本文關(guān)鍵詞：OECs上清液誘導(dǎo)NSCs分化前后膜蛋白質(zhì)復(fù)合物的差異表達(dá)研究　出處：《湖南師范大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 嗅鞘細(xì)胞 神經(jīng)干細(xì)胞 分化 神經(jīng)元 嗅鞘細(xì)胞 神經(jīng)干細(xì)胞 細(xì)胞膜 BN-PAGE 蛋白質(zhì)復(fù)合物

【摘要】：第一章OECs培養(yǎng)及上清液對(duì)NSCs的誘導(dǎo)分化研究 目的：探索小鼠嗅鞘細(xì)胞(olfactory ensheathing cells,OECs)對(duì)神經(jīng)干細(xì)胞(neural stem cells,NSCs)分化的影響。 方法：健康新生昆明小鼠10只,取大腦皮層組織,用含2%B27、1%N2、20ng/ml堿性成纖維生長(zhǎng)因子(basic fibroblast growth factor, bFGF)和20ng/ml表皮生長(zhǎng)因子(epidermal growth factor, EGF)的培養(yǎng)液對(duì)NSCs進(jìn)行懸浮培養(yǎng),每5-7天傳代一次,純化培養(yǎng)2-3代備用,免疫細(xì)胞化學(xué)法對(duì)NSCs進(jìn)行巢蛋白(Nestin)鑒定；取新生昆明小鼠嗅球,0.25%的胰蛋白酶37℃消化,改良Nash差速貼壁培養(yǎng),加阿糖胞苷抑制雜細(xì)胞生長(zhǎng),當(dāng)細(xì)胞數(shù)量達(dá)到大約80%時(shí)吸出上清,加入新鮮培養(yǎng)基(無(wú)血清DMEM),反復(fù)沖洗3遍,收集條件培養(yǎng)液,每隔3天半量換液。-20℃保存條件培養(yǎng)液,免疫細(xì)胞化學(xué)法對(duì)OECs進(jìn)行神經(jīng)生長(zhǎng)因子受體p75(nerve growth factor receptor p75, NGFR p75)鑒定；實(shí)驗(yàn)組NSCs加OECs條件培養(yǎng)液及無(wú)血清DMEM/F12液培養(yǎng),對(duì)照組NSCs只加無(wú)血清DMEM/F12培養(yǎng)液培養(yǎng),每隔3天換液。觀察細(xì)胞生長(zhǎng)及分化情況,神經(jīng)絲蛋白200(neurofilament200,NF200)鑒定神經(jīng)元,膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein,GFAP)鑒定膠質(zhì)細(xì)胞。 結(jié)果：新生昆明小鼠大腦皮層中分離培養(yǎng)出NSCs,這些細(xì)胞具有長(zhǎng)期增殖能力,免疫細(xì)胞化學(xué)鑒定顯示Nestin染色陽(yáng)性；新生昆明小鼠嗅球培養(yǎng)出OECs,7天NGFR p75染色見(jiàn)大量陽(yáng)性細(xì)胞；對(duì)照組NSCs克隆球少部分貼壁,未見(jiàn)增殖分化,細(xì)胞逐漸萎縮,12天時(shí)細(xì)胞死亡,實(shí)驗(yàn)組2天后NSCs克隆球開(kāi)始貼壁分化,7天時(shí)分化較明顯,10天時(shí)免疫細(xì)胞化學(xué)染色,NF200染色顯示NSCs克隆球大部分分化為神經(jīng)元,GFAP染色顯示NSCs克隆球少部分分化為膠質(zhì)細(xì)胞。 結(jié)論：OECs上清液能誘導(dǎo)NSCs向神經(jīng)元方向分化。 目的：探索小鼠嗅鞘細(xì)胞(olfactory ensheathing cells, OECs)上清液誘導(dǎo)神經(jīng)干細(xì)胞(neural stem cells, NSCs)向神經(jīng)細(xì)胞分化的分子機(jī)制,篩選出誘導(dǎo)分化的關(guān)鍵蛋白,為臨床藥物靶向治療神經(jīng)損傷提供實(shí)驗(yàn)依據(jù)。 方法：取新生昆明小鼠嗅球,0.25%的胰蛋白酶37℃消化,改良Nash差速貼壁培養(yǎng),加阿糖胞苷抑制雜細(xì)胞生長(zhǎng),當(dāng)細(xì)胞數(shù)量達(dá)到大約80%時(shí)吸出上清,加入新鮮培養(yǎng)基(無(wú)血清DMEM),反復(fù)沖洗3遍,收集條件培養(yǎng)液,每隔3天半量換液,-20℃保存條件培養(yǎng)液。免疫細(xì)胞化學(xué)法對(duì)OECs進(jìn)行神經(jīng)生長(zhǎng)因子受體p75(nerve growth factorreceptor p75, NGFR p75)鑒定。C172.2小鼠神經(jīng)干細(xì)胞系復(fù)蘇、培養(yǎng)、傳代,37℃快速融化、離心、吹勻,高糖改良Eagle培養(yǎng)基H-DMEM (含15%胎牛血清)培養(yǎng),4-5天傳代一次,擴(kuò)增至第三代(P3)備用。實(shí)驗(yàn)組NSCs加OECs條件培養(yǎng)液及無(wú)血清DMEM液培養(yǎng)誘導(dǎo)分化1/2、1、4小時(shí),對(duì)照組NSCs只加無(wú)血清DMEM培養(yǎng)液培養(yǎng)。應(yīng)用超速離心法提取細(xì)胞膜,藍(lán)綠溫和膠凝膠電泳(Blue Native-PAGE, BN-PAGE)分離膜蛋白質(zhì)復(fù)合物,膠內(nèi)酶切差異蛋白點(diǎn),電噴霧四級(jí)桿時(shí)間飛行質(zhì)譜(ESI-Q-TOF-MS)鑒定各時(shí)間點(diǎn)之間差異蛋白質(zhì)復(fù)合物的表達(dá)。