移植用小鼠胶质前体细胞的培养及其移植后形态和功能初探.docVIP

DOI：10.16016/j.1000-5404.201501052 移植用小鼠胶质前体细胞的培养及其移植后形态和功能初探 杨志奇1，张 宽1，马秦龙2，邓 平2，陈纯海2，周 舟2，谌小维 1 （400038重庆，第三军医大学：基础医学部脑研究中心1,军事预防医学院劳动卫生教研室2） [摘要] 目的 完善体外分离培养移植用胶质前体细胞的方法,并运用双光子显微镜在活体动物水平，对小鼠大脑皮层移植的胶质细胞进行单细胞水平的在体功能研究。方法 体外分离培养小鼠胚胎神经干细胞，并将其诱导为胶质前体细胞及星形胶质细胞。分别用Nestin、A2B5以及GFAP对体外培养的神经干细胞、胶质前体细胞以及星形胶质细胞进行形态学鉴定。体外给予ATP及毒胡萝卜素（Thapsigargin），对体外分化的星形胶质细胞进行功能学鉴定。将以上诱导得到的胶质前体细胞移植入成年小鼠大脑皮层，并在移植12周后对其进行形态学观察。另外，应用双光子活体钙成像技术，观察移植胶质细胞对ATP刺激的反应。结果 ①体外诱导神经干细胞成为胶质前体细胞（A2B5阳性）的比率达到(87.4±3.4)%，继续将其诱导为成熟星形胶质细胞（GFAP阳性）的比率达到(83.4±3.3)%。②体外诱导而来的星形胶质细胞可对ATP及毒胡萝卜素（Thapsigargin）产生钙反应。③移植进入成年小鼠皮层的胶质前体细胞，在移植12周后仍可存活，并可分化为成熟的星形胶质细胞。④双光子活体钙成像实验证实，ATP可诱导活体小鼠皮层内移植细胞产生钙信号。结论 神经干细胞来源的胶质前体细胞在移植进入成年小鼠皮层内可分化为成熟星形胶质细胞，并至少存活12周。另外，胶质前体细胞在移植4周后，便可在活体动物大脑皮层内具备功能活动。 [关键词] 神经干细胞；胶质前体细胞；星形胶质细胞；钙信号；移植 [中图法分类号] [文献标志码] A Culture of mouse glial progenitors for transplantation and preliminary research on their morphology and function Yang Zhiqi1, Zhang Kuan1, Ma Qinlong2, Deng Ping2, Chen Chunhai2, Zhou Zhou2, Chen Xiaowei1 (Brain Research Center of Basic Medical College1, Department of Occupational Health of Preventive Medical College2, Third Military Medical University, Chongqing, 400038, China) [Abstract] Objective To improve a method for isolating and culturing glial progenitors for transplantation, and study in vivo function of the engrafted glial progenitors in cortex in single cell level by two-photon imaging. Methods Neural stem cells (NSCs) were isolated from the embryonic cortex, and cultured in vitro. Then, the cells were induced into glial progenitors and astrocytes. In order to detect the cellular morphology, Nestin, A2B5,and GFAP as markers of NSCs, glial progenitors and astrocytes, respectively, were detected. In order to detect the cellular function, calcium responses of the astrocytes to ATP and Thapsigargin were recorded by confocal imaging. The glial progenitors were engrafted into the cerebral cortex of adult mice, and the morphology of