猪多能干细胞中LAMININ基因表达谱的分析及其可变剪切体的分子克隆

发布时间：2018-06-19 10:32

本文选题：猪 + 层粘连蛋白　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：层粘连蛋白(Laminin,LN)是细胞膜的重要成分,采用LN作为细胞基质培养多能干细胞,可以维持干细胞的自我更新和多能性。LN家族中有15种不同的亚型,每一种亚型由三条肽链(α、β、γ)组成,形成十字架结构的异源三聚体。其中Laminin A5(LAMA5)对于维持多能干细胞的生长有重要作用。本研究旨在检测猪不同组织和细胞中LAMININ的表达情况,研究LN在多能干细胞分化过程中的时空表达特性;通过生物信息学分析和分子克隆,确认猪LAMA5基因的存在和表达变化;发现并分子克隆了与猪多能干细胞特性相关的Laminin B1-a(LAMB1-a)可变剪接体。本研究丰富了对猪多能干细胞培养基质研究的信息,为研究猪干细胞多能性的维持提供了理论依据。本研究获得的主要结果如下:1.LAMININ在猪组织和细胞中的表达谱分析用RT-PCR检测猪不同组织(肝脏、心脏、肺、胃、脑、睾丸、脂肪、颗粒细胞组织)、体细胞(肝细胞、脂肪细胞、猪胎儿成纤维细胞)和多能干细胞(DOX-iPS、PS11、PS23)中LAMININ不同亚型的表达情况。结果表明,不同组织和细胞中LAMININ的表达存在明显差异。通过比对组织来源和细胞来源的样品发现,在细胞中LAMININ的表达特异性更加突出,其中在PEF细胞中LAMININ的表达特征为:LAMA4/5-LAMB1/2-LAMC1/2(即LN-411、LN-421、LN-511和LN-521);在猪iPS细胞中LAMININ的表达特征为:LAMA1/5-LAMB1-a/2-LAMC1(即LN-11a1、LN-121、LN-51a1和LN-521)。2.猪LAMA5的生物学分析和克隆未注释有猪LAMA5基因的序列信息和相关报道。为此我们运用NCBI和UCSC等数据库,分析人、牛和山羊的LAMA5基因序列,预测猪LAMA5基因,以RPS21和CABLES2基因对猪基因组数据库(Sscrofa11.1)中LAMA5的可能序列区域进行定位。通过DNAMAN(V6)软件进行比对,确认了猪LAMA5的序列,并设计特异性引物,从猪肺组织样c DNA中克隆了长度为731 bp的猪LAMA5基因序列。测序结果与人和牛LAMA5序列高度一致,序列上传至GENBANK,获得登录号:KY622023。3.猪多能干细胞中LAMININ的表达特性和LAMB1-a可变剪接体的发现通过检测,发现猪iPS细胞中,LAMB1出现了两条特异性条带,为了进一步研究LAMB1的性质,对克隆的两条特异性条带(LAMB1 505bp、LAMB1-a 639bp)进行测序,结果显示与NCBI中已有的LAMB1-X1和LAMB1-X2序列不一样,发现LAMB1-a是一个新的可变剪接体。为了进一步探讨LAMB1与多能干细胞的相关性,选取猪PEF和猪多能干细胞DOX-iPS及分化的iPS细胞进行了检测。结果显示,随着多能干细胞形态的改变,细胞团形态逐渐消失,LAMB1-a的表达逐渐下降,当再次运用DOX-iPS培养基培养后,当细胞团形态再次恢复的时候,LAMB1-a的表达逐渐恢复。在检测中还发现,在iPS重编程过程中LAMA4的表达随着细胞的分化程度,其表达量明显升高,相反在细胞分化的过程中LAMA1的表达量逐渐降低。总之,本研究确定了猪多能干细胞中LAMININ的表达特征,即LN11a1/LN121和LN51a1/LN521;确认了猪LAMA5基因的存在,并分析和克隆了该基因;发现了LAMB1-a可变剪接体,并验证了该基因与多能干细胞的对应关系。
[Abstract]:Laminin (LN) is an important component of the cell membrane. Using LN as a cell matrix to cultivate pluripotent stem cells, there are 15 different subtypes in the self-renewal of stem cells and the pluripotent.LN family. Each subtype consists of three peptide chains (alpha, beta, and gamma), forming a hetero trimer of the cross structure. Among them, Laminin A5 (LAMA5) The purpose of this study is to maintain the growth of pluripotent stem cells. The purpose of this study is to detect the expression of LAMININ in different tissues and cells of pigs and to study the temporal and spatial expression of LN in the differentiation of pluripotent stem cells. Through bioinformatics analysis and molecular cloning, the existence and expression changes of porcine LAMA5 genes are confirmed; and the molecular cloning of molecular cloning is found. Laminin B1-a (LAMB1-a) variable splicing body related to the characteristics of porcine pluripotent stem cells. This study enriches the information on the study of porcine pluripotent stem cell culture matrix and provides a theoretical basis for the study of the maintenance of the pluripotent activity of pig stem cells. The main results obtained in this study are as follows: the analysis of the expression profiles of 1.LAMININ in pig tissues and cells is RT-PCR The expression of different subtypes of LAMININ in different tissues (liver, heart, lung, stomach, stomach, brain, testicle, fat, granulosa cell tissue), somatic cells (hepatocytes, adipocytes, DOX-iPS, PS11, PS23), and the expression of LAMININ in different tissues and cells showed that the expression of LAMININ in different tissues and cells was significantly different. Samples of tissue source and cell source have found that the expression of LAMININ is more prominent in the cell, and the expression of LAMININ in PEF cells is LAMA4/5-LAMB1/2-LAMC1/2 (LN-411, LN-421, LN-511 and LN-521), and LAMININ expression in the pig iPS cells is LAMA1/5-LAMB1-a/2-LAMC1. 1) the biological analysis and cloning of.2. pig LAMA5 have not been annotated with the sequence information and related reports of the pig LAMA5 gene. Therefore, we use the database of NCBI and UCSC to analyze the LAMA5 gene sequence of human, cattle and goat, predict the pig LAMA5 gene, and take RPS21 and CABLES2 gene to the possible sequence region of the LAMA5 of the pig genome database (Sscrofa11.1). Location. The sequence of pig LAMA5 was confirmed by DNAMAN (V6) software and specific primers were designed. The gene sequence of pig LAMA5 gene was cloned from pig lung tissue like C DNA. The sequence of LAMA5 gene was 731 BP, and the sequence was highly consistent with the sequence of human and bovine LAMA5, and the sequence was uploaded to GENBANK, and LAMININ in KY622023.3. porcine pluripotent stem cells. The discovery of expression characteristics and the discovery of LAMB1-a variable splice body found two specific bands of LAMB1 in pig iPS cells. In order to further study the properties of LAMB1, the two specific bands of the clone (LAMB1 505bp, LAMB1-a 639bp) were sequenced. The results showed that L was different from LAMB1-X1 and LAMB1-X2 sequences in NCBI, and L was found to be L. AMB1-a is a new variable splice body. In order to further explore the correlation between LAMB1 and pluripotent stem cells, the porcine PEF and porcine pluripotent stem cells DOX-iPS and the differentiated iPS cells were detected. The results showed that the morphology of the cells gradually disappeared and the expression of LAMB1-a decreased gradually with the morphologic changes of the pluripotent stem cells. When DOX-iP was used again, the expression of DOX-iP was gradually reduced. After the culture of S medium, the expression of LAMB1-a was gradually restored when the cell morphology was restored again. In the test, the expression of LAMA4 was obviously increased with the degree of cell differentiation in the iPS reprogramming process. On the contrary, the expression of LAMA1 was gradually reduced during the process of cell differentiation. The expression of LAMININ in stem cells, namely, LN11a1/LN121 and LN51a1/LN521, confirmed the existence of the porcine LAMA5 gene, and analyzed and cloned the gene, and found the LAMB1-a variable splice body, and verified the relationship between the gene and the pluripotent stem cells.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q813

【相似文献】