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  本文關(guān)鍵詞：胚胎大鼠神經(jīng)干細胞體外培養(yǎng)鑒定及誘導其分化為類毛細胞的研究　出處：《華中科技大學》2008年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 胚鼠 無血清培養(yǎng)基 神經(jīng)干細胞 分化 神經(jīng)干細胞 分化 基底膜 鼠尾膠原 類毛細胞 神經(jīng)干細胞 基底膜 分化 類毛細胞

【摘要】： 第一部分無血清培養(yǎng)條件下神經(jīng)干細胞的生長及分化的研究 目的:探討體外無血清條件下培養(yǎng)胚鼠神經(jīng)干細胞(NSCs),觀察其生長及分化情況. 方法:取孕16-18天SD系大鼠的胚胎海馬組織,在含EGF,bFGF,B27的DMEM/F12培養(yǎng)基中培養(yǎng),電子顯微鏡下觀察其增殖分化過程,并通過免疫熒光方法鑒定分化后的細胞類型. 結(jié)果:NSCs在無血清培養(yǎng)基中生長旺盛,8天左右就可以形成胞體透亮、折光性好的干細胞球,分化后的細胞顯示NSE,GFAP免疫陽性. 結(jié)論:無血清培養(yǎng)條件下NSCs生長良好,在含血清的培養(yǎng)基中可以分化為神經(jīng)元和星形膠質(zhì)細胞. 第二部分神經(jīng)干細胞與體外培養(yǎng)的乳鼠基底膜上清液共培養(yǎng)分化為類毛細胞的研究 目的:探討體外誘導胚胎大鼠神經(jīng)干細胞(neural stem cells,NSCs)分化為類毛細胞的實驗研究。 方法:從SD系胚鼠大腦分離NSCs,在無血清培養(yǎng)基中培養(yǎng)后將其放到含鼠尾膠原包被的蓋玻片的6孔培養(yǎng)板中培養(yǎng),然后將乳鼠基底膜取出體外培養(yǎng)后汲取其上清液與NSCs一起培養(yǎng),14-21天后通過免疫熒光和免疫組化法檢測毛細胞標志物myosin VIIa和calretinin,并計算分化比例。 結(jié)果: NSCs分化后的細胞約有15.4%呈myosin VIIa免疫熒光陽性,21.7%呈免疫組化calretinin陽性。 結(jié)論:體外培養(yǎng)的乳鼠基底膜上清液能誘導NSCs分化為類毛細胞。 第三部分神經(jīng)干細胞與乳鼠基底膜共培養(yǎng)后分化為類毛細胞的研究 目的:探討體外誘導神經(jīng)干細胞(neural stem cells,NSCs)分化為類毛細胞的實驗研究。 方法:從SD系胚鼠大腦分離NSCs,在無血清培養(yǎng)基中培養(yǎng)。取出生3天內(nèi)乳鼠基底膜與NSCs一起培養(yǎng),14-21天后通過免疫熒光和免疫組化法檢測毛細胞標志物myosin VIIa和calretinin,并計算分化比例。 結(jié)果: NSCs分化后的細胞約有17.1%呈myosin VIIa免疫熒光陽性,27.4%呈免疫組化calretinin陽性。 結(jié)論:乳鼠基底膜與NSCs共培養(yǎng)時能引導其朝毛細胞方向分化。
[Abstract]:Part one: growth and differentiation of neural stem cells in serum-free culture Aim: to investigate the growth and differentiation of embryonic rat neural stem cells (NSCs) cultured in serum-free medium in vitro. Methods: the embryonic hippocampal tissues of SD rats were collected from 16-18 days gestation and cultured in DMEM/F12 medium containing EGFN bFGFFGFB27. The process of proliferation and differentiation was observed under electron microscope, and the differentiated cells were identified by immunofluorescence. Results in serum-free medium, stem cell spheres with bright cell body and good refraction could be formed after 8 days of vigorous growth. The differentiated cells showed positive GFAP immunoreactivity. Conclusion: NSCs grows well in serum-free culture and can differentiate into neurons and astrocytes in serum-containing medium. The second part of the study on the differentiation of neural stem cells into hair-like cells by co-culture of neural stem cells with the supernatant of basal membrane of neonatal rats in vitro Aim: to investigate the differentiation of neural stem cells from embryonic rat neural stem cells into hair-like cells in vitro. Methods: NSCs were isolated from the brains of SD embryos and cultured in serum-free medium. The supernatant of the supernatant was extracted from the basement membrane of the neonatal rat and cultured with NSCs in vitro. After 14-21 days, the hair cell markers myosin VIIa and calretinin were detected by immunofluorescence and immunohistochemistry, and the differentiation ratio was calculated. Results: about 15.4% of the cells differentiated by NSCs showed myosin VIIa immunofluorescence positive and 21. 7% were calretinin positive. Conclusion: the supernatant of basal membrane of neonatal rats can induce the differentiation of NSCs into hair-like cells in vitro. The third part of the study on differentiation of neural stem cells into hair-like cells after co-culture with neonatal rat basement membrane Aim: to investigate the differentiation of neural stem cells into hair-like cells in vitro. Methods: NSCs were isolated from the brains of SD embryos and cultured in serum-free medium. The basement membrane of neonatal rats was cultured with NSCs within 3 days. After 14-21 days, the hair cell markers myosin VIIa and calretinin were detected by immunofluorescence and immunohistochemistry, and the differentiation ratio was calculated. Results: about 17.1% of the cells differentiated by NSCs showed myosin VIIa immunofluorescence positive and 27. 4% were calretinin positive. Conclusion: the basal membrane and NSCs can induce the neonatal rat to differentiate towards the hair cells.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329;R764

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本文編號：1383787