AIM： To identify biomarkers indicating virus-specific hepatocarcinogenic process, differential mRNA expression in 32 patients with hepatitis B virus （HBV）-/hepatitis C virus （HCV）-associated hepatocellular carcin...AIM： To identify biomarkers indicating virus-specific hepatocarcinogenic process, differential mRNA expression in 32 patients with hepatitis B virus （HBV）-/hepatitis C virus （HCV）-associated hepatocellular carcinoma （HCC） were investigated by means of cDNA microarrays comprising of 886 genes. METHODS： Thirty two HCC patients were divided into two groups based on viral markers： hepatitis B virus positive and HCV positive. The expression profiles of 32 pairs of specimens （tumorous and surrounding nontumorous liver tissues）, consisting of 886 genes were analyzed. RESULTS： Seven up-regulated genes in HBV-associated HCC comprised genes involved in protein synthesis （RPSS）, cytoskeletal organization （KRTS）, apoptosis related genes （CFLAR）, transport （ATPSF1）, cell membrane receptor related genes （IGFBP2）, signal transduction or transcription related genes （MAP3KS）, and metastasis-related genes （MMP9）. The up-regulated genes in HCV-infected group included 4 genes： V/M （cell structure）, ACTB （cell structure）, GAPD （glycolysis） and CD58 （cell adhesion）. The expression patterns of the 11 genes, identified by cDNA microarray, were confirmed by quantitative RT-PCR in 32 specimens.CONCLUSION： The patterns of all identified genes were classified based on the viral factor involved in HBV- and HCV-associated HCC. Our results strongly suggest that the pattern of gene expression in HCC is closely associated with the etiologic factor. The present study indicates that HBV and HCV cause hepatocarcinogenesis by different mechanisms, and provide novel tools for the diagnosis and treatment of HBV- and HCV-associated HCC.展开更多
Growth hormone （GH） is a polypeptide hormone secreted by the pituitary, which promotes animal growth, muscle development, metabolism regulation and other important physiological functions. In this study, a pair of m...Growth hormone （GH） is a polypeptide hormone secreted by the pituitary, which promotes animal growth, muscle development, metabolism regulation and other important physiological functions. In this study, a pair of mGH short hairpin RNA （shRNA） was designed according to mouse （ Mus musculus） GH mRNA sequence; pSingle-tTS-mGH shRNA-RFP, an integrated controllable expression vector of mGH shRNA, was constructed successfully. The recombinant vector was transfected into mouse pituitary tumor cell line AtT-20. After addition of doxycyelin （ DOX）, the expression of red fluorescent protein （RFP） was observed un- der a fluorescent microscope. The expression level of mGH in cells was detected by quantitative Realtime PCR （qRT-PCR） and Western blot. After DOX induction, the relative expression level of GH mRNA in cells transfected with GH shRNA was reduced by about 70% compared with that in DOX-free group and other control groups, exhibiting extremely significant differences （P 〈 0.01 ） ; moreover, the relative expression level of GH protein was reduced by about 90% ; the expression level of GH mRNA and GH protein exhibited no significant difference among other groups （P 〉 0.05）. In this study, a controllable expression vector of GH shRNA with high gene silencing efficiency was constructed successfully, which could be used to reveal GH autocfine / paracrine interactions and analyze functions of GH gene in growth, development and disease occurrence of animals by regulating GH expression levels.展开更多
