MicroRNA-223与RhoB在GFP阳性大鼠胚胎脊髓源性神经干细胞中的表达

发布时间：2018-06-08 22:59

本文选题：脊髓神经干细胞 + 无血清培养　；参考：《福建医科大学》2015年硕士论文

【摘要】：目的探讨GFP(绿色荧光蛋白)阳性大鼠胚胎脊髓源性神经干细胞(Neural stem cells,NSCs)的获取,并对所获取细胞进行相关鉴定,为第二步实验提供实验细胞基础。方法显微解剖分离胚胎大鼠脊髓组织,用酶消化和机械分离法制成单细胞悬液,在含有表皮生长因子(EGF)、碱性成纤维生长因子(b FGF)及N2、B27的DMEM/F12无血清培养基中培养,传代后分别在相差显微镜下进行形态学观察并用免疫细胞化学方法鉴定巢蛋白(Nestin)、神经元核蛋白(Neuronal nuclei,Neu N)及胶质纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)的表达。结果分离获取的GFP阳性大鼠胚胎脊髓源神经干细胞在无血清培养基(Serum free medium,SFM)中生长良好,原代及传代培养的神经干细胞表达巢蛋白(nestin);而且用血清诱导分化后神经干细胞均有Neu N和GFAP蛋白阳性表达。结论采用酶消化及机械吹打相结合的方法能从大鼠胚胎脊髓组织中获得神经干细胞(Neural stem cell,NSCs),且保持完整的分化潜能,为下一步实验奠定了基础。目的检测mi R-223及Rho B蛋白在脊髓源性神经干细胞(Neural stem cells,NSCs)中的表达情况,并用统计学方法初步分析两者的关系。方法选取E14天、E16天、E18及E20天的孕鼠,分离培养脊髓来源神经干细胞(Neural stem cell,NSC),对4组细胞利用荧光定量PCR、western blot分别检测mi R-223和Rho B蛋白的表达,并且进行统计学分析。结果荧光定量PCR检测显示mi R-223在4组NSC细胞中都有表达,其中mi R-223在18天组NSCs中的表达明显高于14天组、16天组、20天组,各组间的表达差异具有显著性。利用Western blot检测Rho B蛋白在4组NSC细胞中的表达,结果显示Rho B在4组细胞中均有表达,在14天、16天组细胞中的表达最高,在18天及20天组的表达则相对较低。利用Gel-Pro analyzer灰度软件将Rho B蛋白的Western blot结果进行条带灰度分析,然后采用Spearman相关性统计学方法对mi R-223的荧光定量PCR表达结果进行分析。结果表明在检测的4种NSC细胞之间mi R-223与Rho B的表达存在负相关关系(r=-0.875,P0.001)。结论miR-223及RhoB蛋白在脊髓源性神经干细胞(Neural stem cells,NSCs)中均有表达,并且统计学上显示两者可能存在负性相关。
[Abstract]:Objective to investigate the acquisition of neural stem cells from embryonic spinal cord derived neural stem cells from GFP positive rats, and to identify the obtained cells in order to provide the experimental cell basis for the second step experiment. Methods the spinal cord tissue of embryonic rats was isolated by microdissection. Single cell suspension was prepared by enzyme digestion and mechanical separation. The suspension was cultured in serum-free medium containing epidermal growth factor (EGFN), basic fibroblast growth factor (bFGF) and N2OB27 (DMEM / F12). The expression of nestin, Neuronal nuclein N and glial fibrillary acidic protein (Glial fibrillary acidic protein) were detected by immunocytochemical method after passage under phase contrast microscope (PPM). The expression of nestin, neuronal nuclein and glial fibrillary acidic protein (Glial fibrillary acidic) were detected by immunocytochemistry. Results the neural stem cells derived from the spinal cord of GFP positive rats grew well in Serum free medium SFMs. Nestin protein was expressed in primary and cultured neural stem cells, and Neu N and GFAP protein were positive in all neural stem cells induced by serum. Conclusion Neural stem cells (NSCs) can be obtained from rat embryonic spinal cord by enzyme digestion and mechanical blow, and their differentiation potential can be maintained, which lays a foundation for further experiments. Objective to detect the expression of miR-223 and Rho B proteins in neural stem cells of spinal cord derived neural stem cells (NSCs) and to analyze the relationship between them by statistical method. Methods Neural-derived neural stem cells (NSCs) from spinal cord were isolated and cultured from pregnant mice of E14-day E16-day E18 and E20-day. The expression of miR-223 and Rho B protein was detected by fluorescence quantitative western blot in four groups of cells, and the results were statistically analyzed. Results the expression of miR-223 in NSC cells was detected by fluorescence quantitative PCR. The expression of miR-223 in NSCs of 18 days group was significantly higher than that of 16 days group and 20 days group of 14 days group, and there was significant difference among the four groups. Western blot was used to detect the expression of Rho B protein in NSC cells of 4 groups. The results showed that the expression of Rho B protein was the highest in NSC cells at day 14 and day 16, but relatively low at day 18 and day 20. Western blot results of Rho B protein were analyzed by Gel-Pro analyzer software, and then the results of fluorescent quantitative PCR of miR-223 were analyzed by Spearman correlation statistics. The results showed that there was a negative correlation between the expression of miR-223 and Rho B in the four NSC cells. Conclusion both miR-223 and RhoB proteins are expressed in neural stem cells of spinal cord derived neural stem cells, and there may be a negative correlation between them.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

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