Impact of Double Expression of C-MYC/BCL2 Protein and Cell of Origin Subtypes on the Outcome among Patients with Diffuse Large B-Cell Lymphoma: a Single Asian Center Experience

Background: Diffuse large B-cell lymphoma (DLBCL) with double expression of c-MYC and BCL2 protein isassociated with dismal outcome after treatment with R-CHOP. Local data on disease burden and survival outcome inDLBCL is limited. We investigated the prognostic values of c-MYC/BCL2 protein co-expression and cell of originsubtypes using immunohistochemistry (IHC) and to determine their associations with multiethnic groups underresource limited setting. Methods: This was a retrospective study which recruited 104 patients in between June 2012and December 2015 for IHC review and analysis. Result: We demonstrated that patients with high InternationalPrognostic Index (IPI) (score 3-5) and co-expression of c-MYC/BCL2 protein had significant inferior overall survival(OS) and event free survival (EFS) respectively (P<0.05). c-MYC/BCL2 protein co-expression was more common innon-germinal center B-cell (non-GCB) (P=0.048) and contributed to adverse prognosis in this group of patients (OS,P=0.004; EFS, P=0.005). In multivariate analysis, double-protein co-expression was a significant independent predictorof inferior outcome after adjusted for IPI and cell of origin subtypes (OS hazard ratio [HR], 2.11; 95% CI, 1.01 to 4.04;P=0.048; EFS HR, 2.31; 95% CI, 1.05 to 5.04; P=0.036). In addition, non-GCB subtype was more common than GCBin Malays (60% vs 40%, P=0.106) and Chinese (81.2% vs 18.8%, P=0.042). Indians had more DLBCL without c-MYC/BCL2 protein co-expression compared to double-protein positive cases (66.7% vs 33.3%, P=0.414). Otherwise, theprognostic impact of ethnicity on survival outcome was insignificant (P=0.961). Conclusion: c-MYC/BCL2 proteinco-expression in non-GCB subtype constituted a unique group with extremely inferior outcome regardless of ethnicity.Gene expression profile (GEP) may possibly provide insights into the cause of discrepancies in DLBCL subtypes andprotein expression among the multiethnic groups.     

基于加权基因共表达网络分析（WGCNA）探讨龙葵抗肺腺癌功能基因模块及生物标记识别研究

目的 采用加权基因共表达网络分析（WGCNA）法探讨龙葵抗肺腺癌的潜在生物靶标。方法 基于中药系统药理学技术平台（TCMSP）及文献检索,以口服利用度（OB）、类药性（DL）构建龙葵活性成分数据库,采用DRAR-CPI服务器反向模拟分子-靶蛋白对接龙葵预测成分可能的作用靶标,并结合WGCNA挖掘在美国国立生物技术信息中心（NCBI）的GEO数据库中的GSE10072数据集得到共表达基因模块,并与龙葵预测靶标匹配映射,得到龙葵潜在抗肺腺癌靶点。将预测靶标与抗肺腺癌基因分别利用生物学信息注释数据库（Metascape）进行GO生物学过程富集分析和KEGG通路富集分析,并用STRING数据库结合Cytoscape软件可视化龙葵潜在抗肺腺癌靶点蛋白相互作用网络并进行网络拓扑学分析,同时构建龙葵活性成分-抗癌靶点-通路网络,探讨龙葵抗癌作用的机制。并通过UALCAN及Kaplan Meier plotter数据库分析关键基因在肺腺癌组织中转录水平的变化,通过KM plotter分析关键基因与肺腺癌患者预后的关系。结果 共收集龙葵活性成分9个,包括皮树脂醇、谷甾醇、薯蓣皂苷元、辣茄碱、槲皮素、α-茄碱、澳洲茄碱、澳洲茄边碱、澳洲茄胺,预测成分作用靶标271个,筛选龙葵潜在抗癌靶点41个,其潜在调控通路包括癌症通路、PI3K-Akt信号通路、化学物致癌作用、癌症的中心碳代谢等通路。由蛋白互作网络分析可得关键基因为EGFR、CASP8、HPGDS、FYN,且证实高表达的EGFR和CASP8、低表达的HPGDS及FYN与肺腺癌患者不良预后紧密相关。结论 龙葵抗肺腺癌具有多成分、多靶点、多途径作用的特点,为其抗癌物质基础及作用机制的阐明提供了科学依据。     