【目的】本研究旨在克隆与鉴定家蚕Bombyx mori C类清道夫受体基因BmSR-C,为探析其在家蚕免疫中的功能奠定基础。【方法】利用RACE技术克隆家蚕C类清道夫受体基因全长序列,并对其进行生物信息学分析。利用RT-PCR和qPCR方法对BmSR-C基因...【目的】本研究旨在克隆与鉴定家蚕Bombyx mori C类清道夫受体基因BmSR-C,为探析其在家蚕免疫中的功能奠定基础。【方法】利用RACE技术克隆家蚕C类清道夫受体基因全长序列,并对其进行生物信息学分析。利用RT-PCR和qPCR方法对BmSR-C基因的时空表达情况进行检测。通过原核表达和Ni^(+)亲和层析的方法获得BmSR-C重组蛋白,免疫昆明小鼠制备抗BmSR-C多克隆抗体。利用ELISA法和Western blot分别对鼠抗BmSR-C蛋白多克隆抗体的效价和特异性进行检测。构建家蚕BmSR-C的真核表达载体,转染家蚕BmE细胞,分析该蛋白的亚细胞定位情况。【结果】克隆获得家蚕BmSR-C基因(GenBank登录号:BGIBMGA004577),其开放阅读框(ORF)全长为1821 bp,编码606个氨基酸残基。BmSR-C具有典型的C类清道夫受体家族结构特征,主要由CCP,MAM和SO结构域以及靠近C端的单次跨膜结构域组成。进化分析结果显示鳞翅目昆虫SR-C单独聚为一支,家蚕BmSR-C与同为鳞翅目昆虫的草地贪夜蛾Spodoptera frugiperda和大红斑蝶Danaus plexippus的同源蛋白亲缘关系最为接近。对BmSR-C基因的时空表达分析表明,BmSR-C在家蚕的马氏管和血细胞中高表达,而在其他组织中无明显表达;其在家蚕不同发育时期的血细胞中均有表达,且在4龄眠期的表达量达到峰值。ELISA检测结果显示,所制得抗体效价高达1∶128000;Western blot检测结果显示,该抗体可以特异性识别重组蛋白。家蚕BmE细胞中的亚细胞定位结果表明BmSR-C主要定位于细胞膜。【结论】获得家蚕C类清道夫受体基因BmSR-C的完整cDNA序列及其表达特征;成功制备了BmSR-C的多克隆抗体,利用家蚕BmE细胞在细胞水平上分析了BmSR-C的亚细胞定位情况,推测其参与家蚕的生长发育及病原微生物入侵的免疫反应,为进一步研究BmSR-C的生物学功能奠定了基础。展开更多
文摘AIM： To identify biomarkers indicating virus-specific hepatocarcinogenic process, differential mRNA expression in 32 patients with hepatitis B virus （HBV）-/hepatitis C virus （HCV）-associated hepatocellular carcinoma （HCC） were investigated by means of cDNA microarrays comprising of 886 genes. METHODS： Thirty two HCC patients were divided into two groups based on viral markers： hepatitis B virus positive and HCV positive. The expression profiles of 32 pairs of specimens （tumorous and surrounding nontumorous liver tissues）, consisting of 886 genes were analyzed. RESULTS： Seven up-regulated genes in HBV-associated HCC comprised genes involved in protein synthesis （RPSS）, cytoskeletal organization （KRTS）, apoptosis related genes （CFLAR）, transport （ATPSF1）, cell membrane receptor related genes （IGFBP2）, signal transduction or transcription related genes （MAP3KS）, and metastasis-related genes （MMP9）. The up-regulated genes in HCV-infected group included 4 genes： V/M （cell structure）, ACTB （cell structure）, GAPD （glycolysis） and CD58 （cell adhesion）. The expression patterns of the 11 genes, identified by cDNA microarray, were confirmed by quantitative RT-PCR in 32 specimens.CONCLUSION： The patterns of all identified genes were classified based on the viral factor involved in HBV- and HCV-associated HCC. Our results strongly suggest that the pattern of gene expression in HCC is closely associated with the etiologic factor. The present study indicates that HBV and HCV cause hepatocarcinogenesis by different mechanisms, and provide novel tools for the diagnosis and treatment of HBV- and HCV-associated HCC.
基金Supported by National Natural Science Foundation of China(31101683,31272405)Priority Academic Program Development of Jiangsu Higher Education Institutions(2014)Science and Technology Innovation Fund for Students in Yangzhou University(No.x2015719)
文摘Growth hormone （GH） is a polypeptide hormone secreted by the pituitary, which promotes animal growth, muscle development, metabolism regulation and other important physiological functions. In this study, a pair of mGH short hairpin RNA （shRNA） was designed according to mouse （ Mus musculus） GH mRNA sequence; pSingle-tTS-mGH shRNA-RFP, an integrated controllable expression vector of mGH shRNA, was constructed successfully. The recombinant vector was transfected into mouse pituitary tumor cell line AtT-20. After addition of doxycyelin （ DOX）, the expression of red fluorescent protein （RFP） was observed un- der a fluorescent microscope. The expression level of mGH in cells was detected by quantitative Realtime PCR （qRT-PCR） and Western blot. After DOX induction, the relative expression level of GH mRNA in cells transfected with GH shRNA was reduced by about 70% compared with that in DOX-free group and other control groups, exhibiting extremely significant differences （P 〈 0.01 ） ; moreover, the relative expression level of GH protein was reduced by about 90% ; the expression level of GH mRNA and GH protein exhibited no significant difference among other groups （P 〉 0.05）. In this study, a controllable expression vector of GH shRNA with high gene silencing efficiency was constructed successfully, which could be used to reveal GH autocfine / paracrine interactions and analyze functions of GH gene in growth, development and disease occurrence of animals by regulating GH expression levels.
