本文選題：胚胎干細(xì)胞　切入點(diǎn)：分化　出處：《鄭州大學(xué)》2008年碩士論文

【摘要】：胚胎干細(xì)胞(embryonic stem cells,ESCs)是從早期囊胚的內(nèi)細(xì)胞團(tuán)(innercell mass,ICM)分離出來的一種多能細(xì)胞系;它能在體外長期不斷自我更新,并保持多向分化潛能,可以分化為內(nèi)、中、外三胚層來源的幾乎所有類型的細(xì)胞。已有研究表明,ESCs在特定的誘導(dǎo)培養(yǎng)體系中,可被定向誘導(dǎo)分化為神經(jīng)細(xì)胞,為ESCs移植治療神經(jīng)系統(tǒng)疾病提供了實(shí)踐依據(jù)。迄今為止,體外誘導(dǎo)法主要有4-/4+法、五步法、基質(zhì)細(xì)胞誘導(dǎo)法、單層粘附培養(yǎng)法和基因修飾法等。其中4-/4+法是最經(jīng)典的誘導(dǎo)法,但該方法使用維甲酸(retinoic acid,RA)誘導(dǎo)前需形成類胚體(embryonic bodies,EB),使分化較難控制和分析,且培養(yǎng)時(shí)間長、操作復(fù)雜,對細(xì)胞密度、營養(yǎng)、設(shè)備等培養(yǎng)要求較高。探索新的細(xì)胞誘導(dǎo)方法用以減少誘導(dǎo)時(shí)間、簡化操作將促進(jìn)ESCs體外培養(yǎng)分化的研究。目前對ESCs分化為神經(jīng)細(xì)胞的機(jī)制仍不清。有報(bào)道稱在ESCs向神經(jīng)細(xì)胞分化過程中出現(xiàn)某些蛋白的表達(dá)增高(如PKC_δ和PKC_ε)或者降低(如notch1)。在對大鼠嗜鉻神經(jīng)瘤PC12細(xì)胞分化和小鼠小腦神經(jīng)元發(fā)育的研究中均發(fā)現(xiàn)細(xì)胞骨架蛋白4.1蛋白的表達(dá)量、剪切形式和細(xì)胞定位會隨著神經(jīng)細(xì)胞的分化而改變。但4.1蛋白在ESCs向神經(jīng)細(xì)胞分化過程中的改變尚有待研究。 目的: 1.建立一種新的體外定向誘導(dǎo)ESCs分化為神經(jīng)細(xì)胞的方法,從而縮短誘導(dǎo)分化時(shí)間、簡化體外操作過程。 2.研究在ESCs分化為神經(jīng)細(xì)胞過程中4.1N和4.1B的表達(dá)情況。 方法: 采用鄭州大學(xué)干細(xì)胞中心培育的小鼠胚胎干細(xì)胞系。組織塊原代培養(yǎng)法制備小鼠胚胎成纖維細(xì)胞(MEF)。ESCs復(fù)蘇后直接接種到經(jīng)絲裂霉素C(MMC)處理的MEF上,使用含白血病抑制因子(LIF)的MEF條件化培養(yǎng)基(CM)進(jìn)行培養(yǎng)和傳代。 待ESCs生長穩(wěn)定,以10~5個(gè)/ml接種于培養(yǎng)皿中。分三組:(1)對照組:用EB培養(yǎng)基重懸后貼壁培養(yǎng),不加入任何誘導(dǎo)劑,自然分化。(2)4-/4+誘導(dǎo)組:用EB培養(yǎng)基重懸,懸浮培養(yǎng)4d,每2-3 h搖晃1次,防止EB貼壁,第5d收集細(xì)胞,用誘導(dǎo)培養(yǎng)基(含10~(-6) mol/L RA)重懸,接種至明膠包被的培養(yǎng)皿,4 d后換為EB培養(yǎng)基繼續(xù)培養(yǎng)。(3)RA序貫誘導(dǎo)組:用誘導(dǎo)培養(yǎng)基(含10~(-6) mol/L RA的EB培養(yǎng)基)重懸,貼壁培養(yǎng)48 h后,每個(gè)培養(yǎng)皿加入3 ml神經(jīng)干細(xì)胞培養(yǎng)基(含20μg/L bFGF、2%B27、1%N2的無血清DMEM/F12培養(yǎng)基)培養(yǎng)2 d,此后每天加入1 ml神經(jīng)細(xì)胞培養(yǎng)基(含2%B27、15%FBS的DMEM/F12培養(yǎng)基),直到第7 d用神經(jīng)細(xì)胞培養(yǎng)基換液,繼續(xù)培養(yǎng)。 倒置顯微鏡觀察分化過程中細(xì)胞形態(tài);免疫細(xì)胞化學(xué)法檢測分化第10 d各組成熟神經(jīng)元標(biāo)志性抗原神經(jīng)元特異性烯醇化酶(NSE)和星形膠質(zhì)細(xì)胞標(biāo)志性抗原膠質(zhì)纖維酸性蛋白(GFAP),計(jì)算NSE和GFAP的陽性細(xì)胞率;Western-blot檢測分化前后4.1N和4.1B的表達(dá)量。 結(jié)果: 倒置顯微鏡下滋養(yǎng)層細(xì)胞MEF表現(xiàn)為100%融合的貼壁生長的梭形細(xì)胞。分化前ESCs呈圓形或橢圓形克隆樣生長,折光性強(qiáng),細(xì)胞排列緊密。對照組自然分化10 d后可見克隆團(tuán)邊緣幾乎均為圓形高亮的上皮樣細(xì)胞,外周為少量梭形細(xì)胞;4-/4+誘導(dǎo)組分化第5 d貼壁12 h后即可見梭形細(xì)胞爬出;分化第10 d神經(jīng)樣細(xì)胞突起細(xì)長并形成網(wǎng)絡(luò);RA序貫誘導(dǎo)組分化第5 d出現(xiàn)神經(jīng)細(xì)胞樣改變,細(xì)胞突起細(xì)長;神經(jīng)元樣細(xì)胞逐漸增多,分化第7 d呈雙極性或多極性,形成網(wǎng)絡(luò)。 免疫細(xì)胞化學(xué)法檢測分化第10 d對照組、4-/4+誘導(dǎo)組和RA序貫誘導(dǎo)組的NSE陽性細(xì)胞率分別是11.8%±1.8%、50.6%±2.7%和57.6%±1.4%;GFAP陽性細(xì)胞率分別是19.0%±2.9%、36.0%±2.5%和33.0%±2.0%。單因素方差分析顯示對照組和兩組誘導(dǎo)組之間有顯著性差異(P＜0.01),4-/4+誘導(dǎo)組和RA序貫誘導(dǎo)組之間無顯著性差異(P＞0.05)。 Western-blot檢測顯示:未分化ESCs中表達(dá)4.1N和4.1B蛋白,誘導(dǎo)分化后兩者表達(dá)量都增加。 結(jié)論 1.RA序貫誘導(dǎo)法可在ESCs分化后第7d獲得神經(jīng)元樣細(xì)胞,較4-/4+誘導(dǎo)法提前3d,且兩者的NSE和GFAP陽性細(xì)胞率無差異,因此RA序貫誘導(dǎo)法可作為經(jīng)典的4-/4+誘導(dǎo)法的替代方法。 2.4.1N和4.1B蛋白的表達(dá)量隨著ESCs分化為神經(jīng)細(xì)胞而增加。
