无透明带核移植胚胎干细胞系移植治疗帕金森病分子机制的比较研究

In this study, we combine both Zona-Free Somatic Cell Nuclear Transfer and embryonic stem cell technology advantages. Wuzhishan miniature pig somatic cells will be used as donor cells and Huainan pig oocytes as cytoplasmic receptors, an important native pig breeds in Anhui Province. Firstly, Zona pellucida will be digested using pronase, and removing the nucleus through the Demecolcine (DM) auxiliary and DM-induced trans- nuclear cloning to zona-free nuclear transfer. The effects of zona-free nuclear transfer for embryonic development will be studied by the detection of Morphology of reconstructed embryos, blastocyst rate and embryo apoptosis. Secondly, embryoic stem cell is cultured and ESc cell line is established using a large number of zona-free nuclear transfer blastocyst through naked embryo culture and whole embryo culture methods. And ESc pluripotency are tested using surface antigen, differentiation characteristics, telomerase activity and tumorigenicity experiment in vivo animal model. On the other hand, used the ES cells and ES cells induced NSC cells as the effective transplanting donor, and transplanted the neural stem cell, ES cells, ES cells induced NSC cells into striatum of Parkinson's Wuzhishan miniature pig separately, observed the live, differentiation DA and migration condition, evaluate the transplanting efficacy of theses cells, choose the best transplanting donor and clinical and pathological animal model, discuss the feasibility of the clinical application of ES cells transplantation in the treatment of Parkinson's disease, and provide theoretical and practical basis, which could significantly promote the researches in this area.

本研究结合无透明带核移植与胚胎干细胞培养两方面的优势技术，以五指山猪体细胞作为供体细胞，安徽省重要地方猪种-淮南猪卵母细胞为胞质受体，采用链霉蛋白酶消化透明带，通过DM辅助切割去核及DM诱导反式克隆去核进行无透明带核移植，并对重构胚形态学、囊胚率及胚胎凋亡情况来比较无透明带技术对胚胎发育力的影响；并对获得的大量囊胚，采用裸胚培养法与全胚培养法进行ESc的培育及建系研究，并进行表面抗原、分化特性、端粒酶活性等特征检测以及模式动物体内致瘤实验，检测其全能性。另一方面甄选有效的ES诱导分化NSC移植供体，通过移植帕金森五指山猪纹状体后在宿主脑内的存活、分化及迁移情况，客观的评价两种干细胞的移植功效，探讨ES细胞移植治疗帕金森病的可行性以及为人ES的临床应用提供重要参考数据，建立较完善的猪ES建系、诱导NSC与移植治疗临床应用鉴定理论与实践基础，提升我国本领域的研究具有极其重要意义。

本研究以构建22 个个体的五指山小型猪胚胎成纤维细胞系为供体细胞，淮南猪卵母细胞为胞质受体，对猪无透明带核移植技术体系初步进行建立与优化，并对透明带消化、半卵切割去核、供体细胞与半卵融合参数、核移植前后甲基化、乙酰化研究，无透明带胚胎培养的方式进行较为系统研究；初步探索无透明带核移植囊胚中可以分离类ESCs 的可行性，获得两个原代ESCs 集落，AKP 染色呈阳性，但传代后消失，说明无透明带技术用于胚胎干细胞建系具有可行性，但猪的胚胎干细胞建系影响因素较多需要进一步探索；组合使用6-轻多巴胺(6-OHDA)单侧黑质毁损术技术，结合MPTP毁损模型制作6只帕金森猪实验动物模型与40余只帕金森病SD大鼠模型，并对造模成功的PD大鼠开展行为学评估，通过立体定向技术下移植iNSC与NSC治疗PD实验动物模型，观察其在宿主脑内的存活、分化及迁移情况，确定对帕金森动物模型症状具有明显的修复效果；同时构建了五指山小型猪骨髓间充质干细胞系（BMSC）1个、肺间充质干细胞系（PMSC）1个、神经干细胞系（NSC）1个； BMSC-iPSCs与NSC-iPSCs，iPSCs- iNSC各1个，并对Melatonin、VPA、Reversine与Repsox等小分子化合物对于成纤维细胞重编程机制进行探析，本研究对于诱导分化NSC机制与移植治疗PD临床应用鉴定理论与实践基础。