Wnt信号通路调控脂肪间充质干细胞向神经元样细胞分化在盆底神经损伤修复中的作用和机制研究

Neuropathy of pelvic supportive structure has been the focus of the etiology study in pelvic organ prolaps(POP) these years. Our previous study supported by the National Nature Science Funds Key Program had proved the fact that wnt signaling pathway contributed to a neurodegenerative role in POP development，but the mechanism still requires well defined. Research findings have raised promising hope for the development of stem cell transplantation in human neurodegenerative disorders and also evidences have shown that Wnt signaling pathway is closely involved in neurogenesis. In our study, first we intend to assess the role of Wnt signaling pathway in the differentiation of adipose-derived mesenchymal stem cells(ADSCs) to neuron-like cells in vitro using the RNAi technology, RT-PCR, Western blot and flow cytometry technique. Secondly, a biodegradable polymer PLLA/PLGA will be used as the carrier of the ADSCs, which will be transplanted into the subepithelial space of sheep's vagina along with the sustained-release microspheres containing regulating factors of Wnt signaling pathway. With this research, we expect to investigate the effect of Wnt signaling pathway on the differentiation of the transplanted stem cells in vivo, and also the potential capability in neural repair contributed to the restoration of vaginal biological function. Besides, we anticipated to assess the feasibility of the preparation of sustained-release microspheres of pathway, as well as the biodegradable ploymer PLLA/PLGA as the reliable carrier. In conclusion, this is the first cell signaling research on neuropathic etiology of POP, and is highly expected to shed new light on the therapeutic approach of regenerative medicine in POP regarding the source of stem cells, the technique approach of stem cell transplantation and the in vivo modulation of transplanted cells.

盆腔器官脱垂（POP）盆底支持结构的神经病变是近年的研究热点，前期研究在国家自然科学基金重点项目资助下证实Wnt信号通路相关的神经损伤及退行性变与该病密切相关，但确切机制不明。基于干细胞移植在神经损伤和退行性疾病中的应用，及Wnt信号通路在神经系统发育中的关键作用。本实验先在体外研究Wnt信号通路在脂肪间充质干细胞（ADSCs）向神经元样细胞分化过程中的作用和机制；再用可降解生物材料PLLA/PLGA共聚物为移植载体，将Wnt信号通路调控因子制成缓释微球和ADSCs一起移植入绵羊阴道上皮内，在体内研究信号通路对干细胞定向分化的影响，帮助修复POP神经损伤，重塑阴道生物学功能的作用，探讨信号通路调控因子缓释微球技术作为干细胞载体移植技术的可行性。本课题首次在分子信号转导层面研究POP神经病理学发病机制，将在干细胞来源、移植方法、移植后调控等方面为POP发病机制研究及治疗提供新思路和突破点。

神经损伤机制是近年来盆腔脏器脱垂（POP）病因学研究热点之一。随着以间充质干细胞为主的再生医学在神经损伤及神经退行性疾病中的广泛应用，将间充质干细胞横向诱导分化产生神经干细胞用以修复盆底神经损伤进而改善盆底生物学功能，将会是盆底修复医学中新的亮点和突破点。基于此设想，本研究首先利用小分子法诱导间充质干细胞横向分化为神经干细胞，并通过双侧阴部神经阻断法建立大鼠盆底支持结构神经损伤模型，最终采用多种方案多种途径移植干细胞，以验证对神经损伤的修复作用。实验结果显示，（1）采用九种小分子诱导的方法，成功把脂肪间充质干细胞体外横向诱导分化为神经干细胞，qRT-PCR法、Western Blot及细胞荧光发现在诱导过程中，神经干细胞相关蛋白Sox2、Nestin、Vimentin、Olig2和Ascl1的表达明显升高。（2）采用双侧阴部神经阻断的方法制备阴道前壁神经损伤的动物模型，双侧阴部神经损伤组动物阴道前壁神经纤维数量明显降低，神经标记蛋白UCHL的mRNA的表达量明显降低。（3）通过干细胞对大鼠阴道前壁神经损伤模型的神经修复研究得出，脂肪间充质干细胞可促进大鼠阴道前壁神经损伤模型的神经修复，神经纤维的含量及神经相关蛋白的表达明显升高。负载于细胞支架的脂肪间充质干细胞神经修复的效果好于注射间充质干细胞组，且可能更早地发生修复作用。三个移植细胞组术后1月阴道前壁神经干细胞及早期神经细胞的含量均增加，并且移植间充质干细胞和神经干细胞混合组神经干细胞及早期神经细胞的含量增加最显著。本研究建立了脂肪间充质干细胞横向诱导分化为神经干细胞的方法，首次评估了双侧阴部神经阻断法对阴道前壁神经的损伤作用，及作为盆底神经损伤动物模型建立的可能，并初步证实了干细胞在阴道前壁神经损伤中的修复作用，为今后潜在的临床应用价值提供了依据。基于本课题，已发表核心论文2篇，待发表SCI论文两篇。