[Abstract]:Embryonic stem cells (embryonic stem cells, ESCs) is from the inner cell mass of the early blastocyst (innercell mass ICM) is a kind of pluripotent cell lines isolated in vitro; it can continuously self renew and maintain multilineage differentiation potential and can differentiate into inside, outside the three germ layers of almost all sources cell types. Studies have shown that ESCs in the specific induction culture system, can be induced to differentiate into neural cells and provide a practical basis for ESCs transplantation for the treatment of diseases of the nervous system. So far, the main method in vitro by 4-/4+ method, five step, stromal cells induced by adhesion, monolayer culture and gene modification method. The 4-/4+ method is induced by the most classical method, but this method is the use of retinoic acid (retinoic, acid, RA) before induction to the formation of embryoid bodies (embryonic, bodies, EB), the differentiation is difficult to control and analysis, and the training time is long, complex operation of Cell density, nutrition, equipment and other training requirements higher. To explore new cells induced to reduce the induction time, simplify the operation will promote the differentiation of cultured ESCs in vitro. The mechanism of ESCs differentiation into neural cells is still unclear. Some reports said the expression of some proteins to neural cells during the differentiation process of ESCs. (such as PKC_ PKC_ and delta epsilon) or lower (such as Notch1). The rats addicted to the expression of protein 4.1 were found on PC12 cell differentiation and neuron development of mouse cerebellum chromium neuroma and cell form, shear position will change with the differentiation of neural cells. But 4.1 proteins in ESCs the process of neural cell differentiation changes need to be studied.
Objective:
1. a new method of inducing ESCs to differentiate into nerve cells in vitro is established, thus shortening the induction time and simplifying the operation process in vitro.
2. the expression of 4.1N and 4.1B during the differentiation of ESCs into neural cells was studied.
Method:
The Zhengzhou University Center for stem cell mouse embryonic stem cell lines. Tissue cultured preparation of mouse embryonic fibroblasts (MEF).ESCs after resuscitation to direct inoculation of mitomycin C (MMC) treatment on MEF with leukemia inhibitory factor (LIF) MEF of culture medium (CM) were cultured and passaged.
寰匛SCs鐢熼暱紼沖畾,浠，

本文編號：1667490