本文選題：胚胎成纖維細胞 + 逆轉錄病毒��； 參考：《南方醫(yī)科大學》2012年碩士論文

【摘要】：多潛能干細胞(pluripotent stem cells)是指能夠自我更新并具有分化成多種細胞潛能的細胞,它在細胞替代治療、基因治療、發(fā)育生物學等研究中具有獨特的作用及優(yōu)越性。目前,最為人類所熟知的多潛能性干細胞是胚胎干細胞(embryonic stem cells, ES細胞),它一般是從囊胚的內(nèi)細胞團經(jīng)過適當?shù)呐囵B(yǎng)而產(chǎn)生,也可通過體細胞核轉移或細胞融合等途徑得到多潛能性干細胞。然而胚胎干細胞的研究,尤其是人胚胎干細胞,都需要破壞胚胎或卵細胞,引發(fā)諸多倫理爭議,應用于臨床時還將面臨免疫排斥等問題。誘導性多能干細胞(induced pluripotent stem cells, iPS細胞)的出現(xiàn)為解決上述難題提供了希望。與ESCs相比,iPS細胞有其獨到的優(yōu)勢：①iPS由體細胞誘導產(chǎn)生,不存在ESCs面臨的倫理問題；②iPS細胞來源廣泛,iPS可來自患者體細胞(包括病變體細胞),經(jīng)過克隆增殖分化可滿足臨床移植治療數(shù)量上的要求；③來自患者的iPS避免了一直困擾再生醫(yī)學界的免疫排斥難題。因此自iPS技術誕生以來,其在誘導效率、安全性及臨床應用等相關研究中取得了巨大的進展。本研究利用逆轉錄病毒介導的方法將三個干細胞因子Oct4、Sox2、Klf4導入小鼠胚胎成纖維細胞(mouse embryonic fibroblasts, MEFs),成功獲得了小鼠iPS細胞,并對其干細胞標記的表達及全能性進行了分析鑒定。 目的：1.利用Yamanaka三因子(Oct4、Sox2、Klf4)構建小鼠誘導性多能干細胞系。2.對所獲得的iPS細胞進行鑒定及全能性分析,以期獲得與胚胎干細胞類似的多能干細胞,為本課題后續(xù)將iPS細胞進一步定向分化為胰島樣細胞提供實驗基礎和技術平臺。 方法： 1.逆轉錄病毒的制備：包含Oct4,Sox2和K1f4三個基因的逆轉錄病毒質(zhì)粒pMXs-Oct3、pMXs-Sox2、pMXs-Klf4及參照質(zhì)粒pMXs-EGFP由Addgene公司購買,使用DH5α細菌進行擴增后將4個質(zhì)粒分別轉染至逆轉錄病毒包裝細胞Plat-E,48h后收集病毒上清,過濾、濃縮后用于感染MEFs。 2.鼠胚成纖維細胞的分離與培養(yǎng)：無菌分離孕13.5d的BALB/C鼠胚,消化后接種至平皿培養(yǎng)。1-3代內(nèi)的細胞用于iPS細胞的誘導。 3.逆轉錄病毒感染MEF及iPS細胞的產(chǎn)生：將收集的病毒上清感染生長良好的MEFs,約1周時可首次看到iPS克隆,20d左右iPS克隆可長至做夠大被挑取擴大培養(yǎng)。 4.iPS細胞的生物學特性鑒定：通過鏡下觀察、堿性磷酸酶染色、反轉錄PCR(RT-PCR)、免疫熒光實驗及畸胎瘤形成實驗等對iPS細胞形態(tài)、多能性基因表達情況、干細胞表面標記及全能性等進行鑒定分析。 結果： 1.逆轉錄病毒的制備：逆轉錄病毒質(zhì)粒轉染Plat-E細胞48h后,熒光顯微鏡下可見綠色熒光表達(pMXs-EGFP),轉染效率約在50-70%之間,細胞狀態(tài)良好。 2.鼠胚成纖維細胞的分離與培養(yǎng)：MEFs原代培養(yǎng)在接種后3-4h貼壁,細胞呈長梭形、多邊形或不規(guī)則形,中間1-2個圓形或卵圓形的核,原代MEF雜細胞較多,隨著傳代次數(shù)的增多,其純度增高,一般第二代后即可得到較純的細胞。 3.逆轉錄病毒感染MEFs及iPS細胞的產(chǎn)生：包含三個干細胞基因的逆轉錄病毒感染MEFs后大約一周左右可以在鏡下首次看到iPS克隆,而后克隆繼續(xù)增殖,12d左右形成肉眼可見的iPS集落,16-20d時克隆長至足夠大,此時進行挑取、消化、接種至飼細胞上傳代培養(yǎng)。 4.iPS細胞的生物學特性鑒定：從MEFs獲得的iPSCs鏡下觀察呈典型的克隆狀生長,圓形或橢圓形,與飼細胞分界清楚；RT-PCR、堿性磷酸酶染色及免疫熒光檢測iPS細胞高表達胚胎干細胞相關基因及蛋白；種植在免疫缺陷鼠體內(nèi)能夠形成向內(nèi)中外三個胚層分化的畸胎瘤,表明iPSCs具有多潛能性。未感染逆轉錄病毒的MEFs不具有上述ES相關的細胞特性。 結論： 1.通過轉導三個重編程因子(Oct4、Sox2、Klf4)可以將小鼠胚胎成纖維細胞誘導為多能干細胞,即iPS細胞。 2.該iPS細胞具有ES樣的生物學特性,并能長期多次傳代而維持,表明初步建立了小鼠iPS細胞系。
[Abstract]:Pluripotent stem cells is a cell that is self renewing and has the potential to differentiate into multiple cells. It has a unique role and superiority in cell replacement therapy, gene therapy and developmental biology. At present, the most human known pluripotent stem cells are embryonic stem cells (embryonic stem CE). LLS, ES cells), it is usually produced by proper culture of the inner cell mass of the blastocyst, and can also obtain pluripotent stem cells through the transfer of somatic cell nucleus or cell fusion. However, embryonic stem cells, especially human embryonic stem cells, need to destroy embryo or egg cells and cause a lot of ethical disputes to be applied to the clinic. The emergence of inducible pluripotent stem cells (induced pluripotent stem cells, iPS cells) provides hope for solving the above problems. Compared with ESCs, iPS cells have their unique advantages: (1) iPS is induced by somatic cells, and there is no ethical problem in ESCs face; (2) iPS cells are widely derived and iPS can come. From patients' somatic cells (including somatic cells), cloned proliferation and differentiation can meet the requirements of clinical transplantation treatment. (3) iPS from patients has avoided the problem of immune rejection that has been puzzling the regenerative medicine community. Since the birth of iPS technology, it has made great achievements in the related research of induction efficiency, safety and clinical application. Great progress. In this study, three stem cell factors Oct4, Sox2, and Klf4 were introduced into mouse embryonic fibroblasts (mouse embryonic fibroblasts, MEFs) by retrovirus mediated method. The mouse iPS cells were successfully obtained and the expression and omnipotency of the stem cell markers were analyzed and identified.
Objective: 1. using Yamanaka three factor (Oct4, Sox2, Klf4) to construct mouse induced pluripotent stem cell line.2. to identify and analyze the acquired iPS cells, in order to obtain the pluripotent stem cells similar to embryonic stem cells, and provide experimental basis and technology for further directing the differentiation of iPS cells into islet like cells. Platform.
Method錛，

本文編號：1864694