本文關(guān)鍵詞： 胚胎干細(xì)胞 胚胎后腎間充質(zhì)細(xì)胞 共培養(yǎng) 腎發(fā)生 分化　出處：《廣西醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 背景及目的：胚胎干細(xì)胞(embryonic stem cell, ESC)是指從早期胚胎內(nèi)細(xì)胞團(tuán)或原始生殖細(xì)胞分離出來的一種具有無限增殖能力和全方向分化能力的一種多潛能細(xì)胞,具有修復(fù)甚至替換喪失功能的組織和器官的潛在應(yīng)用價(jià)值。如何誘導(dǎo)胚胎干細(xì)胞向特定細(xì)胞類型的分化是其基礎(chǔ)研究和臨床治療應(yīng)用的關(guān)鍵點(diǎn)。胚胎干細(xì)胞的這種定向分化能力除了細(xì)胞內(nèi)各種轉(zhuǎn)錄因子的內(nèi)在決定因素外,細(xì)胞間因子的分化誘導(dǎo)、抑制作用及細(xì)胞外物質(zhì)的介導(dǎo)作用等外源性因素也是必不可少的。目前,在胚胎干細(xì)胞的眾多誘導(dǎo)分化方式中,采用共培養(yǎng)的方式來誘導(dǎo)其定向分化已經(jīng)成為組織工程領(lǐng)域一種新興而很有發(fā)展前景的技術(shù),在很多醫(yī)學(xué)領(lǐng)域都有成功的報(bào)道,如心血管、呼吸系統(tǒng)和肝臟等領(lǐng)域。在泌尿系統(tǒng)領(lǐng)域,既往研究發(fā)現(xiàn)胚胎干細(xì)胞與離體的胎腎器官共培養(yǎng)能成功誘導(dǎo)其向腎系細(xì)胞分化。但是有關(guān)胚胎干細(xì)胞在體外能否通過和一種特定的細(xì)胞相互作用繼而定向分化成腎系細(xì)胞并未見報(bào)道。為此本實(shí)驗(yàn)嘗試體外通過Transwell雙層培養(yǎng)皿將胚胎干細(xì)胞同大鼠胚胎后腎間充質(zhì)細(xì)胞(metanephric mesenchymal cells MMCs)進(jìn)行共培養(yǎng)的方法,觀察共培養(yǎng)條件下,胚胎干細(xì)胞能否定向分化成腎臟樣細(xì)胞,期望為下一步應(yīng)用于急慢性腎功能衰竭的動(dòng)物實(shí)驗(yàn)?zāi)Ｐ偷闹委煹於ɑA(chǔ)。 方法：1.取妊娠13.5d的胎鼠,采用組織消化法分離培養(yǎng)出小鼠胚胎成纖維細(xì)胞(Mouse Embryonic Fibroblasts MEFs),對(duì)MEFs的生長(zhǎng)形態(tài)、生長(zhǎng)曲線及分裂指數(shù)進(jìn)行觀察,MTT法篩選絲裂霉素C (Mytomycin C, MMC)作用的最佳濃度和時(shí)間。 2.收集昆明系小鼠3.5d的囊胚,培養(yǎng)在經(jīng)MMC處理的第三代小鼠胚胎成纖維細(xì)胞飼養(yǎng)層上,對(duì)分離克隆出的胚胎干細(xì)胞集落的形態(tài)學(xué)、堿性磷酸酶(Alkaline phosphatase, AKP)、全能型因子(OCT-4,NANOG)的表達(dá)、核型分析以及體內(nèi)外分化能力加以鑒定。 3.從孕12.5-14.5d的SD胎鼠中分離培養(yǎng)出后腎間充質(zhì)細(xì)胞,并對(duì)其進(jìn)行形態(tài)學(xué)、細(xì)胞免疫學(xué)和電鏡的鑒定。 4.采用直接懸浮培養(yǎng)法使小鼠胚胎干細(xì)胞形成擬胚體(EBs)后,將擬胚體細(xì)胞與胚胎后腎間充質(zhì)細(xì)胞通過Transwell培養(yǎng)系統(tǒng)共培養(yǎng)7d,RT-PCR檢測(cè)共培養(yǎng)的擬胚體細(xì)胞是否表達(dá)腎臟發(fā)育相關(guān)的特征基因(WT-1,Wnt-4, pax-2,c-Ret)和終末分化的腎細(xì)胞標(biāo)志物(podocalyxin, Nephrin)。 結(jié)果：1.MEFs為一種貼壁生長(zhǎng)且增殖速度較快的細(xì)胞,第三代細(xì)胞增殖旺盛,第5代以后細(xì)胞開始變形并趨于衰老。MMC能抑制胚胎成纖維細(xì)胞的增殖,最佳的作用濃度和時(shí)間是10ug／ml作用2.5-4.0h,20ug/ml作用1.0-2.5h。 2.分離得到的ES細(xì)胞有典型的形態(tài)學(xué)特征：集落呈鳥巢狀,邊緣清楚結(jié)構(gòu)致密,隆起生長(zhǎng)；堿性磷酸酶染色呈強(qiáng)陽性；RT-PCR分析顯示OCT-4,NANOG等全能性因子表達(dá)陽性；具有正常的二倍體核型；可形成擬胚體；皮下注射到免疫缺陷小鼠可形成包括三個(gè)胚層的畸胎瘤。 3.SD大鼠胚胎后腎間充質(zhì)細(xì)胞為一種貼壁生長(zhǎng)、生長(zhǎng)速度較快、呈漩渦樣生長(zhǎng)的細(xì)胞。細(xì)胞體積較小,胞漿少、胞核大、核仁明顯。間質(zhì)細(xì)胞標(biāo)志物波形蛋白陽性,而上皮細(xì)胞標(biāo)志物角蛋白為陰性。電鏡下細(xì)胞呈不規(guī)形,胞核大,內(nèi)有細(xì)胞器,細(xì)胞表面有微絨毛。 4.擬胚體細(xì)胞與胚胎后腎間充質(zhì)細(xì)胞共培養(yǎng)7d后,通過RT-PCR檢測(cè)到共培養(yǎng)后的胚胎干細(xì)胞表達(dá)腎臟發(fā)育相關(guān)標(biāo)志物(WT-1, Wnt-4,pax-2, c-Ret)和終末分化的腎細(xì)胞標(biāo)志物(podocalyxin, Nephrin)。 結(jié)論：采用共培養(yǎng)模型,大鼠胚胎后腎間充質(zhì)細(xì)胞可以誘導(dǎo)昆明小鼠胚胎干細(xì)胞定向分化為腎臟樣細(xì)胞。但是,分化得到的這種細(xì)胞僅僅是一種前祖細(xì)胞,而非一種特定的腎臟細(xì)胞。因此如何得到一種特定的腎臟細(xì)胞,以及任何將分化后的細(xì)胞從未分化的胚胎干細(xì)胞分離出來進(jìn)行純化從而應(yīng)用于急慢性腎功能衰竭動(dòng)物實(shí)驗(yàn)?zāi)Ｐ偷闹委熤袑⑹俏覀兿乱徊揭芯康年P(guān)鍵問題。
