东部马脑炎病毒E2基因与GM-CSF共表达质粒的构建及其免疫原性初步研究

目的:利用粒细胞-巨噬细胞集落刺激因子(GM-CSF)作为基因佐剂,提高东部马脑炎病毒E2基因重组质粒的免疫效果.方法:分别扩增E2基因与GM-CSF基因,连接进入含有内部核糖体插入位点(IRES)的真核表达载体中,构建共表达质粒.Lipofectamine2000介导转染真核细胞BHK-21,反转录PCR检测E2和GM-CSF两个基因的转录,Western印迹法和间接免疫荧光法(IFA)检测细胞内E2基因表达产物的抗原性.通过肌肉注射免疫BALB/c小鼠,IFA法测定血清抗体效价,FACS测定免疫小鼠脾细胞中CD4 /CD8 淋巴细胞构成比,初步观察共表达质粒的免疫原性.结果:经酶切鉴定与序列测定证明重组共表达质粒中含有E2和GM-CSF两个基因且序列正确.转染细胞的反转录PCR可见E2和GM-CSF两个基因的转录.Western印迹结果可见转染细胞的裂解液在相对分子质量50 000位置有特异性条带.将转染细胞制成荧光抗原片,经IFA检测可见胞浆中出现特异荧光.免疫小鼠的最高血清抗体效价为1:160,但细胞免疫未观察到明显的提高.结论:构建的共表达质粒pE2-IRES-mGM-CSF具有良好的免疫原性,能有效刺激小鼠特异性体液免疫应答,提高了E2基因重组质粒诱导的血清抗体效价.     

Comprehensive analysis of key lncRNAs in HCV-positive hepatocellular carcinoma

IntroductionHepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Despite the therapeutic advances in HCC in the past few decades, the mortality rate of HCC is still high. Hepatitis C (HCV) infection is one of the major etiological risk factors of HCCs. However, the underlying mechanisms of HCV-induced hepatocarcinogenesis remain largely unclear.Material and methodsOur study represented the comprehensive analysis of differentially expressed lncRNAs in HCV-positive HCC for the first time by analyzing the public dataset {"type":"entrez-geo","attrs":{"text":"GSE17856","term_id":"17856"}}GSE17856. Co-expression network and gene ontology (GO) analysis revealed the functions of those differentially expressed lncRNAs.ResultsWe identified 256 upregulated lncRNAs and 198 downregulated lncRNAs in HCV- positive HCC compared to the normal liver tissues. Co-expression network and GO analysis showed that these lncRNAs were involved in regulating metabolism, energy pathways, proliferation and the immune response. Seven lncRNAs (LOC341056, CCT6P1, PTTG3P, LOC643387, LOC100133920, C3P1 and C22orf45) were identified as key lncRNAs and co-expressed with more than 100 differentially expressed genes (DEGs) in HCV-related HCC. Kaplan-Meier analysis showed that higher expression levels of LOC643387, PTTG3P, LOC341056, CCT6P1 and lower expression levels of C3P1 and C22orf45 were associated with shorter survival time in the TCGA dataset.ConclusionsWe believe that this study can provide novel potential therapeutic and prognostic biomarkers for HCV-positive HCC.     

Identification of TNIK as a novel potential drug target in thyroid cancer based on protein druggability prediction

Thyroid cancer is a common endocrine malignancy; however, surgery remains its primary treatment option. A novel targeted drug for the development and application of targeted therapy in thyroid cancer treatment remain underexplored.We obtained RNA sequence data of thyroid cancer from The Cancer Genome Atlas database and identified differentially expressed genes (DEGs). Then, we constructed co-expression network with DEGs and combined it with differentially methylation analysis to screen the key genes in thyroid cancer. PockDrug-Server, an online tool, was applied to predict the druggability of the key genes. Finally, we constructed protein-protein interaction (PPI) network to observe potential targeted drugs for thyroid cancer.We identified 3 genes correlated with altered DNA methylation level and oncogenesis of thyroid cancer. According to the druggable analysis and PPI network, we predicted TRAF2 and NCK-interacting protein kinase (TNIK) sever as the drug targeted for thyroid cancer. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that genes in protein-protein interaction network of TNIK enriched in mitogen-activated protein kinase signaling pathway. For drug repositioning, we identified a targeted drug of genes in PPI network.Our study provides a bioinformatics method for screening drug targets and provides a theoretical basis for thyroid cancer targeted therapy.     

