肝细胞癌中转录因子E2F7对Wnt/β-catenin信号通路活性的影响

探讨肝细胞癌(HCC)中非典型性E2F家族成员E2F7在肝癌细胞生长、分化中的作用及可能涉及的分子机制.本研究运用实时荧光定量PCR检测38例原发性肝细胞癌及对应的癌旁组织中E2F7基因mRNA的表达情况;分别通过基因过表达和RNA干扰技术上调或下调E2F7基因表达,并运用实时荧光定量PCR和Western印迹检测肝癌细胞株MHCC-H中β-catenin及其靶基因cmyc的表达情况;双荧光素酶报告基因系统检测E2F7对Wnt/β-catenin信号通路活性的影响;核浆分离实验检测过表达E2F7基因对β-catenin入核的影响;免疫共沉淀实验检测异位表达E2F7与内源β-catenin的相互作用.结果显示,肝细胞癌组织中E2F7基因的表达量显著高于相应的癌旁组织(P0.001);转录因子E2F7可与β-catenin相互作用并促进β-catenin进入细胞核.转录因子E2F7可以促进Wnt/β-catenin信号通路的活性.     

猪链球菌2型全基因组DNA芯片的研制及基因表达谱技术平台的建立

目的：研制猪链球菌2型（SS2）全基因组DNA芯片,建立SS2基因表达谱技术平台。方法：利用SS2全基因组序列,挑选出2194条基因,经PCR扩增出2156条基因并将产物纯化,点样制备芯片;将芯片用于表达谱研究,采用实时定量PCR验证表达谱结果,对芯片进行可靠性分析。结果：芯片杂交数据与实时定量PCR验证显示了较高的相关性,二者相关系数r=0.87。结论：研制了一批SS2全基因组DNA芯片,并建立了基于DNA芯片的表达谱技术平台。     

蛋白酪氨酸激酶在类风湿性关节炎滑膜细胞中的表达和功能

克隆类风湿性关节炎（RA）滑膜细胞（FLS）中的蛋白酪氨酸激酶（PTKs）,并研究它们在RA滑膜细胞异常增殖和侵软骨中的作用。根据巳知的PTKs氨基酸序列保守区设计简并引物,采用3'快速末端扩增法（3'RACE)扩增滑膜细胞中PTKs cDNAs的3'末端,将所得序列与Genebank中序列进行比较,以鉴定其是否为巳知的PTKs或与PTKs同源的新序列,然后通过RNA点杂交方法分别观察这些PTKs在RA和骨性关节炎（OA）病人滑膜细胞中的表达水平。结果表明,从RA FLS中克隆到6种巳知PTKs的cDNAs片段,分别为血小板衍生生长因子受体A（PDGFRA）、胰岛素生长因子-1受体（IGF1R）、含discoidin结构域的受体型酪氨酸激酶（DDR2）、Lyn、Janus激酶1（JAK1）和TYK2。RNA点杂交结果显示,在4个RA病人和2个OA病人的滑膜细胞中,PDGFRA、IGF-1R和DDR2在RA滑膜细胞中的表达水平高于OA滑膜细胞,其它几处激酶在两种细胞中的表达水平相同。说明RI滑膜细胞中至少表达PDGFR、IGF1R、Lyn、DDR2、JAK1和TYK2等6种PTKs,其中PDGFRA、IGF1R和DDR2可能与RA滑膜细胞的过度增殖和对软骨的侵蚀性相关。     

Bio-Plex悬浮芯片系统检测两种实验兔白介素基因的差异表达

目的采用液相悬浮芯片系统同时测定实验兔圆小囊中IL-1β、IL-1R1、IL=8、IL-8RA和IL=15各基因的表达情况,并对该方法进行评价。方法利用Affymetrix的Panomics QuantiGene Plex2．0Assay中bDNA信号放大和多磁珠分析技术,来同时检测两种实验兔圆小囊中多重mRNA并定量。建立实验兔免疫相关白介素基因的液相悬浮芯片检测方法。结果可同时检测IL-1β、IL=1R1、IL-8、IL=8RA和IL-15各基因的含量,并发现WHBE兔IL-15基因的相对表达量显著高于JW兔（P〈0．05）,IL-1R1基因的相对表达量显著高于JW兔（P〈0．01）,IL-8RA基因在WHBE兔中的相对表达量也高于JW兔（P〈0．05）。结论建立了实验兔白介素基因的液相悬浮芯片检测方法,WHBE兔的IL-15、IL-1R1和IL-8RA基因表达量较高,可能与WHBE兔独特的免疫学特性有关。     

