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  本文選題：神經(jīng)干細胞 + 誘導(dǎo)分化　； 參考：《復(fù)旦大學(xué)》2012年碩士論文

【摘要】：目的星形膠質(zhì)細胞是中樞神經(jīng)系統(tǒng)數(shù)量最多的膠質(zhì)細胞并且在中樞的許多功能中發(fā)揮重要作用,星形膠質(zhì)細胞體外獲得有原代培養(yǎng)星形膠質(zhì)細胞和神經(jīng)干細胞誘導(dǎo)分化的星形膠質(zhì)細胞兩種方法,然而兩種方法得到的星形膠質(zhì)細胞其生物學(xué)特性是否等同有待于進一步研究。本研究通過細胞生長、劃傷反應(yīng)及檢測細胞功能及致癌性等,直接驗證原代培養(yǎng)星形膠質(zhì)細胞與神經(jīng)干細胞誘導(dǎo)分化的星形膠質(zhì)細胞生物學(xué)是否等同。 方法通過誘導(dǎo)原代培養(yǎng)的新生大鼠海馬神經(jīng)干細胞分化為星形膠質(zhì)細胞,經(jīng)典方法分離大腦皮層得到星形膠質(zhì)細胞,并運用差速貼壁和恒溫搖床振蕩的方法對兩者進行純化,采用間接免疫熒光方法檢測其純度。將兩種細胞進行細胞生長、細胞劃傷反應(yīng)進行比較。并且采用熒光定量PCR、Western-blot檢測細胞內(nèi)蛋白分子對兩者生物學(xué)性質(zhì)進行對比。應(yīng)用SPSS17.0統(tǒng)計軟件進行分析,所有計量資料以均數(shù)標準差(X±s)表示,兩組組間比較采用獨立樣本的t檢驗,P0.05認為有統(tǒng)計學(xué)意義。 結(jié)果原代培養(yǎng)及神經(jīng)干細胞誘導(dǎo)分化兩種方法,經(jīng)培養(yǎng)都可以得到星形膠質(zhì)細胞。經(jīng)間接細胞免疫熒光實驗證實,神經(jīng)干細胞誘導(dǎo)分化為星形膠質(zhì)細胞方法培養(yǎng)與純化得到的星形膠質(zhì)細胞的純度可以達到99.4±0.5%,而傳統(tǒng)方法培養(yǎng)得到的星形膠質(zhì)細胞純度為94.2±2%,兩種方法培養(yǎng)的星形膠質(zhì)細胞的形態(tài),生長趨勢和對損傷的反應(yīng)方面無明顯差異。采用Western-blot檢測細胞內(nèi)EGFR和P53蛋白的表達,及熒光定量PCR測定GFAP的表達,兩種細胞其表達量基本一致無統(tǒng)計學(xué)差異。 小結(jié)神經(jīng)干細胞誘導(dǎo)分化為星形膠質(zhì)細胞的培養(yǎng)與純化可以得到純凈的星形膠質(zhì)細胞,兩種方法培養(yǎng)的細胞在細胞生長、劃傷反應(yīng)、功能鑒定及致癌性等方面無明顯差異,并且神經(jīng)于細胞誘導(dǎo)分化方法具有易制備易純化的特點,因此可作為制備星形膠質(zhì)細胞的常規(guī)方法。為各種體外星形膠質(zhì)細胞試驗提供細胞基礎(chǔ)。
[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 鹵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 鹵0.5, while the purity of astrocytes cultured by traditional methods was 94.2 鹵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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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