ROCK抑制剂诱导条件性重编程颗粒细胞及其干化机制的研究

The incidence of premature ovarian insufficiency (POI) was increased quickly and women suffering from POI became younger. The low-estrogen related complications seriously affected women’s health. Moreover, there are also many limitations in the usage of traditional hormone replacement therapy regarding its safety and effectiveness. As a result, new treatment options are warranted.In contrast to current methods including iPSCs and ESC, conditional reprogramming is a new method to obtain deficient cells efficiently and safely. Conditional reprogrammed cell(CRC)has the pluripotency to differentiate into tissue specific cells but without stimulating tumors or immunologic rejection. In the previous study, we demonstrated a culture system, which consisted of F medium containing ROCK inhibitor Y27632 and irradiated mouse fibroblast cells, could successfully conditionally reprogrammed adult hepatic cells to an indefinite stem-like state. Moreover, once the ROCK inhibitor Y27632 was added into common culture medium, the growth pattern of primary human granulosa cells changed. However, translation of these findings to conditional reprogram human granulosa cells not yet been reported. In this study, we try to establish stable CRC from adult granulosa cells induced by ROCK inhibitor and further identify its stem-like phenotype, genetic stability, as well as its tumorigenicity. To explore the molecular mechanism for this reprogramming and finally observe its effect on ovarian function improving in a POI mouse model. Our study will provide evidence on cell replacement therapy in treating POI, which would open new therapeutic opportunities for POI patients.

卵巢早衰（POI）发病率逐年升高并有年轻化趋势，严重影响妇女身心健康。传统激素替代治疗在使用安全性、适用范围性及治疗有效性方面均存在局限，因此亟需寻求新的POI治疗方法。条件性重编程细胞(CRC)是一种比诱导性多潜能干细胞及胚胎干细胞更安全、高效的获得缺失细胞的新方法，CRC具有组织特异性的分化潜能而无免疫排斥和致瘤性。课题组前期成功构建了ROCK抑制剂和成纤维细胞为主的CRC培养体系，逆转干化获得了人和小鼠CRC肝细胞；并发现常规培养基中加入ROCK抑制剂可改变颗粒细胞生长状态，但目前尚无条件性重编程颗粒细胞的相关研究。本项目拟通过ROCK抑制剂体外诱导建立稳定的CRC颗粒细胞系，并对其干性、遗传稳定性及致瘤性进行鉴定；探索颗粒细胞向CRC逆转干化的分子机制，并体内观察CRC颗粒细胞对小鼠卵巢功能的修复作用，以期为临床获得安全、高效、便捷的颗粒细胞来源、自体细胞替代治疗POI奠定实验基础

近年来，随着肿瘤早期诊断率的提高以及各种新型化疗药物的上市，肿瘤患者的生存期得以显著延长，但化疗所致卵巢早衰的发生率逐年提高并有年轻化趋势。卵泡是卵巢的主要功能单位，而颗粒细胞又是卵泡中最大的细胞群, 是维持卵巢生殖及分泌功能的关键细胞。本课题成功从超排卵穿刺取卵后所剩余的卵泡液并分离获取人卵巢颗粒细胞，进行体外原代培养。发现，在J2细胞与颗粒细胞的共培养体系中加入10μmol/L ROCK 抑制剂Y27632后，颗粒细胞老化延缓，体外生长时间延长。此共培养条件下得到的颗粒细胞可进一步用于CRC颗粒细胞干性特征的鉴定。上皮性卵巢癌是常见的女性生殖系统肿瘤，死亡率居妇科肿瘤首位。深入研究上皮性卵巢癌的发生和进展、转移机制，对发现新的治疗靶点并改善预后有重要意义。SPTBN1作为接头蛋白参与TGF-β信号通路，招募并活化下游的Smads蛋白，从而参与细胞周期调控、DNA损伤修复、血管新生等。有文献报道，SPTBN1 在多种肿瘤中发挥了重要作用，但SPTBN1表达与上皮性卵巢癌临床分期及预后的关系，以及SPTBN1是否影响上皮性卵巢癌的恶性生物学行为及其具体机制尚未见报道。我们利用在线数据库分析SPTBN1与上皮性卵巢癌临床分期及预后的关系，并利用上皮性卵巢癌细胞系进行细胞学实验，探究了SPTBN1在上皮性卵巢癌增殖、迁移行为调控中的作用，并对其机制进行了深入研究。结果发现：随着卵巢癌分期进展，SPTBN1表达逐步降低；SPTBN1表达水平与卵巢癌PFS呈负相关，但是不影响总生存期。下调SPTBN1的表达可体内外促进卵巢癌细胞株增殖活力及迁移能力；SPTBN1表达下调后，上皮间质转化（EMT）相关蛋白中E-cadherin 表达降低而Vimentin、Slug、Snail等表达升高。而且，SPTBN1对上皮性卵巢癌细胞增殖及迁移的抑制作用是通过SOCS3抑制JAK/STAT信号通路介导的。并且在小鼠MEF 细胞中，SPTBN1亦可以调节SOCS3/JAK/STAT3通路及EMT相关蛋白的表达。我们的研究为上皮性卵巢癌的治疗提供了潜在的新靶点。

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