星形胶质细胞原代培养与神经干细胞诱导分化生物学研究

发布时间：2018-05-26 19:13

本文选题：神经干细胞 + 诱导分化　；参考：《复旦大学》2012年硕士论文

【摘要】：目的星形胶质细胞是中枢神经系统数量最多的胶质细胞并且在中枢的许多功能中发挥重要作用,星形胶质细胞体外获得有原代培养星形胶质细胞和神经干细胞诱导分化的星形胶质细胞两种方法,然而两种方法得到的星形胶质细胞其生物学特性是否等同有待于进一步研究。本研究通过细胞生长、划伤反应及检测细胞功能及致癌性等,直接验证原代培养星形胶质细胞与神经干细胞诱导分化的星形胶质细胞生物学是否等同。方法通过诱导原代培养的新生大鼠海马神经干细胞分化为星形胶质细胞,经典方法分离大脑皮层得到星形胶质细胞,并运用差速贴壁和恒温摇床振荡的方法对两者进行纯化,采用间接免疫荧光方法检测其纯度。将两种细胞进行细胞生长、细胞划伤反应进行比较。并且采用荧光定量PCR、Western-blot检测细胞内蛋白分子对两者生物学性质进行对比。应用SPSS17.0统计软件进行分析,所有计量资料以均数标准差(X±s)表示,两组组间比较采用独立样本的t检验,P0.05认为有统计学意义。结果原代培养及神经干细胞诱导分化两种方法,经培养都可以得到星形胶质细胞。经间接细胞免疫荧光实验证实,神经干细胞诱导分化为星形胶质细胞方法培养与纯化得到的星形胶质细胞的纯度可以达到99.4±0.5%,而传统方法培养得到的星形胶质细胞纯度为94.2±2%,两种方法培养的星形胶质细胞的形态,生长趋势和对损伤的反应方面无明显差异。采用Western-blot检测细胞内EGFR和P53蛋白的表达,及荧光定量PCR测定GFAP的表达,两种细胞其表达量基本一致无统计学差异。小结神经干细胞诱导分化为星形胶质细胞的培养与纯化可以得到纯净的星形胶质细胞,两种方法培养的细胞在细胞生长、划伤反应、功能鉴定及致癌性等方面无明显差异,并且神经于细胞诱导分化方法具有易制备易纯化的特点,因此可作为制备星形胶质细胞的常规方法。为各种体外星形胶质细胞试验提供细胞基础。
[Abstract]:Objective astrocytes are the most numerous glial cells in the central nervous system and play an important role in many central functions. There are two methods to obtain astrocytes from astrocytes in vitro, which are primary cultured astrocytes and neural stem cells. However, whether the biological characteristics of astrocytes obtained by the two methods are equivalent to those of neural stem cells remains to be further studied. Through cell growth, scratch reaction and detection of cell function and carcinogenicity, the biological equivalence of astrocytes induced by primary cultured astrocytes and neural stem cells (NSCs) was directly verified. Methods the primary cultured neural stem cells were induced to differentiate into astrocytes. The astrocytes were isolated from the cerebral cortex by classical method. The astrocytes were purified by differential adhesion and constant temperature shaking. Its purity was detected by indirect immunofluorescence method. The cell growth and scratch reaction of the two kinds of cells were compared. Fluorescence quantitative PCR Western blot was used to detect the biological properties of the two proteins. Using SPSS17.0 statistical software, all the measurement data were expressed as mean standard deviation (X 卤s). The comparison between the two groups was considered statistically significant by t-test of independent samples (P0.05). Results astrocytes could be obtained by primary culture and neural stem cell induction. Indirect immunofluorescence assay confirmed that, The purity of astrocytes cultured and purified by neural stem cells was 99.4 卤0.5, while the purity of astrocytes cultured by traditional methods was 94.2 卤22.The morphology of astrocytes cultured by the two methods. There was no significant difference in growth trend and response to injury. Western-blot was used to detect the expression of EGFR and p53 protein, and fluorescence quantitative PCR was used to detect the expression of GFAP. There was no significant difference in the expression of EGFR and p53 between the two cells. Pure astrocytes could be obtained by the culture and purification of neural stem cells induced to differentiate into astrocytes. There was no significant difference in cell growth, scratch reaction, functional identification and carcinogenicity between the two methods. The method of neuronal differentiation is easy to be prepared and purified, so it can be used as a conventional method for the preparation of astrocytes. To provide a cellular basis for various astrocyte tests in vitro.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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