基于加权基因共表达网络分析阿尔茨海默病相关的核心基因

目的 采用加权基因共表达网络分析（WGCNA）探索阿尔茨海默病（AD）相关的差异基因模块及其枢纽基因,并对差异基因模块进行生物功能注释。方法 从GEO数据库下载转录组测序数据,根据基因的相关性,当关联系数阈值设定为0.85时,参数β=8,以此构建基因共表达网络;采用Pearson相关性检验计算模块基因与临床表型相关性,筛选出与AD显著相关的基因模块,根据模块内的连接性筛选枢纽基因;利用GO功能富集分析和KEGG通路分析对模块进行功能注释。进一步建立β-淀粉样蛋白（Aβ1-42）诱导SH-SY5Y细胞损伤模型,在模型组和对照组中检测枢纽基因的表达水平。结果 根据基因表达的相关性,共构建了10个基因共表达模块,其中brown和turquoise模块与AD组显著相关（brown：r=0.66,P<0.001;turquoise：r=-0.68,P<0.001）;结果显示48个基因在共表达网络中处于核心地位;通过生物注释功能发现,两模块中的基因主要富集在DNA损伤修复通路和代谢相关通路等生物学过程中。基因的差异表达分析显示,DNASE1、TEKT2、MTSS1L等基因在AD组中高表达,ACP2、LANCL2、GMPR2 等基因在 AD 组中低表达;体外实验进一步验证了在 Aβ1-42 诱导的 SH-SY5Y 细胞损伤过程中DNASE1、TEKT2、MTSS1L表达上调（P<0.01）,ACP2、LANCL2、GMPR2表达下调（P<0.01）。结论 brown和turquoise模块与AD高度相关,并从模块中筛选出MTSS1L、GMPR2、ACP2、ACTG1、LANCL2等枢纽基因,可能通过调节DNA损伤和修复参与AD发病机制。     

钠碘转运体报告基因共表达对嵌合型受体T细胞体外增殖及杀伤能力的影响

目的 探讨钠碘转运体（NIS）报告基因的共表达对嵌合型受体T（CAR-T）细胞体外增殖和杀伤活性的影响。方法 慢病毒感染法制备CAR-T细胞（仅表达CD19 CAR的T细胞）、NIS-T细胞(仅表达NIS报告基因的T细胞)和NIS-CAR-T细胞（共表达NIS和CD19 CAR的T细胞）,然后按T细胞蛋白表达情况分为4组∶T细胞组（未转染的T细胞）、CAR-T组、NIS-T组、NIS-CART组。流式细胞术检测各组NIS和CAR转染率。各组细胞常规培养24、48、72 h,CCK-8法检测增殖能力。各组T细胞为效应细胞,Nalm6肿瘤细胞为靶细胞,按效靶比0.5∶1、1∶1、2∶1、4∶1共培养24、48、72 h,乳酸脱氢酶细胞毒性检测法（LDH）检测各组细胞对肿瘤细胞的杀伤率,酶联免疫吸附试验（ELISA）法检测共培养上清液中各组细胞因子人干扰素-γ（IFN-γ）和人肿瘤坏死因子-β（TNF-β）释放水平。摄碘实验检测表达NIS蛋白各组细胞的NIS功能。结果 CAR-T细胞、NIS-CAR-T细胞的CAR转染率分别为91.91%、99.41%,NIS-T细胞、NIS-CAR-T细胞的NIS转染率分别为47.83%、50.24%。常规培养24、48、72 h CAR-T细胞与NIS-CAR-T细胞增殖率差异无统计学意义（P>0.05）。0.5∶1、1∶1、2∶1和4∶1效靶比作用24 h时CAR-T细胞杀伤率（%）均高于NIS-CAR-T细胞（P<0.05）,作用48 h和72 h时两组杀伤率差异均无统计学意义（P>0.05）。CAR-T细胞与NIS-CAR-T细胞均存在IFN-γ和TNF-β释放现象,释放水平均高于对照组（P<0.05）。NIS-T细胞与NIS-CAR-T细胞均具有特异性摄碘能力,二者间摄碘能力差异无统计学意义（P>0.05）,但均高于对照组T细胞（P<0.05）。结论 NIS报告基因的共表达不影响CAR-T细胞中CAR转染率,对细胞增殖和杀伤活性无抑制现象。     