[Abstract]:Background and purpose: embryonic stem cells (embryonic stem cell, ESC) is separated from the inner cell mass of early embryos or primordial germ cells which proliferate and differentiate the ability of a pluripotent cells, tissues and organs with repair or replace the loss of function of the potential value. How to induce embryonic stem cells to differentiate into specific cell types is the key point of the basic research and clinical application. The orientation of embryonic stem cell differentiation in addition to internal factors of various transcription factors in cell differentiation, cell factor induced inhibition of extracellular substances mediated by exogenous factors is also essential at present, in embryonic stem cell differentiation in many ways, the co culture way to induce the differentiation of tissue engineering has become a The emerging and promising technology, has been reported in many medical fields such as cardiovascular, respiratory and liver and other fields. In the field of urinary system, previous studies have found that cells and isolated fetal kidney were cultured successfully induce the differentiation of embryonic stem cells into the kidney. But the embryonic stem cells in vitro and whether through a specific interaction between cells differentiate into renal cells and have not been reported. Therefore this experiment in vitro by Transwell double culture embryonic stem cells with rat metanephric mesenchymal cells (metanephric mesenchymal cells MMCs) by co culture method, co culture observation conditions that embryonic stem cells can differentiate into renal like cells, expected to lay the foundation treatment for experimental animal model for the next step in the application of acute and chronic renal failure.
Methods: 1. pregnant 13.5d fetal rats by tissue digestion of isolated mouse embryonic fibroblasts (Mouse Embryonic, Fibroblasts MEFs) on MEFs morphology, growth curve and mitotic index were observed by MTT screening of mitomycin C (Mytomycin C MMC) the optimal concentration and time.
2. Kunming mice were collected 3.5D blastocysts cultured fibroblast feeder cells in MMC treated mice on the third generation of cloned embryos, embryonic stem cell colony morphology, alkaline phosphatase (Alkaline phosphatase, AKP), universal factors (OCT-4, NANOG) expression, karyotype analysis and in vitro and in vivo the differentiation ability were identified.
3. the mesrenal mesenchyme cells were isolated and cultured from the pregnant 12.5-14.5d SD fetal rats, and their morphology, cell immunology and electron microscopy were identified.
4. by direct suspension culture method of mouse embryonic stem cells formed embryoid bodies (EBs), the embryoid body cells and rimm-18 cells cultured by Transwell co culture systems of 7D, RT-PCR detection of co cultured to whether embryo cells expression of kidney development characteristic base related (WT-1, Wnt-4, Pax-2, c-Ret) and terminally differentiated renal cell markers (podocalyxin, Nephrin).
緇撴灉錛，

本文編號(hào)：1514865