本文選題：基因克隆 + JHDM2A��； 參考：《廣西大學(xué)》2017年碩士論文

【摘要】：iPS技術(shù)為構(gòu)建疾病模型、研制評(píng)估新藥、基因治療及再生醫(yī)學(xué)等研究奠定了基礎(chǔ),是干細(xì)胞研究領(lǐng)域的里程碑。豬在生理及體型上與人相似,因此研究豬的iPS細(xì)胞意義重大。但目前豬成纖維細(xì)胞誘導(dǎo)為iPS的重編程效率不足1%。研究發(fā)現(xiàn),組蛋白H3K9甲基化阻礙體細(xì)胞重編程,降低H3K9甲基化水平可促進(jìn)iPS誘導(dǎo)。組蛋白甲基化水平受組蛋白甲基轉(zhuǎn)移酶與組蛋白去甲基化酶共同調(diào)節(jié)。JHDM2A,即JmjC結(jié)構(gòu)域組蛋白去甲基化酶2,可使H3K9me1/2發(fā)生去甲基化。過(guò)表達(dá)JHDM2A基因可促進(jìn)與ESCs融合的小鼠NSCs重編程為iPS,促進(jìn)iPSCs誘導(dǎo);還可調(diào)節(jié)胚胎干細(xì)胞的自我更新。本研究首先采用DOX誘導(dǎo)型的慢病毒載體體系,誘導(dǎo)獲得豬多能干細(xì)胞(piPSCs),在此基礎(chǔ)上通過(guò)過(guò)表達(dá)JHDM2A基因,探究組蛋白H3K9me1/2甲基化水平對(duì)豬iPSCs誘導(dǎo)效率的影響,并對(duì)其分子機(jī)制進(jìn)行初步探究,以便為進(jìn)一步提高iPSCs誘導(dǎo)效率、建立大家畜ESCs系奠定基礎(chǔ)。本研究主要結(jié)果如下:1、豬JHDM2A基因克隆及其表達(dá)載體構(gòu)建根據(jù)NCBI上公布的豬JHDM2A基因序列,設(shè)計(jì)特異性擴(kuò)增引物,克隆得到豬JHDM2A基因,其編碼區(qū)長(zhǎng)度為3945bp,編碼1315個(gè)氨基酸。多重氨基酸序列比對(duì)發(fā)現(xiàn),豬JHDM2A基因與黃牛、水牛、綿羊和人相應(yīng)氨基酸序列的同源性分別為93.5%、94.7%、94.7%、93.8%。蛋白質(zhì)分子系統(tǒng)進(jìn)化樹分析結(jié)果表明,JHDM2A基因在物種進(jìn)化過(guò)程中高度保守。免疫組化結(jié)果顯示,JHDM2J蛋白在不同發(fā)育階段豬卵泡中均有表達(dá)。進(jìn)一步構(gòu)建并驗(yàn)證了 pLVX-IRES-ZsGreen1-JHDM2A真核表達(dá)載體。2、過(guò)表達(dá)JHDM2A基因?qū)ωiiPSCs形成及相關(guān)基因表達(dá)的影響首先利用DOX誘導(dǎo)型慢病毒載體體系,將OSKM因子組合導(dǎo)入豬成纖維細(xì)胞,將其誘導(dǎo)為iPSCs,在此基礎(chǔ)上過(guò)表達(dá)JHDM2A基因,并分別對(duì)獲得的iPSCs進(jìn)行鑒定。當(dāng)過(guò)表達(dá)JHDM2A基因時(shí),細(xì)胞在第3 d出現(xiàn)形變,第8 d形成piPSCs克隆,分別比未處理組提前了 1d、2d。AP檢測(cè)陽(yáng)性克隆數(shù)結(jié)果發(fā)現(xiàn),與未處理組相比,過(guò)表達(dá)JHDM2A基因?qū)嶒?yàn)組的誘導(dǎo)效率提高了 8倍,且iPSCs克隆大小更均勻、邊緣更整齊、細(xì)胞更致密,AP染色著色更深。RT-PCR分析結(jié)果顯示,所檢測(cè)的三種細(xì)胞均表達(dá)Klf、c-Myc、Nanog多能性基因,弱表達(dá)Sox2基因;過(guò)表達(dá)JHDM2A實(shí)驗(yàn)組和未處理組的piPSCs均表達(dá)Oct4,而PFF弱表達(dá)表達(dá)Oct4。定量PCR結(jié)果顯示,與未處理組相比,過(guò)表達(dá)JHDM2A實(shí)驗(yàn)組 piPSCs 多能性基因 Sox2、klf4、c-Myc、Nanog表達(dá)顯著升高(P0.05),其中對(duì)Oct4的表達(dá)影響極顯著(P0.01)。去DOX后,過(guò)表達(dá)JHDM2A實(shí)驗(yàn)組細(xì)胞中的外源基因沉默,內(nèi)源性O(shè)ct4基因表達(dá)逐代幾乎沒(méi)有變化,Sox2、Nanog基因表達(dá)逐代降低,Klf4、c-Myc基因表達(dá)先升高后降低。與未處理組和PFF相比,過(guò)表達(dá)JHDM2A實(shí)驗(yàn)組piPSC組蛋白甲基化基因Tcll的表達(dá)顯著提高(P0.05),TcfTp2L1和Zfp57的表達(dá)顯著降低(P0.05)以上結(jié)果表明,克隆獲得了豬JHDM2A基因并構(gòu)建了其慢病毒表達(dá)載體,過(guò)表達(dá)JHDM2A基因可通過(guò)調(diào)控組蛋白甲基化水平提高piPSCs的誘導(dǎo)效率。
[Abstract]:IPS technology is a milestone in stem cell research, which has laid a foundation for the establishment of disease models, the development and evaluation of new drugs, gene therapy and regenerative medicine. Pigs are physiologically and physically similar to humans, so it is of great significance to study iPS cells in pigs. However, the reprogramming efficiency of inducing iPS from pig fibroblasts is less than 1%. It was found that histone H 3K 9 methylation hinders somatic reprogramming and promotes iPS induction by reducing H 3K 9 methylation level. Histone methylation level is regulated by histone methyltransferase and histone demethylase. JHDM2A, or JmjC domain histone demethylase 2, can demethylate H3K9me1 / 2. Overexpression of JHDM2A gene could promote the reprogramming of mouse NSCs fused with ESCs into iPSs, promote the induction of iPSCs, and regulate the self-renewal of embryonic stem cells. In this study, porcine pluripotent