significantly.Conclusion:The application of GnRHa with CTX can protect ovarian reserve by reducing the level of AMH retained in the cells caused by CTX,and increasing the concentration of AMH outside the KGN cells and in the serum without interfering with the efficacy of chemotherapy.