發(fā)布時(shí)間：2019-06-22 13:17

【摘要】： 2006年8月,Yamanaka小組將24種轉(zhuǎn)錄因子基因排列組合導(dǎo)入小鼠成纖維細(xì)胞,最終確定最少有4種轉(zhuǎn)錄因子組合——3ct4、Sox2、c-Myc和Klf4即可將成纖維細(xì)胞重編程為誘導(dǎo)性多潛能干(iPS)細(xì)胞,2007年11～12月,Yamanaka小組和Thomson小組先后將人的體細(xì)胞重編程為iPS細(xì)胞。這之后iPS細(xì)胞的研究和關(guān)注度呈爆炸式增長(zhǎng),且取得了一些突破性進(jìn)展,如建立了疾病特異的人iPS細(xì)胞、借助轉(zhuǎn)座子介導(dǎo)的轉(zhuǎn)基因方法高效制備了virus-free iPS細(xì)胞以及成功地從所獲得的iPS細(xì)胞中移除先前導(dǎo)入的轉(zhuǎn)錄因子基因。在利用特定小分子化合物的情況下,更少的外源基因?qū)爰纯筛咝诗@得iPS細(xì)胞,這向制備無(wú)遺傳修飾的iPS細(xì)胞方面邁出了一大步；此外,亦建立了大鼠和猴等的iPS細(xì)胞系,這些成績(jī)將iPS細(xì)胞在臨床上的實(shí)際應(yīng)用又大大向前推進(jìn)了一步,iPS細(xì)胞研究和應(yīng)用將有望成為21世紀(jì)最偉大的醫(yī)學(xué)生物學(xué)成就之一。 研究腫瘤細(xì)胞重編程的經(jīng)典方法有：①利用胚胎微環(huán)境,②囊胚腔注射,③利用胚胎干細(xì)胞生長(zhǎng)微環(huán)境。iPS細(xì)胞技術(shù)的問(wèn)世為研究腫瘤細(xì)胞重編程提供了一種新的思路和手段。利用Oct4、Sox2、C-myc和Klf4即可將腫瘤細(xì)胞重編程,2009年7月Hochedlinger課題組用Oct4、Sox2、c-Myc和Klf4將小鼠黑色素瘤細(xì)胞重編程為iPS細(xì)胞。iPS技術(shù)為在體外研究腫瘤細(xì)胞重編程提供了重要的技術(shù)平臺(tái),為研究腫瘤發(fā)病機(jī)制及腫瘤治療等方面提供一種新思路。 本課題借助慢病毒基因投遞法將Oct4、Sox2、Klf4和c-Myc四種基因?qū)肴似つw成纖維(CCD)細(xì)胞,進(jìn)而將其重編程為iPS細(xì)胞,從而實(shí)現(xiàn)以下目的：①為體外研究腫瘤細(xì)胞重編程構(gòu)筑技術(shù)平臺(tái)；②為后續(xù)研究[如iPS細(xì)胞生物學(xué)特性和行為(如自我復(fù)制、增殖和分化等)調(diào)控機(jī)制研究]奠定基礎(chǔ)。 目的： 借助慢病毒載體法將Oct4、Sox2、Klf4和c-Myc四種基因?qū)隒CD細(xì)胞,進(jìn)而將其重編程為iPS細(xì)胞,以實(shí)現(xiàn)多種目的(見(jiàn)上)。 方法： 1)攜帶Oct4、Sox2、C-Myc和Klf4基因的慢病毒載體鑒定 酶切鑒定分別用BamHⅠ/KpnⅠ和EcoRⅠ/EcoRⅤ進(jìn)行酶切,酶切產(chǎn)物進(jìn)行1%瓊脂糖凝膠電泳。 PCR鑒定根據(jù)Oct4、Klf4、c-Myc和Sox2序列設(shè)計(jì)引物分別擴(kuò)增Oct4、Klf4、c-Myc和Sox2基因；PCR擴(kuò)增后,取5μl反應(yīng)液進(jìn)行2%瓊脂糖凝膠電泳。 2)慢病毒生產(chǎn)與滴度測(cè)定 慢病毒包裝按標(biāo)準(zhǔn)程序進(jìn)行慢病毒包裝(脂質(zhì)體介導(dǎo)的轉(zhuǎn)染)。 慢病毒滴度測(cè)定用5μl病毒上清感染15萬(wàn)293T細(xì)胞,48h后4%多聚甲醛固定,DAPI染色,熒光顯微鏡下計(jì)數(shù)EGFP陽(yáng)性細(xì)胞和總細(xì)胞數(shù),將其帶入公式：病毒滴度(IU/ml)=EGFP陽(yáng)性細(xì)胞數(shù)／總細(xì)胞數(shù)÷5×1.5x105×103。 3)慢病毒載體法重編程CCD細(xì)胞為iPS細(xì)胞 a)取一定量的病毒上清過(guò)濾后懸浮感染CCD細(xì)胞。 b)病毒感染24小時(shí)后,將CCD細(xì)胞消化鋪于feeder細(xì)胞上,換為hES細(xì)胞培養(yǎng)基,第十天開(kāi)始換為條件培養(yǎng)基。 c)20天左右開(kāi)始挑取克隆,選擇AP陽(yáng)性細(xì)胞繼續(xù)后續(xù)工作。 4)人iPS細(xì)胞建系及其生物學(xué)特性鑒定 a)iPS細(xì)胞克隆形態(tài)觀察； b)細(xì)胞免疫熒光檢測(cè)人ES細(xì)胞標(biāo)志性抗原表達(dá)； c)提取iPS細(xì)胞總RNA,進(jìn)而RT-PCR檢測(cè)人ES細(xì)胞標(biāo)志性基因表達(dá)； d)人iPS細(xì)胞核型分析； e)懸浮培養(yǎng)觀察擬胚體形成及體外分化； f)iPS細(xì)胞接種至SCID小鼠皮下,觀察畸胎瘤形成及體內(nèi)分化情況。 結(jié)果： 1)攜帶Oct4、Sox2、C-Myc和Klf4基因的慢病毒載體鑒定 酶切鑒定攜帶Oct4、Sox2、C-Myc和Klf4基因的慢病毒載體分別經(jīng)BamH I/KpnⅠ和EcoRⅠ／EcoRⅤ酶切,酶切產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳均可見(jiàn)兩條預(yù)期大小的條帶。 PCR鑒定分別以攜帶Oct4、Sox2、C-Myc和Klf4基因的慢病毒載體為模板,擴(kuò)增Oct4、Klf4、c-Myc和Sox2基因,PCR產(chǎn)物大小均與預(yù)期值相符。 2)慢病毒生產(chǎn)與滴度測(cè)定 攜帶Oct4、Sox2、C-Myc和Klf4基因的慢病毒載體與病毒包裝質(zhì)粒共轉(zhuǎn)染293T細(xì)胞,24h后倒置熒光顯微鏡下可見(jiàn)綠色熒光,預(yù)示轉(zhuǎn)染成功。按照上述.方法進(jìn)行慢病毒滴度測(cè)定,病毒滴度值都在1×106IU/ml以上。 3)慢病毒載體法重編程CCD細(xì)胞為iPS細(xì)胞 用攜帶Oct4、Sox2、C-Myc和Klf4基因的慢病毒感染CCD細(xì)胞,24h后種植于飼養(yǎng)層(feeder)細(xì)胞上,并換為hES細(xì)胞培養(yǎng)基,48-72h在倒置熒光顯微鏡下觀察到綠色熒光,第十天左右開(kāi)始換為條件培養(yǎng)基。 4)人iPS細(xì)胞建系及其生物學(xué)特性鑒定 a)第7-8天可見(jiàn)CCD細(xì)胞形態(tài)發(fā)生變化,由長(zhǎng)梭形變?yōu)閳A形并聚集；第20天左右挑取克��； b)其中只有1株iPS細(xì)胞AP染色呈陽(yáng)性,且具有典型的hES細(xì)胞克隆形態(tài)； c)1株人iPS細(xì)胞系表達(dá)hES細(xì)胞特有的標(biāo)志性基因； d)1株人iPS細(xì)胞系表達(dá)hES細(xì)胞特有的標(biāo)志性抗原：SSAE-4(+), TRA-1-60(+),TRA-1-81(+),Oct4(+),SSAE-1(-)； e) 1株人iPS細(xì)胞核型分析未見(jiàn)異常； f) 1株人iPS細(xì)胞系具有體外分化形成囊性擬胚體的能力,并能表達(dá)各胚層的標(biāo)志性基因； g)畸胎瘤實(shí)驗(yàn)?zāi)壳斑�在進(jìn)行中。 結(jié)論： 1.運(yùn)用Oct4、Sox2、c-Myc和Klf4四種基因成功將CCD細(xì)胞重編程為iPS細(xì)胞。 2.1株人iPS細(xì)胞系具有人ES細(xì)胞的一些生物學(xué)特,且在體外長(zhǎng)期傳代中能夠維持此特性,初步證明建立了人iPS細(xì)胞系。
