【摘要】： 目的本實(shí)驗(yàn)?zāi)康脑谟诮⑸窠?jīng)干細(xì)胞培養(yǎng)方法,觀察人工腦脊液(ACSF)中不同濃度的苯巴比妥鈉(PBS)對新生大鼠腦源性神經(jīng)干細(xì)胞分化成熟的影響。 方法從SD新生大鼠大腦分離、培養(yǎng)神經(jīng)干細(xì)胞,采用無血清培養(yǎng)。免疫細(xì)胞化學(xué)方法檢測神經(jīng)干細(xì)胞特征性標(biāo)志神經(jīng)上皮干細(xì)胞蛋白(Nestin)表達(dá),用5-溴尿嘧啶核苷(BrdU)檢測細(xì)胞增殖活性。用四甲基偶氮唑鹽法(MTT)觀察不同濃度人工腦脊液對神經(jīng)干細(xì)胞存活率的影響。不同濃度苯巴比妥鈉在神經(jīng)干細(xì)胞分化中的作用采用免疫組化來檢測,并采用掃描電鏡對分化后的細(xì)胞形態(tài)進(jìn)行觀察。 結(jié)果(1)經(jīng)過體外培養(yǎng)獲得的神經(jīng)干細(xì)胞能夠分化成為神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞,具有多向分化能力,同時(shí)有很強(qiáng)的自我更新能力;神經(jīng)球能夠表達(dá)Nestin和BrdU,傳代培養(yǎng)的神經(jīng)干細(xì)胞性能穩(wěn)定,經(jīng)過長期培養(yǎng)后能夠保持神經(jīng)干細(xì)胞特點(diǎn)。(2)MTT檢測顯示,≥40%的人工腦脊液能夠明顯降低神經(jīng)干細(xì)胞的存活率。(3)免疫細(xì)胞化學(xué)結(jié)果顯示,隨著人工腦脊液中的苯巴比妥鈉濃度的增高,誘導(dǎo)分化后的細(xì)胞中神經(jīng)元百分比逐漸降低,神經(jīng)膠質(zhì)細(xì)胞百分比逐漸升高,當(dāng)ACSF中的苯巴比妥鈉濃度為10μg/ml時(shí),可顯著降低NSCs分化為MAP 2染色陽性細(xì)胞的百分率(P＜0.01),當(dāng)ACSF中的苯巴比妥鈉濃度為25μg/ml時(shí),可顯著增高NSCs分化為GFAP染色陽性細(xì)胞的百分率(P＜0.01)。(4)掃描電鏡觀察實(shí)驗(yàn)組誘導(dǎo)分化后的神經(jīng)元不具備正常神經(jīng)元行使功能的形態(tài)結(jié)構(gòu)。 結(jié)論建立了體外培養(yǎng)大鼠神經(jīng)干細(xì)胞的方法,培養(yǎng)的神經(jīng)干細(xì)胞表達(dá)Nestin,有自我更新、多向分化的特點(diǎn)。人工腦脊液中的苯巴比妥鈉能夠抑制神經(jīng)干細(xì)胞向神經(jīng)元方向分化,促進(jìn)其向星型膠質(zhì)方向分化,且降低了分化后的正常神經(jīng)元比率。
[Abstract]:Objective to establish a culture method of neural stem cells and to observe the effects of different concentrations of phenobarbital sodium (PBS) in artificial cerebrospinal fluid (ACSF) on the differentiation and maturation of brain-derived neural stem cells in neonatal rats. Methods Neural stem cells were isolated from the brain of neonatal SD rats and cultured without serum. Immunocytochemical method was used to detect the expression of neural epithelial stem cell protein (Nestin), and 5-brominouracil nucleoside (BrdU) was used to detect the proliferative activity of neural stem cells (NSCs). The effects of different concentrations of artificial cerebrospinal fluid (CSF) on the survival rate of neural stem cells were observed by (MTT) method. The effects of phenobarbital sodium at different concentrations on the differentiation of neural stem cells were detected by immunohistochemistry and the morphology of differentiated cells was observed by scanning electron microscope (SEM). Results (1) the neural stem cells cultured in vitro were able to differentiate into neurons and glial cells. The neural stem cells cultured by Nestin and BrdU, have stable performance and can maintain the characteristics of neural stem cells after long term culture. (2) MTT detection showed that, (3) Immunocytochemistry showed that the concentration of phenobarbital sodium increased with the increase of the concentration of phenobarbital sodium in artificial cerebrospinal fluid. The percentage of neurons in the cells induced by differentiation gradually decreased, and the percentage of glial cells increased gradually. When the concentration of phenobarbital sodium in ACSF was 10 渭 g/ml, The percentage of NSCs differentiated into MAP 2 positive cells was significantly decreased (P < 0. 01). When the concentration of phenobarbital sodium in ACSF was 25 渭 g/ml, The percentage of NSCs differentiated into GFAP staining positive cells was significantly increased (P < 0. 01). (4). SEM observation showed that the differentiated neurons in the experimental group did not have the function of normal neurons. Conclusion the method of cultured rat neural stem cells in vitro was established. The cultured neural stem cells expressed Nestin, with the characteristics of self-renewal and multi-differentiation. Phenobarbital sodium in artificial cerebrospinal fluid can inhibit the neural stem cells to differentiate into neurons, promote the differentiation to astrocytes, and decrease the ratio of normal neurons after differentiation.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

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