采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转...采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转化DH 5α感受态细菌,大量扩繁后提取过表达载体pCDH-TRPV1的质粒,与psPAX2和pMD两种含有慢病毒包装所必需元件的质粒混合,再与脂质体混合制备脂质体-载体混合液.将脂质体-载体混合液转染至单层的293T细胞中,培养48h进行病毒包装.收集富含慢病毒颗粒的293T细胞上清液,超离心纯化成浓缩病毒,然后再与polybrene一起感染单层Caco-2细胞,通过GFP绿荧光信号来筛选获得TRPV1基因过表达的稳定细胞株.通过Realtime PCR方法和Western-blot检测TRPV1的mRNA表达量及蛋白表达量,结果表明,Caco-2-TRPV1重组细胞株的TRPV1的mRNA表达量及蛋白表达量均显著高于Caco-2-GFP对照细胞(P<0.05).成功构建了TRPV1基因过表达的稳定细胞株,为后续辣椒素降脂机理的研究提供了正向调控细胞模型.展开更多
背景:溃疡性结肠炎是一种以结肠黏膜的慢性炎症为特征的疾病。克罗恩病是一种炎症性肠道疾病。GBP2、CASP1基因在溃疡性结肠炎和克罗恩病及其护理中的作用尚不清楚。方法:溃疡性结肠炎数据集GSE11223和克罗恩病数据集GSE186582配置文件...背景:溃疡性结肠炎是一种以结肠黏膜的慢性炎症为特征的疾病。克罗恩病是一种炎症性肠道疾病。GBP2、CASP1基因在溃疡性结肠炎和克罗恩病及其护理中的作用尚不清楚。方法:溃疡性结肠炎数据集GSE11223和克罗恩病数据集GSE186582配置文件是从GPL1708、GPL570生成的基因表达综合数据库(GEO)中下载的。进行差异表达基因(DEGs)的筛选,加权基因共表达网络分析(WGCNA),蛋白质–蛋白质相互作用(PPI)网络的构建与分析,功能富集分析,基因集合富集分析(GSEA),免疫浸润分析。绘制基因表达量热图。结果:鉴定出474个DEGs。根据基因本体论(GO)分析,它们主要富集在细胞因子介导的信号通路、细胞内囊泡、细胞表面、信号受体结合。京都基因和基因组百科全书(KEGG)分析结果显示,靶细胞主要富集在趋化因子信号通路、TNF信号通路、IL-17信号通路。WGCNA中的软阈值功率设置为5。获得了7个核心基因(GBP2, GBP1, GBP5, SAMD9L, CASP1, IFITM3, IFITM2)。基因表达量热图发现3个核心基因(GBP5, GBP2, CASP1)在溃疡性结肠炎样本中高表达,在正常组织样本中低表达。对于克罗恩病,GBP5基因在疾病和正常样本中都是低表达,GBP2、CASP1基因高表达。结论:GBP2和CASP1在溃疡性结肠炎和克罗恩病中高表达,可能通过细胞调节等途径在溃疡性结肠炎和克罗恩病的护理中发挥重要作用。Background: Ulcerative colitis is characterized by chronic inflammation of the colon mucosa. Crohn’s disease is an inflammatory bowel disease. The role of GBP2 and CASP1 genes in ulcerative colitis and Crohn’s disease and their care remains unclear. Methods: Ulcerative colitis dataset GSE11223 and Crohn’s disease dataset GSE186582 profiles were downloaded from the Gene Expression Omnibus (GEO) generated from GPL1708, GPL570. Differentially expressed genes (DEGs) were screened, followed by weighted gene co-expression network analysis (WGCNA), construction and analysis of protein-protein interaction (PPI) networks, functional enrichment analysis, gene set enrichment analysis (GSEA), and immune infiltration analysis. Heatmaps of gene expression were plotted. Results: 474 DEGs were identified. According to Gene Ontology (GO) analysis, they were mainly enriched in cytokine-mediated signaling pathways, intracellular vesicles, cell surface, and signal receptor binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the target cells were primarily enriched in chemokine signaling pathways, TNF signaling pathway, and IL-17 signaling pathway. The soft-thresholding power in WGCNA was set to 5. Seven core genes (GBP2, GBP1, GBP5, SAMD9L, CASP1, IFITM3, IFITM2) were obtained. The heatmap of gene expression revealed that three core genes (GBP5, GBP2, CASP1) were highly expressed in ulcerative colitis samples and lowly expressed in normal tissue samples. For Crohn’s disease, the GBP5 gene was lowly expressed in both disease and normal samples, while GBP2 and CASP1 genes were highly expressed. Conclusion: GBP2 and CASP1 are highly expressed in ulcerative colitis and Crohn’s disease, and may play an important role in the care of ulcerative colitis and Crohn’s disease through cellular regulation and other pathways.展开更多
文摘采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转化DH 5α感受态细菌,大量扩繁后提取过表达载体pCDH-TRPV1的质粒,与psPAX2和pMD两种含有慢病毒包装所必需元件的质粒混合,再与脂质体混合制备脂质体-载体混合液.将脂质体-载体混合液转染至单层的293T细胞中,培养48h进行病毒包装.收集富含慢病毒颗粒的293T细胞上清液,超离心纯化成浓缩病毒,然后再与polybrene一起感染单层Caco-2细胞,通过GFP绿荧光信号来筛选获得TRPV1基因过表达的稳定细胞株.通过Realtime PCR方法和Western-blot检测TRPV1的mRNA表达量及蛋白表达量,结果表明,Caco-2-TRPV1重组细胞株的TRPV1的mRNA表达量及蛋白表达量均显著高于Caco-2-GFP对照细胞(P<0.05).成功构建了TRPV1基因过表达的稳定细胞株,为后续辣椒素降脂机理的研究提供了正向调控细胞模型.