stem cells (piPSCs) were induced by DOX-induced lentivirus vector system, and the effect of histone H3K9me1 / 2 methylation level on the induction efficiency of porcine iPSCs was investigated by overexpressing JHDM2A gene. In order to improve the induction efficiency of iPSCs and to establish the ESCs system of large livestock, the molecular mechanism of IPSCs was preliminarily explored. The main results were as follows: 1: 1, pig JHDM2A gene clone and its expression vector were constructed according to the sequence of porcine JHDM2A gene published on NCBI. Specific primers were designed and cloned into porcine JHDM2A gene. The length of encoding region was 3945 BP, encoding 1315 amino acids. Multiple amino acid sequence alignment showed that the homology of pig JHDM2A gene with corresponding amino acid sequences of yellow cattle, buffalo, sheep and human was 93.50.94. 774 and 94.794. 775, respectively. The results of phylogenetic tree analysis showed that JHDM2A gene was highly conserved during species evolution. Immunohistochemical results showed that JHDM2J protein was expressed in pig follicles at different stages of development. Furthermore, the eukaryotic expression vector of pLVX-IRES-ZsGreen1-JHDM2A was constructed and verified. The effect of overexpression of JHDM2A gene on the formation of porcine iPSCs and the expression of related genes was confirmed. Firstly, the combination of OSKM factors was introduced into porcine fibroblasts by DOX-induced lentivirus vector system. The JHDM2A gene was overexpressed and the obtained iPSCs were identified. When the JHDM2A gene was overexpressed, the cells deformed on the 3rd day and formed the piPSCs clone on the 8th day. The induction efficiency of overexpression of JHDM2A gene in the experimental group was increased by 8 times, and the cloning size of iPSCs was more uniform, the edges were more neat, and the cells were denser and stained with AP. RT-PCR analysis showed that all the three kinds of cells expressed KlfCc-MycNa-Nanog multipotent gene. PSCs of JHDM2A experimental group and untreated group expressed Oct4, while PFF expressed Oct4 weakly. The results of quantitative PCR showed that the overexpression of JHDM2A gene in piPSCs was significantly higher than that in untreated group (P0.05), and the expression of Oct4 was significantly affected (P0.01). After DOX was removed, the expression of exogenous gene was silenced in JHDM2A experimental group, and the expression of endogenous Oct4 gene was almost unchanged from generation to generation. Compared with untreated group and PFF group, the expression of histone methylation gene Tcll of piPSC in the experimental group of JHDM2A was significantly increased (P0.05) and the expression of TcfTp2L1 and Zfp57 was significantly decreased (P0.05). The results showed that the porcine JHDM2A gene was cloned and its lentivirus expression vector was constructed. Overexpression of JHDM2A gene can improve the induction efficiency of piPSCs by regulating histone methylation level.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78

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