本文選題：脂肪基質(zhì)細(xì)胞 + 細(xì)胞誘導(dǎo)�。� 參考：《南方醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 成體脂肪基質(zhì)細(xì)胞(adipose-derived stromal cells,ADSCs)是一類存在于脂肪組織中、具有多向分化潛能的干細(xì)胞,研究發(fā)現(xiàn),它具有幾乎和胚胎干細(xì)胞(embryonic stem cells,ESCs)一樣具有較強(qiáng)的分化潛能,在一定誘導(dǎo)條件下,既可以向中胚層起源的成骨細(xì)胞、成軟骨細(xì)胞、成肌細(xì)胞和脂肪細(xì)胞分化,也可以向外胚層起源的神經(jīng)細(xì)胞分化,且至今為止尚未發(fā)現(xiàn)致瘤性。因其來(lái)源豐富且易于取材,將可彌補(bǔ)胚胎組織來(lái)源和成體中樞來(lái)源神經(jīng)干細(xì)胞(neuralstem cells,NSCs)在應(yīng)用上的困難如來(lái)源不足、采集困難、免疫排斥、法律倫理限制等,顯現(xiàn)了在中樞神經(jīng)系統(tǒng)(central nerve system,CNS)疾病細(xì)胞移植治療領(lǐng)域的巨大潛能。本課題以大鼠ADSCs為研究對(duì)象,主要從ADSCs的分離培養(yǎng)和向神經(jīng)細(xì)胞定向誘導(dǎo)分化的方法學(xué)上進(jìn)行研究,探討建立一種簡(jiǎn)便高效的由成體ADSCs在體外誘導(dǎo)培養(yǎng)獲得NSCs的方法,以期優(yōu)化成體ADSCs誘導(dǎo)分化實(shí)驗(yàn)流程、提高定向分化比例,從而解決NSCs移植的種子細(xì)胞來(lái)源不足、體外定向分化率低等問(wèn)題,為細(xì)胞移植治療CNS疾病的臨床應(yīng)用提供實(shí)驗(yàn)基礎(chǔ)。 第一部分:大鼠ADSCs的體外培養(yǎng)擴(kuò)增及其生物學(xué)特性的觀察 目的:分離、純化、體外培養(yǎng)擴(kuò)增大鼠ADSCs,并觀察其生物學(xué)特性。 方法:無(wú)菌麻醉狀態(tài)下取SD大鼠的腹膜后脂肪組織,用剪刀剪碎并以Ⅰ型膠原酶消化離心,以含10%胎牛血清的DMEM/F12培養(yǎng)基重懸細(xì)胞,接種于培養(yǎng)瓶,置于37℃、5%CO_2培養(yǎng)箱中培養(yǎng)并傳代,CK2型倒置光學(xué)相差顯微鏡下追蹤觀察活細(xì)胞培養(yǎng)生長(zhǎng)情況并拍照;四唑鹽(methyl thiazolyl tetrazolium,MTT)法檢測(cè)細(xì)胞生長(zhǎng)增殖情況,繪制細(xì)胞生長(zhǎng)曲線;流式細(xì)胞儀檢測(cè)及免疫熒光細(xì)胞化學(xué)染色(間接法)檢測(cè)CD34、CD106、CD44、HLA-1等細(xì)胞表面標(biāo)志。 結(jié)果:倒置顯微鏡下觀察,剛接種的細(xì)胞大多呈圓球狀,懸浮在培養(yǎng)基中,可見(jiàn)少數(shù)脂滴懸在最上層;ADSCs原代有多種形態(tài),有圓球狀、長(zhǎng)梭形及成纖維樣。通過(guò)貼壁傳代法除去雜細(xì)胞,傳至第五代,細(xì)胞形態(tài)較為一致,呈長(zhǎng)梭形,細(xì)胞排列整齊,有漩渦狀特征;在10%胎牛血清存在的條件下,傳代細(xì)胞每24h增殖1倍,并表達(dá)較強(qiáng)的CD44免疫細(xì)胞化學(xué)陽(yáng)性染色。流式細(xì)胞儀檢測(cè)細(xì)胞表面標(biāo)記表達(dá)率分別為CD34 0.8%,CD106 13.6%,CD44 99.7%,HLA-1 96.7%,符合ADSCs的表型特征。 結(jié)論:所采用的細(xì)胞分離培養(yǎng)方法簡(jiǎn)便可行,所獲得的ADSCs純度較高,細(xì)胞活力旺盛,自我更新能力強(qiáng)。 第二部分:大鼠ADSCs向神經(jīng)細(xì)胞的二步法誘導(dǎo)分化 目的:探索以“ADSCs→神經(jīng)(干細(xì)胞)球(第一步)→神經(jīng)組織細(xì)胞(第二步)”的二步法誘導(dǎo)大鼠ADSCs向神經(jīng)細(xì)胞定向分化的方法。 方法:取第五代培養(yǎng)細(xì)胞進(jìn)行誘導(dǎo)分化:無(wú)血清Neurobasal培養(yǎng)基聯(lián)合應(yīng)用堿性成纖維生長(zhǎng)因子(basic fibroblast growth factor,bFGF)20ng/ml、表皮生長(zhǎng)因子(epithelial growth factor,EGF)20ng/ml和N2(1:100)添加劑,將細(xì)胞先誘導(dǎo)成神經(jīng)球,當(dāng)形成的神經(jīng)球達(dá)到15～20個(gè)/低倍視野時(shí),機(jī)械法輕柔吹打神經(jīng)球使之離散,再以Neurobasal培養(yǎng)基重懸細(xì)胞,按1:3比例進(jìn)行傳代。取第二代神經(jīng)球加入1%胎牛血清、5%馬血清、0.5μmol/L的維甲酸(retinoic acid,RA)和50ng/mL(brain-derived neurotrophic factor,BDNF),促進(jìn)其向神經(jīng)元和膠質(zhì)細(xì)胞方向分化。免疫細(xì)胞化學(xué)檢測(cè)巢蛋白(Nestin)、微管聯(lián)合蛋白2(MAP2)、β-神經(jīng)微管蛋白(β-tubulinⅢ)、神經(jīng)膠質(zhì)纖維酸性蛋白(gliafibrillary acidic protein,GFAP)及半乳糖苷酶(Galactocerebroside,GalC)等標(biāo)志性蛋白的表達(dá)情況。熒光顯微鏡下觀察細(xì)胞形態(tài)、拍照記錄,并進(jìn)行陽(yáng)性細(xì)胞記數(shù),掃描電鏡下觀察分化細(xì)胞的表面形貌。 結(jié)果:傳代純化后的ADSCs貼壁生長(zhǎng),呈梭形,增殖旺盛,幾乎不表達(dá)NSCs標(biāo)志性蛋白Nestin。無(wú)血清Neurobasal培養(yǎng)基誘導(dǎo)第3～4天時(shí),細(xì)胞開(kāi)始向中央聚集,形成多個(gè)克隆團(tuán),第5～7天時(shí),可見(jiàn)許多小的懸浮的神經(jīng)球出現(xiàn)。神經(jīng)球迅速增殖,14天的直徑可達(dá)100um。神經(jīng)球樣結(jié)構(gòu)在添加了血清及神經(jīng)營(yíng)養(yǎng)物的Neurobasal培養(yǎng)基中逐漸展開(kāi)并呈貼壁生長(zhǎng),細(xì)胞形態(tài)各異,生長(zhǎng)旺盛。于誘導(dǎo)培養(yǎng)后的第4～5天開(kāi)始,部分呈現(xiàn)典型的神經(jīng)元樣改變。掃描電鏡下觀察ADSCs在未分化狀態(tài)下,細(xì)胞呈扁平成纖維樣,細(xì)胞表面分泌物不多;ADSCs分化為神經(jīng)元后,細(xì)胞立體感增強(qiáng),細(xì)胞胞體變得梭長(zhǎng),并發(fā)出突起,胞體表面分泌物增多。免疫熒光染色顯示神經(jīng)球高度表達(dá)NSCs標(biāo)志性蛋白Nestin;自神經(jīng)球誘導(dǎo)分化而來(lái)的細(xì)胞大部分呈β-tubulinⅢ(早期神經(jīng)元標(biāo)志性蛋白)、MAP2(成熟神經(jīng)元標(biāo)志性蛋白)陽(yáng)性表達(dá)、有少量表達(dá)GFAP(神經(jīng)膠質(zhì)細(xì)胞標(biāo)志性蛋白)和GalC(少突膠質(zhì)細(xì)胞標(biāo)志性蛋白)。 結(jié)論:應(yīng)用本實(shí)驗(yàn)方法,以無(wú)血清Neurobasal培養(yǎng)基添加N2、bFGF、EGF等作為NSCs增殖培養(yǎng)基,可獲得較高比例的克隆形成。將ADSCs誘導(dǎo)成神經(jīng)球后再進(jìn)行傳代,可穩(wěn)定表達(dá)細(xì)胞特性,且在連續(xù)傳代培養(yǎng)中依舊維持這種克隆能力。光鏡及掃描電鏡下可見(jiàn)分化細(xì)胞呈現(xiàn)典型的神經(jīng)元樣形態(tài),免疫細(xì)胞化學(xué)證實(shí)分化細(xì)胞可表達(dá)高比率的神經(jīng)組織細(xì)胞特別是神經(jīng)元的特異性標(biāo)志,表明了本實(shí)驗(yàn)方案的高效性和可行性。
[Abstract]:Adipose - derived stromal cells ( ADSCs ) are a kind of stem cells which exist in adipose tissue and have multi - directional differentiation potential .



