褪黑素对环磷酰胺所致卵巢早衰中原始卵泡过度激活的抑制作用及机制研究

Chemotherapy is the cause of excessive activation of primordial follicle, which leads to premature ovarian failure. However, how to protect the fertility of tumor patients is a new challenge in the field of oncofertility. As an anti-oxidant and anticancer drug, melatonin is considered as a potential chemotherapy adjuvant. Our previous results showed that melatonin obviously protected the number of primordial follicles, reduces the apoptosis of granulosa cell, and maintain the level of serum anti muller's tube hormone (AMH) after cyclophosphamide chemotherapy. Therefore, we hypothesized that melatonin inhibited the activation of primordial follicles through inhibiting of granulosa cell apoptosis and PTEN/AKT/FOXO3a pathway in premature ovarian failure induced by cyclophosphamide chemotherapy. So, we firstly will study the role of P53 pathway, mitochondrial apoptosis pathway, sirt1 pathway and PTEN pathway in inhibiting the excessive activation of primary follicles in the process of cyclophosphamide chemotherapy with melatonin intervention. Secondly, the inhibition mechanism of apoptosis in granular cells will be study by techniques of protein sequencing and CRISPR/Cas9, et. al. Finally, the effect of AMH on the regulation of melatonin function will be studied by ovarian in situ AMH over-expression. Our study will clarify the mechanism of melatonin on inhibition of excessive activation of primordial follicles induced by cyclophosphamide chemotherapy, and will provide the basis for the adjuvant selection and gene therapy for fertility preservation of female cancer patients during chemotherapy.

化疗过程中原始卵泡过度激活导致卵巢早衰，而如何保护卵巢储备是肿瘤生育领域一个难题。褪黑素作为一种抗氧化剂具有卵泡保护作用，同时兼抗癌作用，被认为是一种潜在的化疗辅助治疗药物。我们前期研究结果显示：环磷酰胺化疗后褪黑素干预明显保护了原始卵泡数量，降低了颗粒细胞凋亡和维持了血清抗穆勒氏管激素（AMH）水平。因此假设：褪黑素通过抗颗粒细胞凋亡维持较高AMH及抑制PTEN/AKT/FOXO3a信号通路抑制了环磷酰胺化疗所致原始卵泡过度激活。故拟在环磷酰胺化疗过程中进行褪黑素干预研究P53通路、线粒体凋亡通路、sirt1通路、PTEN通路等在抑制原始卵泡过度激活中的作用；采用蛋白组测序、CRISPR/Cas9等技术研究颗粒细胞抑凋亡机制；采用卵巢原位过表达研究AMH对褪黑素级联调控作用，最终明确褪黑素对环磷酰胺诱发原始卵泡过渡激活抑制作用及机制，为女性肿瘤患者生育力保存药物选择和基因治疗提供依据。

褪黑素是一种潜在的女性肿瘤患者生育力保存的化疗辅助治疗药物，但其对环磷酰胺化疗的辅助治疗作用与机制不清，本研究发现：①褪黑素干预（腹腔注射，50mg /kg /day，连续干预14天）抑制小鼠环磷酰胺化疗所致原始卵泡过度激活，维持了化疗后原始卵泡数量和生育能力。环磷酰胺化疗后14d小鼠卵巢中原始卵泡数量显著减少，而初级卵泡数量显著增多，经褪黑素干预后小鼠原始卵泡数量显著增多，初级卵泡数量却显著减少。化疗后第115天至170天本交期内（持续55天），环磷酰胺化疗小鼠平均产仔数和累计产仔数都显著减少，经褪黑素干预后这种下降趋势都得到显著抑制；②褪黑素干预维持环磷酰胺化疗后小鼠卵巢颗粒细胞AMH的表达，通过AMH介导的间接途径抑制原始卵泡过度激活。环磷酰胺化疗后14d小鼠血清AMH水平显著下降，褪黑素干预后AMH水平下降趋势显著逆转。采用慢病毒卵巢原位转染法过表达AMH能够显著提高环磷酰胺化疗后小鼠血清AMH水平和维持卵巢原始卵泡数量。环磷酰胺化疗和褪黑素干预都对原始卵泡直接激活通路PTEN/AKT/FOXO3a中关键蛋白磷酸化水平没有影响；③褪黑素干预抑制环磷酰胺化疗后颗粒细胞凋亡。环磷酰胺化疗促进卵巢颗粒细胞凋亡，凋亡高峰出现在化疗后12h。褪黑素干预后显著降低了环磷酰胺化疗后12h生长卵泡TUNEL阳性的凋亡颗粒细胞数量；④褪黑素干预通过线粒体凋亡途径抑制环磷酰胺诱导的颗粒细胞凋亡。环磷酰胺化疗后显著上调了线粒体凋亡通路关键蛋白cleaved-Caspase3和Bax的表达，促进细胞色素C从线粒体释放入胞质中，而褪黑素干预后可显著抑制cleaved-Caspase3、Bax的表达和细胞色素C的释放。综上所述，褪黑素作为环磷酰胺化疗辅助治疗药物，其主要作用机制是抑制环磷酰胺化疗后卵巢颗粒细胞凋亡和维持颗粒细胞AMH表达，防止化疗所致原始卵泡过度激活，从而维持化疗后小鼠生育能力。