星形膠質(zhì)細(xì)胞原代培養(yǎng)與神經(jīng)干細(xì)胞誘導(dǎo)分化生物學(xué)研究
發(fā)布時間:2018-05-26 19:13
本文選題:神經(jīng)干細(xì)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(xì)胞是中樞神經(jīng)系統(tǒng)數(shù)量最多的膠質(zhì)細(xì)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(xì)胞體外獲得有原代培養(yǎng)星形膠質(zhì)細(xì)胞和神經(jīng)干細(xì)胞誘導(dǎo)分化的星形膠質(zhì)細(xì)胞兩種方法,然而兩種方法得到的星形膠質(zhì)細(xì)胞其生物學(xué)特性是否等同有待于進(jìn)一步研究。本研究通過細(xì)胞生長、劃傷反應(yīng)及檢測細(xì)胞功能及致癌性等,直接驗證原代培養(yǎng)星形膠質(zhì)細(xì)胞與神經(jīng)干細(xì)胞誘導(dǎo)分化的星形膠質(zhì)細(xì)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(xì)胞分化為星形膠質(zhì)細(xì)胞,經(jīng)典方法分離大腦皮層得到星形膠質(zhì)細(xì)胞,并運用差速貼壁和恒溫?fù)u床振蕩的方法對兩者進(jìn)行純化,采用間接免疫熒光方法檢測其純度。將兩種細(xì)胞進(jìn)行細(xì)胞生長、細(xì)胞劃傷反應(yīng)進(jìn)行比較。并且采用熒光定量PCR、Western-blot檢測細(xì)胞內(nèi)蛋白分子對兩者生物學(xué)性質(zhì)進(jìn)行對比。應(yīng)用SPSS17.0統(tǒng)計軟件進(jìn)行分析,所有計量資料以均數(shù)標(biāo)準(zhǔn)差(X±s)表示,兩組組間比較采用獨立樣本的t檢驗,P0.05認(rèn)為有統(tǒng)計學(xué)意義。 結(jié)果原代培養(yǎng)及神經(jīng)干細(xì)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(xì)胞。經(jīng)間接細(xì)胞免疫熒光實驗證實,神經(jīng)干細(xì)胞誘導(dǎo)分化為星形膠質(zhì)細(xì)胞方法培養(yǎng)與純化得到的星形膠質(zhì)細(xì)胞的純度可以達(dá)到99.4±0.5%,而傳統(tǒng)方法培養(yǎng)得到的星形膠質(zhì)細(xì)胞純度為94.2±2%,兩種方法培養(yǎng)的星形膠質(zhì)細(xì)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(xì)胞內(nèi)EGFR和P53蛋白的表達(dá),及熒光定量PCR測定GFAP的表達(dá),兩種細(xì)胞其表達(dá)量基本一致無統(tǒng)計學(xué)差異。 小結(jié)神經(jīng)干細(xì)胞誘導(dǎo)分化為星形膠質(zhì)細(xì)胞的培養(yǎng)與純化可以得到純凈的星形膠質(zhì)細(xì)胞,兩種方法培養(yǎng)的細(xì)胞在細(xì)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(xì)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(xì)胞的常規(guī)方法。為各種體外星形膠質(zhì)細(xì)胞試驗提供細(xì)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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