人胚胎脉络丛上皮细胞移植介导脑出血后神经再生作用及机制的研究

Intracerebral Hemorrhage (ICH) is a common and frequently encountered disease, which severely harms human health, however, rarely any effective treatment for neurological function injury after ICH is available at present. Not only can choroid plexus epithelial cells secrete endogenous neurotrophic factors, but also hold the characteristics of stem cell and potential to differentiate into astrocytes, which improves local microenvironment in brain and favors neuron regeneration. Therefore, choroid plexus epithelial cells is hopeful to become a novel treatment for neurological injury, yet the application about transplatation of which in ICH treatment has never been reported. Our previous study demonstrated that choroid plexus epithelial cell transplantation are able to improve nerve regeneration capability after ICH, with its advantages of high stability, non tumorigenecity and high survival rate, and can be chosen as seed cells in cerebral transplantaion, however, the detailed action mechanism still calls for further exploration. Based on our previous animal experiment concerning "choroid plexus cells mediate neuron regeneration after cerebral hemorrhage" (Natural Science Foundation of China, Grant No.30973099), the present project observes the related cytokine and gene expression in host choroid plexus and related brain areas before and after transplantation with human embryonic choroid plexus epithelial cell, and evaluates corresponding neuron function to demonstrate the function and related mechanism underlying the human embryonic choroid plexus epithelial cell transplatation in nerve regeneration after ICH. It provides experimental foundation for application of human embryonic choroid plexus epithelial cell transplantation in ICH treatment.

脑出血(ICH)是严重危害人类健康的常见病和多发病，目前对ICH后神经功能损害尚无确切有些的治疗手段。脉络丛上皮细胞不仅能分泌内源性神经营养因子，而且具有干细胞的特性和分化成星形胶质细胞的能力，可改善脑局部微环境，有助于神经再生，期望成为一种新的神经损害治疗方法，但脉络丛上皮细胞移植治疗ICH至今一直未见报道。本课题前期研究结果表明，脉络丛细胞移植能促进ICH后神经再生，并有稳定性强、无致瘤性与存活率高等优点，可作为脑内移植的种子细胞，但其具体作用机制尚待进一步研究。本课题在原有脉络丛细胞移植促进ICH后神经再生动物实验研究工作（国科金：30973099）的基础上，通过移植人胚胎脉络丛上皮细胞，观察移植前后宿主脉络丛及相关脑区细胞因子和基因的表达情况，并对其神经功能作评估，研究人胚胎脉络丛上皮细胞介导ICH后神经再生的作用及机制，为ICH采用人胚胎脉络丛上皮细胞移植治疗的应用提供实验依据。

目的：探索人胚胎脉络丛上皮细胞移植促进脑出血后神经再生的作用及机制，为今后人胚胎脉络丛上皮细胞移植治疗脑出血提供理论基础和实验依据。方法：1.分离人胚胎脉络丛组织，获取人胚胎脉络丛上皮细胞，并进行鉴定和观察。2.对人胚胎脉络丛上皮细胞进行食管癌相关基因4（ECRG4）表达检测。3.采用qRT-PCR法、 Western Blot法和Elisa法对人胚胎脉络丛上皮细胞的脑源性神经营养因子（BDNF）和神经生长因子（NGF）的表达进行检测。4.采用胶原酶诱导法建立大鼠脑出血模型，在1天及7天后采用Bederson评分和平衡木行走实验对模型作行为学评分。5.大鼠血肿腔进行人胚胎脉络丛上皮细胞移植，进行神经行为学评分和大鼠海马caspase-3表达的检测。结果：1.人胚胎脉络丛上皮细胞生长良好，呈蜂窝状紧密连接，TTR大量表达。2.ECRG4在人胚胎脉络丛上皮细胞中明显表达。3.BDNF和NGF在人胚胎脉络丛上皮细胞中表达明显。4.对细胞移植前后脑出血大鼠进行神经行为学评分，差异具有统计学意义（P＜0.05）。5.移植前后脑出血大鼠海马caspase-3表达具有统计学意义（P＜0.05）。结论：首次使用改良机械分散法成功地进行人胚胎脉络丛上皮细胞的体外培养，并对其分泌BDNF和NGF的能力和趋势进行研究，证实人胚胎脉络丛上皮细胞在脑出血早期移植可以改善大鼠神经功能缺失。本研究初步表明，人胚胎脉络丛上皮细胞移植在脑出血早期对神经功能的修复作用，为促进神经再生作用及机制的深入研究提供重要的理论基础和实验依据。