基于加权基因共表达网络分析运动性精子结构域包含蛋白2在类风湿关节炎中的表达及与疾病活动度的相关性

目的利用加权基因共表达网络分析(WGCNA)及实验验证寻找RA相关的关键基因。方法从GEO数据库下载了RA患者基因芯片数据,构建基因网络,利用WGCNA将基因划分为不同的模块,将与RA临床症状相关的模块中的关键基因进行了基因本体论分析。随后使用GEO不同的数据集用受试者工作特征曲线(ROC)评价关键基因对RA诊断的准确性。此外,还通过实时荧光定量反转录PCR(RT-PCR)及蛋白质印迹法验证关键基因在RA中的表达,分析其与DAS28的关系。采用配对样本t检验和Pearson相关性分析对结果进行分析。结果共筛选出5413个基因构建了加权基因共表达网络,将基因分为23个模块。其中,黑色模块与RA临床症状密切,包含346个基因。富集分析及京都基因与基因组百科全书(KEGG)信号通路分析显示其要富集于对IL-6的正调控、IL-1β分泌、破骨细胞分化、NOD样受体信号通路、辅助性T细胞(Th)17细胞分化等多个与RA密切相关的通路。其中运动性精子结构域包含蛋白2(MOSPD2)与临床症状具有明显相关性,在血液单核细胞、骨髓单核细胞中高表达(t=2.238,P=0.032;t=3.153,P=0.006),在RA关节滑膜液中与血液中表达呈正相关(r=0.683,P=0.03)。ROC曲线分析表明,MOSPD2能区分RA和对照组(曲线下面积分别为0.855和0.726)。RT-PCR及蛋白质印迹法结果显示,MOSPD2在RA患者中表达上调(t=-3.96,P=0.02)。MOSPD2在血液中的表达水平与RA患者的DAS28呈正相关(r=0.8846,P=0.0462)。结论MOSDP2与RA患者临床症状密切相关,可能是诊断及治疗RA的靶点之一。     

基于加权基因共表达网络的膀胱尿路上皮癌预后及疗效相关基因筛选

目的构建加权基因共表达网络分析（weighed gene co-expression network analysis,WGCNA）,鉴定出膀胱尿路上皮癌（bladder urothelial carcinoma,BLCA）中与预后及治疗效果相关的基因,并分析其在肿瘤相关通路中的活性。方法在癌症基因组图谱数据库获取膀胱尿路上皮癌的转录组测序及相应临床数据;选择年龄、性别、身高、体重、人种、生存时间、吸烟年数、治疗效果、病理分级、TNM分期及淋巴结浸润为临床表型进行加权基因共表达网络分析;利用R语言进行生存分析;利用GSCALite工具进行肿瘤相关的通路活性分析;利用基因表达汇编数据库中的GSE13507和GSE3167数据集进行验证。结果本研究鉴定出与预后及治疗效果均显著相关的基因模块及模块关键基因GPHN、CPSF2、PRMT5、EIF2S1、KLC1、SLC39A9、SRP54及CNIH,上述8个基因在肿瘤相关通路中有一定活性。结论GPHN、CPSF2、PRMT5、EIF2S1、KLC1、SLC39A9、SRP54及CNIH基因在BLCA中与预后及治疗效果均显著相关,并且参与了肿瘤相关通路,可能成为BLCA的生物标志物或潜在治疗靶点。     

Dual expression of Epstein-Barr virus,latent membrane protein-1 and human papillomavirus-16 E6 transform primary mouse embryonic fibroblasts through NF-κB signaling

