【摘要】：Lgr5(Leucine-rich repeat-containing G-protein coupled receptor 5)是腸上皮干細(xì)胞(intestinal epithelial stem cells,IESCs)的標(biāo)記基因,它標(biāo)記的是隱窩基底柱狀(crypt-base columnar,CBC)細(xì)胞。為了研究豬Lgr5對腸上皮細(xì)胞增殖的影響,本試驗通過克隆豬Lgr5基因,運用生物信息學(xué)方法分析預(yù)測了其結(jié)構(gòu)與功能;通過構(gòu)建Lgr5-pc DNA3.1+載體,獲取Lgr5過表達(dá)的IPEC-J2穩(wěn)轉(zhuǎn)細(xì)胞株。運用細(xì)胞計數(shù)和MTT法檢測Lgr5過表達(dá)后對IPEC-J2細(xì)胞增殖的影響,應(yīng)用Western blot法檢測Wnt/β-catenin信號通路關(guān)鍵蛋白的表達(dá)水平。利用Wnt/β-catenin信號通路激動劑Wnt3a處理對照組細(xì)胞和Wnt/β-catenin信號通路抑制劑XAV939處理Lgr5過表達(dá)組細(xì)胞后,分別檢測其對細(xì)胞增殖與Wnt/β-catenin信號通路關(guān)鍵蛋白表達(dá)水平的影響,解析豬Lgr5促進(jìn)腸上皮細(xì)胞增殖的機制。為開展Lgr5標(biāo)記的豬腸上皮干細(xì)胞研究奠定基礎(chǔ)。試驗結(jié)果如下:(1)豬Lgr5 c DNA的克隆運用3'RACE(Rapid amplification of c DNA ends,RACE)、同源性克隆和重疊PCR的方法,克隆獲得了豬Lgr5 c DNA序列,全長2824 bp,其中編碼區(qū)(coding sequence,CDS)長2724 bp,3'非編碼區(qū)(untranslated region,UTR)長108 bp。(2)豬Lgr5的生物信息學(xué)分析豬Lgr5蛋白經(jīng)預(yù)測為膜蛋白,含有信號肽和7次α螺旋跨膜結(jié)構(gòu),與人Lgr5蛋白氨基酸序列的同源性高達(dá)90.30%;進(jìn)化樹分析發(fā)現(xiàn)豬Lgr5蛋白是一個種保守性蛋白。(3)Lgr5在IPEC-J2細(xì)胞中過表達(dá)的鑒定構(gòu)建過表達(dá)載體Lgr5-pc DNA3.1+,并獲得穩(wěn)定表達(dá)Lgr5的IPEC-J2細(xì)胞株。Real-time PCR和Western blot檢測結(jié)果顯示,在接種72 h,Lgr5過表達(dá)組Lgr5 m RNA豐度和蛋白表達(dá)水平均顯著高于(P0.05)對照組。(4)Lgr5過表達(dá)促進(jìn)IPEC-J2細(xì)胞增殖細(xì)胞計數(shù)和MTT檢測結(jié)果顯示,與對照組相比,Lgr5過表達(dá)組的細(xì)胞在接種48h后增殖能力顯著提高(P0.05)。(5)Lgr5過表達(dá)激活Wnt/β-catenin信號通路收集細(xì)胞生長72 h的細(xì)胞樣品。Western blot檢測結(jié)果顯示,與對照組相比,Lgr5過表達(dá)組Axin2、GSK-3β蛋白表達(dá)水平顯著降低(P0.05),β-catenin、c-Myc和cyclin D1蛋白表達(dá)水平顯著升高(P0.05)。(6)Lgr5激活Wnt/β-catenin信號通路促進(jìn)IPEC-J2細(xì)胞增殖Wnt3a組細(xì)胞增殖能力在Wnt3a處理48 h顯著高于對照組(P0.05);與對照組相比,在Wnt3a處理48 h,Wnt3a組的Axin2和GSK-3β蛋白表達(dá)水平顯著降低(P0.05),β-catenin、c-Myc、cyclin D1和Lgr5蛋白表達(dá)水平顯著升高(P0.05);XAV939組細(xì)胞增殖能力在XAV939處理24 h和48 h后顯著低于(P0.05)Lgr5過表達(dá)組;與Lgr5過表達(dá)組相比,在XAV939處理48 h,XAV939組的Axin2和GSK-3β蛋白表達(dá)水平顯著升高(P0.05),β-catenin、c-Myc、cyclin D1和Lgr5蛋白表達(dá)水平顯著降低(P0.05)。結(jié)論:(1)首次克隆了豬Lgr5含有完整CDS的c DNA序列;(2)豬Lgr5可通過激活Wnt/β-catenin信號通路促進(jìn)豬腸上皮細(xì)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 偽 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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S828

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