过表达JHDM2A基因对猪iPSCs诱导效率影响的初步研究

发布时间：2018-06-26 12:49

  本文选题：基因克隆 + JHDM2A　； 参考：《广西大学》2017年硕士论文

【摘要】：iPS技术为构建疾病模型、研制评估新药、基因治疗及再生医学等研究奠定了基础,是干细胞研究领域的里程碑。猪在生理及体型上与人相似,因此研究猪的iPS细胞意义重大。但目前猪成纤维细胞诱导为iPS的重编程效率不足1%。研究发现,组蛋白H3K9甲基化阻碍体细胞重编程,降低H3K9甲基化水平可促进iPS诱导。组蛋白甲基化水平受组蛋白甲基转移酶与组蛋白去甲基化酶共同调节。JHDM2A,即JmjC结构域组蛋白去甲基化酶2,可使H3K9me1/2发生去甲基化。过表达JHDM2A基因可促进与ESCs融合的小鼠NSCs重编程为iPS,促进iPSCs诱导;还可调节胚胎干细胞的自我更新。本研究首先采用DOX诱导型的慢病毒载体体系,诱导获得猪多能干细胞(piPSCs),在此基础上通过过表达JHDM2A基因,探究组蛋白H3K9me1/2甲基化水平对猪iPSCs诱导效率的影响,并对其分子机制进行初步探究,以便为进一步提高iPSCs诱导效率、建立大家畜ESCs系奠定基础。本研究主要结果如下:1、猪JHDM2A基因克隆及其表达载体构建根据NCBI上公布的猪JHDM2A基因序列,设计特异性扩增引物,克隆得到猪JHDM2A基因,其编码区长度为3945bp,编码1315个氨基酸。多重氨基酸序列比对发现,猪JHDM2A基因与黄牛、水牛、绵羊和人相应氨基酸序列的同源性分别为93.5%、94.7%、94.7%、93.8%。蛋白质分子系统进化树分析结果表明,JHDM2A基因在物种进化过程中高度保守。免疫组化结果显示,JHDM2J蛋白在不同发育阶段猪卵泡中均有表达。进一步构建并验证了 pLVX-IRES-ZsGreen1-JHDM2A真核表达载体。2、过表达JHDM2A基因对猪iPSCs形成及相关基因表达的影响首先利用DOX诱导型慢病毒载体体系,将OSKM因子组合导入猪成纤维细胞,将其诱导为iPSCs,在此基础上过表达JHDM2A基因,并分别对获得的iPSCs进行鉴定。当过表达JHDM2A基因时,细胞在第3 d出现形变,第8 d形成piPSCs克隆,分别比未处理组提前了 1d、2d。AP检测阳性克隆数结果发现,与未处理组相比,过表达JHDM2A基因实验组的诱导效率提高了 8倍,且iPSCs克隆大小更均匀、边缘更整齐、细胞更致密,AP染色着色更深。RT-PCR分析结果显示,所检测的三种细胞均表达Klf、c-Myc、Nanog多能性基因,弱表达Sox2基因;过表达JHDM2A实验组和未处理组的piPSCs均表达Oct4,而PFF弱表达表达Oct4。定量PCR结果显示,与未处理组相比,过表达JHDM2A实验组 piPSCs 多能性基因 Sox2、klf4、c-Myc、Nanog表达显著升高(P0.05),其中对Oct4的表达影响极显著(P0.01)。去DOX后,过表达JHDM2A实验组细胞中的外源基因沉默,内源性Oct4基因表达逐代几乎没有变化,Sox2、Nanog基因表达逐代降低,Klf4、c-Myc基因表达先升高后降低。与未处理组和PFF相比,过表达JHDM2A实验组piPSC组蛋白甲基化基因Tcll的表达显著提高(P0.05),TcfTp2L1和Zfp57的表达显著降低(P0.05)以上结果表明,克隆获得了猪JHDM2A基因并构建了其慢病毒表达载体,过表达JHDM2A基因可通过调控组蛋白甲基化水平提高piPSCs的诱导效率。
[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.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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