The prevalence of Epstein-Barr virus (EBV) and high-risk human papilloma virus (HPV) infections in patients with oral cancer in Okinawa, southwest islands of Japan, has led to the hypothesis that carcinogenesis is related to EBV and HPV co-infection. To explore the mechanisms of transformation induced by EBV and HPV co-infection, we analyzed the transformation of primary mouse embryonic fibroblasts (MEFs) expressing EBV and HPV-16 genes, alone or in combination. Expression of EBV latent membrane protein-1 (LMP-1) alone or in combination with HPV-16 E6 increased cell proliferation and decreased apoptosis, whereas single expression of EBV nuclear antigen-1 (EBNA-1), or HPV-16 E6 did not. Co-expression of LMP-1 and E6 induced anchorage-independent growth and tumor formation in nude mice, whereas expression of LMP-1 alone did not. Although the singular expression of these viral genes showed increased DNA damage and DNA damage response (DDR), co-expression of LMP-1 and E6 did not induce DDR, which is frequently seen in cancer cells. Furthermore, co-expression of LMP-1 with E6 increased NF-κB signaling, and the knockdown of LMP-1 or E6 in co-expressing cells decreased cell proliferation, anchorage independent growth, and NF-κB activation. These data suggested that expression of individual viral genes is insufficient for inducing transformation and that co-expression of LMP-1 and E6, which is associated with suppression of DDR and increased NF-κB activity, lead to transformation. Our findings demonstrate the synergistic effect by the interaction of oncogenes from different viruses on the transformation of primary MEFs.     

hBcl-2和hVEGF165双基因共表达重组腺病毒载体的构建及鉴定

目的构建hBcl-2和hVEGF165双基因共表达的重组腺病毒载体,为后续基因转染和动物实验提供实验基础。方法通过RT-PCR从A549细胞中分别获得hBcl-2和hVEGF165基因片段,并克隆到pMD-19T载体。将目的基因hBcl-2、IRES元件和hVEGF165依次定向克隆到腺病毒穿梭载体质粒pAd-Track-CMV上。线性化重组穿梭质粒后通过电穿孔转化含有腺病毒骨架质粒pAd-easy-1的感受态大肠杆菌BJ5183构建重组腺病毒载体质粒。脂质体转染线性化的重组腺病毒载体质粒于HEK293细胞以包装制备重组腺病毒。重组腺病毒经PCR鉴定之后进一步扩增、纯化。通过荧光计数确定病毒感染滴度。结果克隆的hBcl-2和hVEGF165基因测序正确,酶切重组穿梭载体质粒和腺病毒载体质粒得到特异酶切产物,重组腺病毒载体质粒转染HEK293细胞3 d后观察到GFP的表达,重组腺病毒的PCR得到hBcl-2和hVEGF165基因的PCR产物,滴度测定为5×109pfu/mL。结论成功构建制备了高滴度的hBcl-2和hVEGF165双基因共表达的重组腺病毒载体,为联合基因治疗心肌梗死的研究提供实验基础。     

Transcription and DNA Methylation Patterns of Blood-Derived CD8+ T Cells Are Associated With Age and Inflammatory Bowel Disease But Do Not Predict Prognosis

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Co-expression of mouse and rat haemoglobins in interspecific hybrids of mouse and rat erythroleukemia cells

A modified technique for cell fusion with lysolecithin-lipid emulsions was used to generate hybrid erythroleukemia cell lines from Friend leukemia mouse cells (FLC) and chemically transformed rat erythroleukemia cells. Chromosome analysis of the hybrid cells showed the presence of both parental genomes even after long culture periods. The hybrids were still able to undergo erythroid differentiation after dimethylsulphoxide (DMSO) stimulation. Analysis of the globin chains from the DMSO-stimulated cells showed that both the rat and the mouse erythroid phenotypes were expressed. This demonstrates the compatibility of the regulatory genetic elements for the control of erythroid differentiation in cell hybrids of erythroleukemic populations from different species.