小鼠胚胎干细胞定向分化为神经细胞的研究进展
5 遗传工程介导的神经细胞分化
2002年Chung等人[25]将Nurr1(nur相关因子1)基因导入小鼠ES细胞,使其过表达,从而诱导ES细胞分化为具有中脑多巴胺能神经元表型的细胞。Nurr1是一种转录因子,直接结合于TH基因的启动子区域,调节维持中脑多巴胺能神经元表型的蛋白质的表达,如L-芳香族氨基酸脱羧酶(aromatic L-amino acid decarboxylase, AADC)、囊泡单胺转运蛋白2(vesicular monoamine transporter 2, VMAT2)[26]和多巴胺转运蛋白(dopamine transporter, DAT)[27]。将这种方法得到的多巴胺能神经元注射到脑内可以改善帕金森模型大鼠的运动缺陷[28]。该方法存在的问题是,在神经细胞分化之前过表达Nurr1会上调其他多巴胺能神经元分子标志的表达,而且由ES细胞分化的其他细胞也会出现这种情况[29]。
2004年Ikeda等人[30]将MASH1基因导入小鼠ES细胞,该ES细胞多数分化为表达βⅢtubulin和泛NCAM(pan NCAM)的神经元样细胞,其中半数进一步分化为Islet阳性的运动神经元。MASH1是一种激活型碱性螺旋-环-螺旋结构的转录因子,表达于腹侧端脑,调节GABA能中间神经元和分支运动神经元的发育[31]。将得到的运动神经元样细胞注射到偏瘫小鼠脑室周围的运动皮层,可明显改善运动障碍。
遗传工程介导的神经细胞分化方法的优势是可以较高效率地得到感兴趣的特定的神经细胞。但是由于导入基因的功能多样性和不确定性,较难判断该基因对细胞的整体影响和长远影响。
6 问题与展望
虽然小鼠胚胎干细胞诱导分化为神经细胞的方法取得了较大进展,目前可以较高效率地得到中枢神经系统的各种神经元和神经胶质细胞,但是仍有许多问题亟待解决。首先,虽然“五步法”方案解决了“4-/4+”方案中分化细胞不易纯化的问题,但是其分子机制尚待进一步阐明、操作步骤需要进一步优化;其次,SDIA诱导方法的作用机制和单层粘附培养的作用机制需要进一步阐明,这将会有利于我们对神经细胞分化过程中信号通路的掌握;再次,遗传工程方法导入的基因对细胞的长远影响需要进一步观察和研究;还有,现在所得到的神经细胞类型还不很明确,因为神经元和神经胶质细胞还可以分为很多亚型。例如中枢神经系统的胆碱能神经元根据形态和生化特性就可以分为不同亚型,它们的分布和生理功能也不尽相同。因此有必要获得特定部位和类型的神经细胞。
ES细胞向神经细胞分化的研究有利于人们了解胚胎神经细胞发育的机制、发现ES细胞向神经细胞分化的关键调节基因;建立体外定向诱导神经细胞分化体系将为神经疾病治疗药物的筛选以及基因工程药物的开发等提供有用的材料;体外大量有功能的特定神经元的获得使神经再生医学正一步步从理论走向实践。
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