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

【摘要】：多潛能干細(xì)胞(pluripotent stem cells)是指能夠自我更新并具有分化成多種細(xì)胞潛能的細(xì)胞,它在細(xì)胞替代治療、基因治療、發(fā)育生物學(xué)等研究中具有獨(dú)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(xì)胞是胚胎干細(xì)胞(embryonic stem cells, ES細(xì)胞),它一般是從囊胚的內(nèi)細(xì)胞團(tuán)經(jīng)過(guò)適當(dāng)?shù)呐囵B(yǎng)而產(chǎn)生,也可通過(guò)體細(xì)胞核轉(zhuǎn)移或細(xì)胞融合等途徑得到多潛能性干細(xì)胞。然而胚胎干細(xì)胞的研究,尤其是人胚胎干細(xì)胞,都需要破壞胚胎或卵細(xì)胞,引發(fā)諸多倫理爭(zhēng)議,應(yīng)用于臨床時(shí)還將面臨免疫排斥等問(wèn)題。誘導(dǎo)性多能干細(xì)胞(induced pluripotent stem cells, iPS細(xì)胞)的出現(xiàn)為解決上述難題提供了希望。與ESCs相比,iPS細(xì)胞有其獨(dú)到的優(yōu)勢(shì)：①iPS由體細(xì)胞誘導(dǎo)產(chǎn)生,不存在ESCs面臨的倫理問(wèn)題；②iPS細(xì)胞來(lái)源廣泛,iPS可來(lái)自患者體細(xì)胞(包括病變體細(xì)胞),經(jīng)過(guò)克隆增殖分化可滿足臨床移植治療數(shù)量上的要求；③來(lái)自患者的iPS避免了一直困擾再生醫(yī)學(xué)界的免疫排斥難題。因此自iPS技術(shù)誕生以來(lái),其在誘導(dǎo)效率、安全性及臨床應(yīng)用等相關(guān)研究中取得了巨大的進(jìn)展。本研究利用逆轉(zhuǎn)錄病毒介導(dǎo)的方法將三個(gè)干細(xì)胞因子Oct4、Sox2、Klf4導(dǎo)入小鼠胚胎成纖維細(xì)胞(mouse embryonic fibroblasts, MEFs),成功獲得了小鼠iPS細(xì)胞,并對(duì)其干細(xì)胞標(biāo)記的表達(dá)及全能性進(jìn)行了分析鑒定。 目的：1.利用Yamanaka三因子(Oct4、Sox2、Klf4)構(gòu)建小鼠誘導(dǎo)性多能干細(xì)胞系。2.對(duì)所獲得的iPS細(xì)胞進(jìn)行鑒定及全能性分析,以期獲得與胚胎干細(xì)胞類似的多能干細(xì)胞,為本課題后續(xù)將iPS細(xì)胞進(jìn)一步定向分化為胰島樣細(xì)胞提供實(shí)驗(yàn)基礎(chǔ)和技術(shù)平臺(tái)。 方法： 1.逆轉(zhuǎn)錄病毒的制備：包含Oct4,Sox2和K1f4三個(gè)基因的逆轉(zhuǎn)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(gòu)買,使用DH5α細(xì)菌進(jìn)行擴(kuò)增后將4個(gè)質(zhì)粒分別轉(zhuǎn)染至逆轉(zhuǎn)錄病毒包裝細(xì)胞Plat-E,48h后收集病毒上清,過(guò)濾、濃縮后用于感染MEFs。 2.鼠胚成纖維細(xì)胞的分離與培養(yǎng)：無(wú)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(xì)胞用于iPS細(xì)胞的誘導(dǎo)。 3.逆轉(zhuǎn)錄病毒感染MEF及iPS細(xì)胞的產(chǎn)生：將收集的病毒上清感染生長(zhǎng)良好的MEFs,約1周時(shí)可首次看到iPS克隆,20d左右iPS克隆可長(zhǎng)至做夠大被挑取擴(kuò)大培養(yǎng)。 4.iPS細(xì)胞的生物學(xué)特性鑒定：通過(guò)鏡下觀察、堿性磷酸酶染色、反轉(zhuǎn)錄PCR(RT-PCR)、免疫熒光實(shí)驗(yàn)及畸胎瘤形成實(shí)驗(yàn)等對(duì)iPS細(xì)胞形態(tài)、多能性基因表達(dá)情況、干細(xì)胞表面標(biāo)記及全能性等進(jìn)行鑒定分析。 結(jié)果： 1.逆轉(zhuǎn)錄病毒的制備：逆轉(zhuǎn)錄病毒質(zhì)粒轉(zhuǎn)染Plat-E細(xì)胞48h后,熒光顯微鏡下可見(jiàn)綠色熒光表達(dá)(pMXs-EGFP),轉(zhuǎn)染效率約在50-70%之間,細(xì)胞狀態(tài)良好。 2.鼠胚成纖維細(xì)胞的分離與培養(yǎng)：MEFs原代培養(yǎng)在接種后3-4h貼壁,細(xì)胞呈長(zhǎng)梭形、多邊形或不規(guī)則形,中間1-2個(gè)圓形或卵圓形的核,原代MEF雜細(xì)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(xì)胞。 3.逆轉(zhuǎn)錄病毒感染MEFs及iPS細(xì)胞的產(chǎn)生：包含三個(gè)干細(xì)胞基因的逆轉(zhuǎn)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(jiàn)的iPS集落,16-20d時(shí)克隆長(zhǎng)至足夠大,此時(shí)進(jìn)行挑取、消化、接種至飼細(xì)胞上傳代培養(yǎng)。 4.iPS細(xì)胞的生物學(xué)特性鑒定：從MEFs獲得的iPSCs鏡下觀察呈典型的克隆狀生長(zhǎng),圓形或橢圓形,與飼細(xì)胞分界清楚；RT-PCR、堿性磷酸酶染色及免疫熒光檢測(cè)iPS細(xì)胞高表達(dá)胚胎干細(xì)胞相關(guān)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(gè)胚層分化的畸胎瘤,表明iPSCs具有多潛能性。未感染逆轉(zhuǎn)錄病毒的MEFs不具有上述ES相關(guān)的細(xì)胞特性。 結(jié)論： 1.通過(guò)轉(zhuǎn)導(dǎo)三個(gè)重編程因子(Oct4、Sox2、Klf4)可以將小鼠胚胎成纖維細(xì)胞誘導(dǎo)為多能干細(xì)胞,即iPS細(xì)胞。 2.該iPS細(xì)胞具有ES樣的生物學(xué)特性,并能長(zhǎng)期多次傳代而維持,表明初步建立了小鼠iPS細(xì)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(hào)：1864694