猪Lgr5基因克隆及其对肠上皮细胞增殖的影响

发布时间：2018-07-16 07:31

【摘要】：Lgr5(Leucine-rich repeat-containing G-protein coupled receptor 5)是肠上皮干细胞(intestinal epithelial stem cells,IESCs)的标记基因,它标记的是隐窝基底柱状(crypt-base columnar,CBC)细胞。为了研究猪Lgr5对肠上皮细胞增殖的影响,本试验通过克隆猪Lgr5基因,运用生物信息学方法分析预测了其结构与功能;通过构建Lgr5-pc DNA3.1+载体,获取Lgr5过表达的IPEC-J2稳转细胞株。运用细胞计数和MTT法检测Lgr5过表达后对IPEC-J2细胞增殖的影响,应用Western blot法检测Wnt/β-catenin信号通路关键蛋白的表达水平。利用Wnt/β-catenin信号通路激动剂Wnt3a处理对照组细胞和Wnt/β-catenin信号通路抑制剂XAV939处理Lgr5过表达组细胞后,分别检测其对细胞增殖与Wnt/β-catenin信号通路关键蛋白表达水平的影响,解析猪Lgr5促进肠上皮细胞增殖的机制。为开展Lgr5标记的猪肠上皮干细胞研究奠定基础。试验结果如下:(1)猪Lgr5 c DNA的克隆运用3'RACE(Rapid amplification of c DNA ends,RACE)、同源性克隆和重叠PCR的方法,克隆获得了猪Lgr5 c DNA序列,全长2824 bp,其中编码区(coding sequence,CDS)长2724 bp,3'非编码区(untranslated region,UTR)长108 bp。(2)猪Lgr5的生物信息学分析猪Lgr5蛋白经预测为膜蛋白,含有信号肽和7次α螺旋跨膜结构,与人Lgr5蛋白氨基酸序列的同源性高达90.30%;进化树分析发现猪Lgr5蛋白是一个种保守性蛋白。(3)Lgr5在IPEC-J2细胞中过表达的鉴定构建过表达载体Lgr5-pc DNA3.1+,并获得稳定表达Lgr5的IPEC-J2细胞株。Real-time PCR和Western blot检测结果显示,在接种72 h,Lgr5过表达组Lgr5 m RNA丰度和蛋白表达水平均显著高于(P0.05)对照组。(4)Lgr5过表达促进IPEC-J2细胞增殖细胞计数和MTT检测结果显示,与对照组相比,Lgr5过表达组的细胞在接种48h后增殖能力显著提高(P0.05)。(5)Lgr5过表达激活Wnt/β-catenin信号通路收集细胞生长72 h的细胞样品。Western blot检测结果显示,与对照组相比,Lgr5过表达组Axin2、GSK-3β蛋白表达水平显著降低(P0.05),β-catenin、c-Myc和cyclin D1蛋白表达水平显著升高(P0.05)。(6)Lgr5激活Wnt/β-catenin信号通路促进IPEC-J2细胞增殖Wnt3a组细胞增殖能力在Wnt3a处理48 h显著高于对照组(P0.05);与对照组相比,在Wnt3a处理48 h,Wnt3a组的Axin2和GSK-3β蛋白表达水平显著降低(P0.05),β-catenin、c-Myc、cyclin D1和Lgr5蛋白表达水平显著升高(P0.05);XAV939组细胞增殖能力在XAV939处理24 h和48 h后显著低于(P0.05)Lgr5过表达组;与Lgr5过表达组相比,在XAV939处理48 h,XAV939组的Axin2和GSK-3β蛋白表达水平显著升高(P0.05),β-catenin、c-Myc、cyclin D1和Lgr5蛋白表达水平显著降低(P0.05)。结论:(1)首次克隆了猪Lgr5含有完整CDS的c DNA序列;(2)猪Lgr5可通过激活Wnt/β-catenin信号通路促进猪肠上皮细胞增殖。
[Abstract]:Lgr5 (Leucine-rich repeat-containing G-protein coupled receptor 5) is a marker gene of intestinal epithelial stem cells (intestinal epithelial stem cells / IESCs). It is labeled with crypt-base columnarnar cells. In order to study the effect of porcine Lgr5 on the proliferation of intestinal epithelial cells, we cloned porcine Lgr5 gene and predicted its structure and function by bioinformatics, and constructed Lgr5-pc DNA3.1 vector to obtain Lgr5 overexpression IPEC-J2 stable cell line. The effect of Lgr5 overexpression on the proliferation of IPEC-J2 cells was detected by cell count and MTT assay. The expression of key proteins in Wnt- 尾 -catenin signaling pathway was detected by Western blot assay. The effects of Wnt3a, a signal agonist of Wnt/ 尾 -catenin pathway, on cell proliferation and expression of key proteins in Wnt/ 尾 -catenin signaling pathway were detected after treated with Wnt- 尾 -catenin signal pathway inhibitor XAV939, and the Lgr5 overexpression group was treated with Wnt3a / 尾 -catenin signal pathway inhibitor XAV939. The mechanism of porcine Lgr5 promoting the proliferation of intestinal epithelial cells was analyzed. To lay a foundation for the study of Lgr5 labeled porcine