將儀器自動(dòng)產(chǎn)生的數(shù)據(jù)文件用Mascot軟件(www. matrixscience.com)中的串聯(lián)質(zhì)譜數(shù)據(jù)庫(kù)搜尋(MS/Ms Ion Search)功能進(jìn)行搜索。 結(jié)果：新生小鼠嗅球培養(yǎng)出OECs,7天NGFR p75染色見(jiàn)大量陽(yáng)性細(xì)胞;C17.2NSCs復(fù)蘇后1天內(nèi)可見(jiàn)細(xì)胞貼壁生長(zhǎng),培養(yǎng)4-5天后細(xì)胞大量生長(zhǎng),進(jìn)行傳代,傳代2天后可見(jiàn)大量細(xì)胞,形態(tài)呈梭形,折光性強(qiáng)；BN-PAGE顯示出11個(gè)條帶,每個(gè)時(shí)間點(diǎn)膠條酶切這11個(gè)條帶。ESI-Q-TOF-MS鑒定到79個(gè)肽段相匹配的蛋白質(zhì),其中53個(gè)蛋白有顯著差別的表達(dá),另外26個(gè)蛋白有相同豐度的表達(dá)。我們共鑒定到5個(gè)蛋白質(zhì)復(fù)合物的差異表達(dá)。 結(jié)論：1.0ECs上清液誘導(dǎo)NSCs分化前期的4個(gè)時(shí)間點(diǎn)間膜蛋白質(zhì)復(fù)合物的表達(dá)有差異； 2.我們發(fā)現(xiàn)5個(gè)差異蛋白質(zhì)復(fù)合物：mRNP granule complex、 ATP synthase complex、eukaryotic translation initiation factor3(eIF-3) complex、BBS/CCT complex及Hsp90complex,通過(guò)各自功能的搜庫(kù)查詢,其中Hsp90復(fù)合物可能在分化過(guò)程中起關(guān)鍵啟動(dòng)作用,為篩選NSCs分化過(guò)程中的關(guān)鍵蛋白提供數(shù)據(jù)。
[Abstract]:First chapter OECs culture and supernatant fluid induced differentiation of neural stem cells Objective : To explore the effect of olfactory ensheathing cells ( OECs ) on the differentiation of neural stem cells . Methods : Ten healthy new Kunming mice were cultured in vitro . The cultured cells were cultured in DMEM / F12 medium . The cells were cultured in DMEM / F12 medium . The cells were cultured in DMEM / F12 medium without serum . Results : The cells were isolated and cultured in the cerebral cortex of Kunming mice . The cells had long - term proliferative ability , and the cells showed Nestin staining positive . The cells of the control group had no proliferation and differentiation . The cells died after 2 days . After 2 days in the experimental group , the cells died . After 2 days in the experimental group , the cells were differentiated into neurons . The expression of NF200 showed that the majority of the cells differentiated into neurons . The GFAP staining showed that there was a small partial differentiation of the subclones into glial cells . Conclusion : The supernatant of OECs can induce the differentiation of neural stem cells into neurons . Objective : To explore the molecular mechanism of the differentiation of neural stem cells and neural stem cells from olfactory ensheathing cells ( OECs ) from mouse olfactory ensheathing cells ( OECs ) into neural cells , and to screen out the key proteins for inducing differentiation and provide experimental basis for the targeted treatment of nerve injury . Methods : The cells were cultured in DMEM culture medium containing 15 % fetal bovine serum at 37 鈩，

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