结核分枝杆菌Ag85A和Ag85B基因真核共表达载体的构建与表达

目的 Ag85A和Ag85B是结核分枝杆菌的主要优势保护抗原,为提高编码Ag85A和Ag85B DNA疫苗的免疫原性和免疫效果,特构建真核共表达Ag85A和Ag85B抗原的真核表达载体pcDNA-Ag85A IRES-Ag85B,并利用293T细胞对其表达进行检测.方法 PCR扩增Ag85A和Ag85B基因,逐步将Ag85A和Ag85B基因定向插入至质粒pcDNA3.1(+)多克隆位点CMV启动子与加A信号BGH poly(A)之间,并将Ag85A和Ag85B基因以内部核糖体进入位点(internal ribo some entry site,IRES)序列连接,酶切和测序验证后将重组质粒转染至293T细胞中进行真核表达,48 h后提取细胞总蛋白,表达产物经SDS-PAGE分离和Western blot分析.结果 限制性内切酶酶切及测序结果表明,重组质粒pcDNA-Ag85AIRES-Ag85B基因序列和阅读框架正确.将重组质粒转入293T细胞后,利用Ag85A和Ag85B特异性抗体Western blot检测证实两种抗原可在同一载体中各自独立表达.结论 成功构建了能够同时独立表达结核分枝杆菌Ag85A和Ag85B抗原基因的真核表达载体pcDNA-Ag85A IRES-Ag85B,为下一步研究它们的免疫原性和免疫效果奠定了基础.     

tPA和VEGF165基因共表达质粒对人血管细胞增殖的影响和纤溶作用

目的：观察组织纤溶酶原激活物（tPA）和血管内皮细胞生长因子165（VEGF165）基因共表达质粒在血管平滑肌细胞（VSMC）中的表达,并研究表达产物对血管内皮细胞（VEC）和VSMC增殖的影响和纤溶作用。方法：用脂质体转染法将tPA和VEGF165的共表达质粒pBudCE4.1/tPA-VEGF165转染VSMC,逆转录－聚合酶链式反应（RT-PCR）和酶联免疫吸附实验（ELISA）分别从mRNA水平和蛋白质水平检测tPA和VEGF165的表达;纤溶蛋白板法检测转染基因的VSMC培养基中tPA的纤溶活性;取转染pBudCE4.1/tPA-VEGF165质粒的VSMC培养基培养VEC和VSMC,用四甲基偶氮唑盐（MTT法）和流式细胞技术检测转基因VSMC的细胞培养基对VEC和VSMC增殖的影响。结果：pBudCE4.1/tPA-VEGF165转染VSMC后,RT-PCR和ELISA检测发现,tPA和VEGF165在mRNA和蛋白质水平均有表达;转基因培养基有明显促进纤溶和促VEC增殖作用,而对VSMC增殖无作用。结论：tPA和VEGF165基因共表达质粒pBudCE4.1/tPA-VEGF165能在VSMC表达有生物学活性的tPA和VEGF165,为tPA和VEGF165基因转染防治移植心脏内血管狭窄奠定了基础。     

Co-evolution analysis on endocrine research: a methodological approach

The rapid growth of different kinds of biological information allows a good opportunity to analyze the co-evolutionary characteristics in endocrine regulatory pathways. Data ranging from kinds of species’ genome, gene sequence, protein structure, and expression profile of different organisms can reveal the inner co-evolutionary relationship of ligands, receptors, and other related molecules. In return, these co-evolutionary characteristics can help us determine uncharacterized ligands and receptors, annotate gene functions, highlight amino acid residues with biochemical significance, and identify regulated genes in the endocrine process. Encouraging examples in this field, although at their starting stage, have emerged. Here we focus on recent progress in endocrine-related co-evolution research from a methodological approach.     

Glial fibrillary acidic protein immunoreactivity in normal and diseased human breast

Summary Immunostaining for glial fibrillary acidic protein (GFAP) identifies a minor subpopulation of immunoreactive myoepithelial cells in the normal resting human breast. The GFAP-immunoreactive cells also express a panel of myoepithelial cell markers, including cytokeratin 14 (CK 14), vimentin, smooth-muscle-specific actin isoforms, nerve growth factor receptor (NGFR) and common acute lymphoblastic leukaemia antigen (CALLA). The percentage of GFAP-immunoreactive myoepithelial cells is greatly increased in various neoplastic and non-neoplastic diseases of the breast, being highest in adenomyoepitheliomas. Furthermore, in all the instances of fibroadenoma, phyllodes tumour, epitheliosis and gynaecomastia, a variable number of epithelial cells also acquires immunoreactivity for GFAP, vimentin, CK 14, NGFR and, to a lesser extent, for CALLA. Conversely, GFAP immunoreactivity has never been encountered in the malignant cells of the different types of breast carcinoma. These findings suggest that the expression of GFAP might be a (possibly transient) feature of proliferating epithelial and myoepithelial cells in breast diseases other than carcinomas.