本文選題：脊髓神經(jīng)干細(xì)胞 + 無(wú)血清培養(yǎng)　； 參考：《福建醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的探討GFP(綠色熒光蛋白)陽(yáng)性大鼠胚胎脊髓源性神經(jīng)干細(xì)胞(Neural stem cells,NSCs)的獲取,并對(duì)所獲取細(xì)胞進(jìn)行相關(guān)鑒定,為第二步實(shí)驗(yàn)提供實(shí)驗(yàn)細(xì)胞基礎(chǔ)。方法顯微解剖分離胚胎大鼠脊髓組織,用酶消化和機(jī)械分離法制成單細(xì)胞懸液,在含有表皮生長(zhǎng)因子(EGF)、堿性成纖維生長(zhǎng)因子(b FGF)及N2、B27的DMEM/F12無(wú)血清培養(yǎng)基中培養(yǎng),傳代后分別在相差顯微鏡下進(jìn)行形態(tài)學(xué)觀察并用免疫細(xì)胞化學(xué)方法鑒定巢蛋白(Nestin)、神經(jīng)元核蛋白(Neuronal nuclei,Neu N)及膠質(zhì)纖維酸性蛋白(Glial fibrillary acidic protein,GFAP)的表達(dá)。結(jié)果分離獲取的GFP陽(yáng)性大鼠胚胎脊髓源神經(jīng)干細(xì)胞在無(wú)血清培養(yǎng)基(Serum free medium,SFM)中生長(zhǎng)良好,原代及傳代培養(yǎng)的神經(jīng)干細(xì)胞表達(dá)巢蛋白(nestin);而且用血清誘導(dǎo)分化后神經(jīng)干細(xì)胞均有Neu N和GFAP蛋白陽(yáng)性表達(dá)。結(jié)論采用酶消化及機(jī)械吹打相結(jié)合的方法能從大鼠胚胎脊髓組織中獲得神經(jīng)干細(xì)胞(Neural stem cell,NSCs),且保持完整的分化潛能,為下一步實(shí)驗(yàn)奠定了基礎(chǔ)。目的檢測(cè)mi R-223及Rho B蛋白在脊髓源性神經(jīng)干細(xì)胞(Neural stem cells,NSCs)中的表達(dá)情況,并用統(tǒng)計(jì)學(xué)方法初步分析兩者的關(guān)系。方法選取E14天、E16天、E18及E20天的孕鼠,分離培養(yǎng)脊髓來(lái)源神經(jīng)干細(xì)胞(Neural stem cell,NSC),對(duì)4組細(xì)胞利用熒光定量PCR、western blot分別檢測(cè)mi R-223和Rho B蛋白的表達(dá),并且進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果熒光定量PCR檢測(cè)顯示mi R-223在4組NSC細(xì)胞中都有表達(dá),其中mi R-223在18天組NSCs中的表達(dá)明顯高于14天組、16天組、20天組,各組間的表達(dá)差異具有顯著性。利用Western blot檢測(cè)Rho B蛋白在4組NSC細(xì)胞中的表達(dá),結(jié)果顯示Rho B在4組細(xì)胞中均有表達(dá),在14天、16天組細(xì)胞中的表達(dá)最高,在18天及20天組的表達(dá)則相對(duì)較低。利用Gel-Pro analyzer灰度軟件將Rho B蛋白的Western blot結(jié)果進(jìn)行條帶灰度分析,然后采用Spearman相關(guān)性統(tǒng)計(jì)學(xué)方法對(duì)mi R-223的熒光定量PCR表達(dá)結(jié)果進(jìn)行分析。結(jié)果表明在檢測(cè)的4種NSC細(xì)胞之間mi R-223與Rho B的表達(dá)存在負(fù)相關(guān)關(guān)系(r=-0.875,P0.001)。結(jié)論miR-223及RhoB蛋白在脊髓源性神經(jīng)干細(xì)胞(Neural stem cells,NSCs)中均有表達(dá),并且統(tǒng)計(jì)學(xué)上顯示兩者可能存在負(fù)性相關(guān)。
[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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R651.2

【共引文獻(xiàn)】

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