intestinal epithelial stem cells. The results were as follows: (1) Porcine Lgr5c DNA sequence was cloned by rapid amplification of c race (Rapid amplification of c DNA endsRACE), homologous cloning and overlapping PCR. The total length of 2824 BP, in which the length of the coding sequence region is 2724 BP ~ (3'). (2) the bioinformatics of porcine Lgr5 protein is predicted to be membrane protein, which contains signal peptide and seven 伪 helix transmembrane structure. Phylogenetic tree analysis showed that porcine Lgr5 protein was a conserved protein. (3) the overexpression of Lgr5 protein in IPEC-J2 cells was identified and the expression vector Lgr5-pc DNA3.1 was constructed. The stable expression of Lgr5 in IPEC-J2 cell line. Real-time PCR and Western blot analysis showed that, Lgr5 mRNA abundance and protein expression were significantly higher in Lgr5 overexpression group than in control group at 72 h after inoculation. (4) Lgr5 overexpression promoted proliferation of IPEC-J2 cells and MTT assay showed that Lgr5 overexpression promoted IPEC-J2 cell proliferation. Compared with the control group, the proliferative ability of Lgr5 overexpression group increased significantly after 48 h inoculation (P0.05). (5). Wnt5 / 尾 -catenin signaling pathway was used to collect cell samples for 72 h of cell growth. Western blot analysis showed that Lgr5 overexpression activated Wnt5 / 尾 -catenin signal pathway for 72 h after inoculation. Compared with the control group, the expression of axin2t2 GSK-3 尾 protein decreased significantly (P0.05), and the expression of 尾 -cateninc-Myc and cyclin D1 protein increased significantly (P0.05). (P0.05) Lgr5 activated Wnt / 尾 -catenin signaling pathway to promote the proliferation of IPEC-J2 cells in Wnt3a group after 48 h treatment with Wnt3a significantly higher than that in Wnt3a group. The exposure group (P0.05), compared with the control group, The expression of Axin2 and GSK-3 尾 in Wnt3a group decreased significantly (P0.05), and the expression levels of 尾 -cateninine c-Myct3a cyclin D1 and Lgr5 protein increased significantly (P0.05). The proliferative ability of XAV939 group was significantly lower than that of Lgr5 overexpression group after XAV939 treatment for 24 h and 48 h, compared with Lgr5 overexpression group. The expression of Axin2 and GSK-3 尾 in XAV939 group was significantly higher than that in XAV939 group (P0.05), while the expression levels of 尾 -cateninc-Mycncyclin D1 and Lgr5 protein were significantly decreased (P0.05). Conclusion: (1) the cDNA sequence of porcine Lgr5 containing complete CDS was cloned for the first time, and (2) porcine Lgr5 could promote the proliferation of porcine intestinal epithelial cells by activating Wnt- 尾 -catenin signaling pathway.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S828

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