文摘背景:溃疡性结肠炎是一种以结肠黏膜的慢性炎症为特征的疾病。克罗恩病是一种炎症性肠道疾病。GBP2、CASP1基因在溃疡性结肠炎和克罗恩病及其护理中的作用尚不清楚。方法:溃疡性结肠炎数据集GSE11223和克罗恩病数据集GSE186582配置文件是从GPL1708、GPL570生成的基因表达综合数据库(GEO)中下载的。进行差异表达基因(DEGs)的筛选,加权基因共表达网络分析(WGCNA),蛋白质–蛋白质相互作用(PPI)网络的构建与分析,功能富集分析,基因集合富集分析(GSEA),免疫浸润分析。绘制基因表达量热图。结果:鉴定出474个DEGs。根据基因本体论(GO)分析,它们主要富集在细胞因子介导的信号通路、细胞内囊泡、细胞表面、信号受体结合。京都基因和基因组百科全书(KEGG)分析结果显示,靶细胞主要富集在趋化因子信号通路、TNF信号通路、IL-17信号通路。WGCNA中的软阈值功率设置为5。获得了7个核心基因(GBP2, GBP1, GBP5, SAMD9L, CASP1, IFITM3, IFITM2)。基因表达量热图发现3个核心基因(GBP5, GBP2, CASP1)在溃疡性结肠炎样本中高表达,在正常组织样本中低表达。对于克罗恩病,GBP5基因在疾病和正常样本中都是低表达,GBP2、CASP1基因高表达。结论:GBP2和CASP1在溃疡性结肠炎和克罗恩病中高表达,可能通过细胞调节等途径在溃疡性结肠炎和克罗恩病的护理中发挥重要作用。Background: Ulcerative colitis is characterized by chronic inflammation of the colon mucosa. Crohn’s disease is an inflammatory bowel disease. The role of GBP2 and CASP1 genes in ulcerative colitis and Crohn’s disease and their care remains unclear. Methods: Ulcerative colitis dataset GSE11223 and Crohn’s disease dataset GSE186582 profiles were downloaded from the Gene Expression Omnibus (GEO) generated from GPL1708, GPL570. Differentially expressed genes (DEGs) were screened, followed by weighted gene co-expression network analysis (WGCNA), construction and analysis of protein-protein interaction (PPI) networks, functional enrichment analysis, gene set enrichment analysis (GSEA), and immune infiltration analysis. Heatmaps of gene expression were plotted. Results: 474 DEGs were identified. According to Gene Ontology (GO) analysis, they were mainly enriched in cytokine-mediated signaling pathways, intracellular vesicles, cell surface, and signal receptor binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the target cells were primarily enriched in chemokine signaling pathways, TNF signaling pathway, and IL-17 signaling pathway. The soft-thresholding power in WGCNA was set to 5. Seven core genes (GBP2, GBP1, GBP5, SAMD9L, CASP1, IFITM3, IFITM2) were obtained. The heatmap of gene expression revealed that three core genes (GBP5, GBP2, CASP1) were highly expressed in ulcerative colitis samples and lowly expressed in normal tissue samples. For Crohn’s disease, the GBP5 gene was lowly expressed in both disease and normal samples, while GBP2 and CASP1 genes were highly expressed. Conclusion: GBP2 and CASP1 are highly expressed in ulcerative colitis and Crohn’s disease, and may play an important role in the care of ulcerative colitis and Crohn’s disease through cellular regulation and other pathways.
