诱导性多潜能干细胞向卵巢颗粒细胞定向分化的分子机制

The incidence of premature ovarian insufficiency (POI) was gradually increased, and supplements of exogenous estrogen was the principal method to reduce the long-term complications. However, exogenous estrogen won't be regulated by hypothalamus-pituitary-gland gonad, and long-term clinical monitoring is needed to ensure the dose rationality and avoid the increasing risks of endometrial carcinoma and breast cancer. So looking for security and effective treatments are urgent to do. In the previous study, our research group obtained induced granulosa cell-like cells, which derived from mouse induced pluripotent stem cells (iPSCs) co-cultured with ovarian granulosa cells and had the endocrine secrete ability. But the mechanisms is still unknown. Our project propose to explore its molecular mechanism of granulosa cells differentiation from iPSCs, by analyzing granulosa cells differentiation condition and researching on key functional genes. And the induced granulosa cells phenotype will be idetified according to hormone secretion capacity, receptor expression and gene expression spectrum. Finally, the induced granulosa cells stimulated by FSH are transplanted to POI mice model to alleviate symptoms of low estrogen for a long-term interval, and the POI mice could get get stable estrogen level regulated by hypothalamus-pituitary-gland gonad.

卵巢早衰(POI)发病率日趋升高，其治疗主要靠外源性补充雌激素以降低患者远期并发症。然而，外源性补充雌激素并不受性腺轴调节，需长期临床监控，患者依从性差，可能增加子宫内膜癌、乳腺癌等肿瘤发病率，因此亟需寻求针对POI安全有效的治疗措施。课题组前期将小鼠诱导性多潜能干细胞(iPSCs)和小鼠颗粒细胞间接共培养，成功诱导iPSCs向颗粒细胞定向分化，并具有分泌雌激素的能力；但是iPSCs向颗粒细胞分化的分子机制尚不明确。故本项目拟通过分析研究颗粒细胞分化条件，选择分析关键功能基因，探究iPSCs向颗粒细胞定向分化的分子机制，并从激素分泌能力、受体表达、基因表达谱等系统鉴定诱导性颗粒细胞功能表型，进一步通过FSH作用训导出分泌功能可受调控的诱导性颗粒细胞，移植给POI小鼠模型，以达到稳定分泌雌激素并受机体性腺轴调节，长期稳定地缓解低雌激素症状的目的。

诱导性多潜能干细胞（induced pluripotent stem cells, iPSCs）可由自身体细胞通过重组获得，具有来源丰富、无伦理问题、无免疫排斥等优势，已成为临床多个学科研究疾病发病机制、筛选药物、治疗相关疾病的新型干细胞。近年来，早发性卵巢功能不全（premature ovarian insufficiency, POI）患者逐渐增多，发病率约为1%，而化疗药物是导致临床上POI发生的常见原因，其中环磷酰胺（Cyclophosphamide, CTX）是卵巢损伤较大的药物之一。环磷酰胺可损害颗粒细胞DNA及激活凋亡通路使卵巢颗粒细胞大量凋亡，继而引起卵泡闭锁，卵巢功能下降。研究证实iPSCs可分化为多种体细胞，具有治疗相关疾病的潜能，但直接移植治疗相关疾病可能存在成瘤问题。因此，将iPSCs体外分化为成熟颗粒细胞后再移植入POI小鼠体内，可能为POI的治疗提供新的思路。.本课题利用间接共培养体系诱导iPSCs向卵巢颗粒细胞分化，于共培养第7天发现诱导后细胞形态与颗粒细胞相似；表达前体颗粒细胞相关基因FOXL2、CTGF、LGR5、KITL、SMOC1，颗粒细胞相关基因FSHR、AMHR、LHR、CYP19A1；表达颗粒细胞标记蛋白FSHR；具有分泌雌激素的能力，且随培养时间延长，诱导后细胞分泌雌激素逐渐升高。由此，体外证实iPSCs可诱导分化为具有颗粒细胞形态特征、相关基因水平、蛋白水平及分泌E2功能的颗粒样细胞（granulosa cells like-cells, GCL）。进一步采用腹腔注射环磷酰胺建立POI小鼠模型，发现GCL移植具有改善POI小鼠AMH、FSH水平及促进各级生长卵泡发育的功能，验证颗粒样细胞的体内功能。进而通过对iPSCs、小鼠卵巢颗粒细胞、颗粒样细胞培养上清液进行质谱检测，分析上清液蛋白组分，初步探索iPSCs向颗粒细胞分化的可能作用分子。.综上所述，本研究利用共培养技术诱导iPSCs向颗粒细胞定向分化，获得诱导性颗粒样细胞，并从体外、体内两方面鉴定颗粒样细胞的特性及功能，研究GCL治疗POI小鼠的可能性，并通过质谱分析初步探索iPSCs向颗粒细胞分化的可能作用分子，为iPSCs治疗POI提供新的实验基础。