南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (12): 1850-1856.doi: 10.12122/j.issn.1673-4254.2021.12.14

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GnRH激动剂和经血源性干细胞联合治疗对小鼠卵巢功能的影响

孙 芳，韦 伟   

  1. 绍兴第二医院医共体总院药剂科，浙江 绍兴 312000；浙江大学医学院附属邵逸夫医院妇产科，浙江 杭州 310000
  • 出版日期:2021-12-20 发布日期:2022-01-05

Effect of GnRH agonist combined with menstrual blood-derived stem cell transplantation on ovarian function in mice

SUN Fang, WEI Wei   

  1. Department of Pharmacy, General Hospital of Shaoxing Second Hospital, Shaoxing 312000, China; Department of Obstetrics and Gynecology, Run Run Shaw Hospital Affiliated to Zhejiang University School of Medicine, Hangzhou 310000, China
  • Online:2021-12-20 Published:2022-01-05

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摘要： 目的 探究促性腺激素释放激素（GnRH）激动剂和经血源性干细胞（MenSCs）联合治疗对小鼠卵巢功能的影响。方法 将45只SPF级实验小鼠随机分为Control组（腹腔注射生理盐水）、POF组（腹腔注射120 mg·kg-1·d-1 CTX）、MenSC+POF组（CTX损伤处理后注射移植含2×106 MenSCs的细胞悬浮液）、GnRHa+POF组（造模前14 d注射丙氨瑞林0.1 mg·kg-1·d-1，第15天开始CTX注射）、MenSC+POF+GnRHa组（按MenSC+CTX组和GnRHa+CTX组方案联合用药），每组9只。HE染色观察小鼠卵巢组织病理变化情况，ELISA法检测小鼠血清中黄体生成素（LH）、卵泡刺激素（FSH）、抗苗勒管激素（AMH）和雌二醇（E2）的含量水平，Western blot检测小鼠卵巢颗粒细胞中KI-67、Bcl-2、BAX、caspase 9、caspase 3、cleaved-caspase 9、cleaved-caspase 3、p-PI3K、PI3K、p-Akt及Akt蛋白的表达情况。结果 与Control组相比，POF组小鼠卵巢纤维化明显，闭锁卵泡数增多，血清中LH和FSH的含量水平极升高（P<0.01），AMH和E2含量水平降低（P<0.01），BAX、cleaved-caspase 9/caspase 9、cleaved-caspase 3/caspase 3蛋白表达水平升高（P<0.01），KI-67、Bcl-2、p-PI3K、p-Akt蛋白的表达水平降低（P<0.01）；与POF组比较，GnRH激动剂和经血源性干细胞联合治疗可抑制小鼠卵巢闭锁卵泡的产生，降低小鼠血清中LH和FSH的含量水平（P<0.01），提高AMH和 E2的含量水平（P<0.05或P<0.01），抑制小鼠卵巢颗粒细胞中BAX、cleaved-caspase 9、cleaved-caspase 3蛋白的表达（P<0.05或 P<0.01），促进KI-67、Bcl-2、p-PI3K、p-Akt蛋白的表达（P<0.05或P<0.01）。结论 GnRH激动剂和经血源性干细胞联合治疗可能通过上调PI3K-Akt信号通路表达，减少卵巢闭锁卵泡的产生，提高小鼠卵巢的储备功能，从而达到对卵巢修复和保护的目的。

关键词: 促性腺激素释放激素激动剂；卵巢早衰；经血源性间充质干细胞；凋亡；激素水平

Abstract: Objective To investigate the effect of combined treatment with gonadotropin-releasing hormone (GnRH) agonist and menstrual blood-derived stem cell (MenSC) transplantation on ovarian function in mice. Methods We tested the effect of GnRH agonist and MenSC transplantation, either alone or in combination, in a mouse model of premature ovarian failure (POF) induced by daily intraperitoneal injection of 120 mg/kg cyclophosphamide (CTX) for 15 days. Venirelin was administered at the daily dose of 0.1 mg via intravenous infusion, starting at 14 days before modeling; MenSC suspension (2×106 cells) was injected into the ovarium 1 day after modeling. HE staining was used to observe ovarian pathologies of the mice. ELISA was used to detect serum levels of LH, FSH, AMH and E2. The expression levels of ki-67, Bcl-2, BAX, caspase 9, caspase 3, their cleavages, P-PI3K, PI3K, P-Akt and Akt proteins in ovarian granulosa cells were determined with Western blotting. Results The mouse models of POF showed obvious ovarian fibrosis with increased atresia follicles and had significantly elevated serum levels of LH and FSH (P<0.01), decreased serum levels of AMH and E2 (P<0.01), significantly increased ovarian expression levels of caspase 9, caspase 3, and their cleavages (P<0.01), and decreased expression levels of Ki-67, Bcl-2, P-PI3K and P-Akt (P<0.01). The combined treatment with GnRH agonist and MenSC transplantation obviously inhibited the production of ovarian atresia follicles, lowered serum levels of LH and FSH (P<0.01), and increased the levels of AMH and E2 (P<0.05 or 0.01) in the mouse models. The combined treatment also inhibited the expressions of BAX and cleaved caspase 9 and caspase 3 proteins (P<0.05 or 0.01) and enhanced the expressions of Ki-67, Bcl-2, P-PI3K, and p-Akt proteins (P<0.05 or 0.01) in ovarian granulococcus cells. Conclusion In mouse models of POF, GnRH agonist combined with MenSC transplantation can reduce the generation of ovarian atresia and improve ovarian reserve by up-regulating PI3K-Akt signaling pathway to achieve ovarian repair and protection.

Key words: gonadotropin-releasing hormone agonist; premature ovarian failure; menstrual blood-derived stem cells; apoptosis; hormone levels