[Abstract]:In August 2006, the Yamanaka group introduced 24 transcription factor genes into the mouse fibroblast, and finally determined the least four transcription factor combinations _ 3ct4, Sox2, c-Myc and Klf4 to reprogram the fibroblasts to the induced pluripotent stem (iPS) cells, in November to December 2007, The Yamanaka group and the Thomson group have reprogrammed human body cells to iPS cells. After this, the research and attention of iPS cells is explosive, and some breakthrough progress has been made, such as the establishment of a disease-specific human iPS cell, The virus-free iPS cells were efficiently prepared by the transposon-mediated transgene method and the previously introduced transcription factor genes were successfully removed from the obtained iPS cells. In the case of using a specific small molecule compound, an iPS cell can be obtained with high efficiency with less foreign gene introduction, which is a significant step in the preparation of iPS cells without genetic modification; in addition, an iPS cell line such as a rat and a monkey is also established, These results have further advanced the practical application of iPS cells, and the research and application of iPS cells will be expected to be one of the greatest medical biological achievements in the 21 st century. The classical methods to study the reprogramming of the tumor cells are: the use of the embryo microenvironment, the injection of the embryo cavity, the growth of the embryo stem cells, The advent of iPS cell technology provides a new way to study the reprogramming of tumor cells And the tumor cells can be reprogrammed by using Oct4, Sox2, C-myc and Klf4, and the mouse melanoma cells are reprogrammed into the iP by the Hochedlinger's research group in July 2009 with Oct4, Sox2, c-Myc and Klf4. The S-cell. iPS technique provides an important technical platform for the in vitro study of the reprogramming of the tumor cells, and provides a method for studying the pathogenesis of the tumor and the treatment of the tumor. In this paper, the four genes of Oct4, Sox2, Klf4 and c-Myc were introduced into human skin fibroblast (CCD) cells by means of slow viral gene delivery, and then the cells were reprogrammed to iPS cells. A platform for the construction of a technology; a regulatory mechanism for follow-up studies, such as the biological characteristics and behavior of iPS cells, such as self-replication, proliferation and differentiation. [Study] The purpose of this paper is to introduce the four genes of Oct4, Sox2, Klf4 and c-Myc into CCD cells by means of lentiviral vector, and then to re-program them as iP. PS緇，

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