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

【摘要】：iPS技術(shù)為構(gòu)建疾病模型、研制評估新藥、基因治療及再生醫(yī)學等研究奠定了基礎,是干細胞研究領域的里程碑。豬在生理及體型上與人相似,因此研究豬的iPS細胞意義重大。但目前豬成纖維細胞誘導為iPS的重編程效率不足1%。研究發(fā)現(xiàn),組蛋白H3K9甲基化阻礙體細胞重編程,降低H3K9甲基化水平可促進iPS誘導。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。過表達JHDM2A基因可促進與ESCs融合的小鼠NSCs重編程為iPS,促進iPSCs誘導;還可調(diào)節(jié)胚胎干細胞的自我更新。本研究首先采用DOX誘導型的慢病毒載體體系,誘導獲得豬多能干細胞(piPSCs),在此基礎上通過過表達JHDM2A基因,探究組蛋白H3K9me1/2甲基化水平對豬iPSCs誘導效率的影響,并對其分子機制進行初步探究,以便為進一步提高iPSCs誘導效率、建立大家畜ESCs系奠定基礎。本研究主要結(jié)果如下:1、豬JHDM2A基因克隆及其表達載體構(gòu)建根據(jù)NCBI上公布的豬JHDM2A基因序列,設計特異性擴增引物,克隆得到豬JHDM2A基因,其編碼區(qū)長度為3945bp,編碼1315個氨基酸。多重氨基酸序列比對發(fā)現(xiàn),豬JHDM2A基因與黃牛、水牛、綿羊和人相應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)進化樹分析結(jié)果表明,JHDM2A基因在物種進化過程中高度保守。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。進一步構(gòu)建并驗證了 pLVX-IRES-ZsGreen1-JHDM2A真核表達載體。2、過表達JHDM2A基因?qū)ωiiPSCs形成及相關基因表達的影響首先利用DOX誘導型慢病毒載體體系,將OSKM因子組合導入豬成纖維細胞,將其誘導為iPSCs,在此基礎上過表達JHDM2A基因,并分別對獲得的iPSCs進行鑒定。當過表達JHDM2A基因時,細胞在第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檢測陽性克隆數(shù)結(jié)果發(fā)現(xiàn),與未處理組相比,過表達JHDM2A基因?qū)嶒灲M的誘導效率提高了 8倍,且iPSCs克隆大小更均勻、邊緣更整齊、細胞更致密,AP染色著色更深。RT-PCR分析結(jié)果顯示,所檢測的三種細胞均表達Klf、c-Myc、Nanog多能性基因,弱表達Sox2基因;過表達JHDM2A實驗組和未處理組的piPSCs均表達Oct4,而PFF弱表達表達Oct4。定量PCR結(jié)果顯示,與未處理組相比,過表達JHDM2A實驗組 piPSCs 多能性基因 Sox2、klf4、c-Myc、Nanog表達顯著升高(P0.05),其中對Oct4的表達影響極顯著(P0.01)。去DOX后,過表達JHDM2A實驗組細胞中的外源基因沉默,內(nèi)源性Oct4基因表達逐代幾乎沒有變化,Sox2、Nanog基因表達逐代降低,Klf4、c-Myc基因表達先升高后降低。與未處理組和PFF相比,過表達JHDM2A實驗組piPSC組蛋白甲基化基因Tcll的表達顯著提高(P0.05),TcfTp2L1和Zfp57的表達顯著降低(P0.05)以上結(jié)果表明,克隆獲得了豬JHDM2A基因并構(gòu)建了其慢病毒表達載體,過表達JHDM2A基因可通過調(diào)控組蛋白甲基化水平提高piPSCs的誘導效率。
[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.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78

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