The first part : In vitro culture and amplification of ADSCs and their biological characteristics in rats



Objective : To isolate , purify and culture ADSCs in vitro and to observe their biological characteristics .



Methods : The retroperitoneal adipose tissue of SD rats was removed under sterile anesthesia . The cells were cultured in DMEM / F12 medium containing 10 % fetal bovine serum , cultured in DMEM / F12 medium containing 10 % fetal bovine serum and cultured in culture flask and photographed . The cell growth curve was detected by MTT assay , and the cell growth curve was drawn . Flow cytometry and immunofluorescence staining ( indirect method ) were used to detect the surface markers of CD34 , CD106 , CD44 and HLA - 1 .



Results : Under the microscope , most of the cells were spherical and suspended in the medium . There were a variety of morphology in the primary culture medium of ADSCs . The cells were characterized by a long shuttle shape , a long shuttle shape , a long shuttle shape and a fibroblast . The expression of the cells was detected by the flow cytometry . The expression of the markers was CD34 0.8 % , CD106 13.6 % , CD44 99.7 % , HLA - 1 95.7 % , which were in accordance with the phenotypic characteristics of ADSCs .



Conclusion : The cell isolation and culture method is simple and feasible , and the obtained ADSCs have high purity , vigorous cell viability and strong self - renewal ability .



Part Two : Differentiation of ADSCs into Neural Cells by Two - step Method



Objective : To explore the method of directional differentiation of ADSCs from ADSCs to neural stem cells ( first step ) 鈫，

本文編號(hào)：1794397