利用表达谱芯片数据筛选不同表达丰度的内参基因

目的基于表达谱芯片的检测结果,筛选多个内参基因,用于小家鼠肝脏组织中具有不同表达丰度基因的定量检测。方法应用表达谱芯片技术,完成小鼠肝脏组织的表达谱检测;依据基因表达丰度将基因分为3组,并进一步通过变异系数(coefficient of variation,CV)组内筛选候选内参基因;采用实时荧光定量PCR技术(realtime quantitative polymerase chain reaction,q PCR)和ge Norm软件确定内参基因。结果表达谱芯片成功采集超过60000个小鼠肝脏组织中转录本的表达量数据,并将之分为低、中、高3个组合。最终筛选了低表达Casp2和Lrrc14、中表达Nrd1和Trpc4ap、高表达Atp5a1和Clu,共6个内参基因。结论基于表达谱芯片数据筛选的6个内参基因,可适用于q PCR技术准确定量小家鼠肝组织转录组中不同表达丰度基因的表达量。     

用改良消减杂交筛选类风湿性关节炎滑膜细胞中高表达基因

 为了研究类风湿性关节炎 (rheumatoid arthritis,RA)滑膜细胞 (fibroblast- like synovialcells,FLS)过度增殖和破坏软骨的分子机理 ,利用改良消减杂交法以骨性关节炎 (osteoarthritis,OA)病人滑膜细胞为对照 ,筛选 RA滑膜细胞中的高表达基因 .将得到的基因片段克隆入质粒载体 ,通过反向点杂交排除假阳性克隆后 ,将阳性克隆进行核酸序列分析 ,最后用 Northern杂交方法检测一些高表达基因在 RA和 OA病人滑膜细胞中的表达水平 .结果显示 ,共分离到 1 50个 RA高表达基因片段 ,其中长于 1 0 0 0 bp的片段占 8% (1 2 /1 50 ) ,长于 40 0 bp的片段占 36.7% (55/1 50 ) ,在大于 40 0 bp的片段中 ,假阳性率为 2 3.7% (1 3/55) .在测序的 1 8个片段中 ,已知基因有 1 2个 ,其中包括 IGF- 1结合蛋白 (IGFBP)特异性丝氨酸蛋白酶、层粘连蛋白受体和组织蛋白酶 B等 .新序列有 6个 ,其中两个序列分别与 Ring- box蛋白 1和 SON DNA结合蛋白同源 .对 IGFBP特异性丝氨酸蛋白酶、层粘连蛋白受体和组织蛋白酶 B基因的 Northern杂交分析显示 ,在 RA病人滑膜细胞中 ,这些基因的表达水平高于 OA病人滑膜细胞 .这些结果提示 ,这种改良消减杂交法是一种简便有效的分离差异表达基因的方法 ;IGF- 1结合蛋白特异性丝氨酸蛋白酶、层粘     

利用表达谱基因芯片筛选溃疡性结肠炎差异表达基因

目的:利用人类全基因组表达谱芯片技术,分析溃疡性结肠炎患者和健康者基因表达谱差异,筛选出溃疡性结肠炎相关基因。方法:采用Trizol法提取8例溃疡性结肠炎患者和8例健康对照者结肠粘膜组织总RNA并纯化,逆转录合成c DNA,利用荧光染料Cy3标记aa UTP,转录合成标记的c RNA,并与Agilent人类全基因组表达谱芯片杂交,扫描荧光信号图像,对芯片原始数据进行归一化处理,利用倍数差异和t检验计算筛选出相关差异表达基因,采用DAVID在线分析系统进行基因的功能注释和关联分析,明确差异基因的生物学功能,并对部分差异表达基因进行实时荧光定量PCR验证。结果:筛查出溃疡性结肠炎结肠粘膜组织差异表达基因4132个,其中上调基因2004个,下调基因2128个。选取6条差异表达基因进行PCR验证,结果有3条基因表达上调,3条基因表达下调,表达趋势与芯片结果一致。结论:溃疡性结肠炎患者与健康对照者基因表达存在明显差异,分析这些差异表达基因有助于我们探索溃疡性结肠炎的发病机制,为疾病的治疗提供理论依据。     

m6A甲基化修饰识别蛋白YTHDF2在肝癌组织中的表达及临床意义

摘要 目的：探讨m6A甲基化修饰结合蛋白YTHDF2(YTH domain-containing family protein 2)在肝癌（Hepatocellular carcinoma, HCC）组织中的表达及临床意义。方法：1）采用Western blot和实时荧光定量PCR（Quantitative Real Time PCR, qRT-PCR）检测20对肝癌和癌旁组织中YTHDF2在蛋白和mRNA水平的表达情况。2）通过免疫组织化学（Immunochemistry, IHC）检测40对肝癌和癌旁组织芯片YTHDF2的表达情况,并以H-score评分法进行半定量分析。3）通过基因表达谱数据动态分析数据库GEPIA（http://gepia.cancer-pku.cn/）分析YTHDF2在肝癌组织中的表达及对患者生存预后的影响;通过肿瘤免疫评估资源数据库TIMER(https://cistrome.shinyapps.io/timer/)分析YTHDF2与肝癌免疫微环境的相关性。结果：相比于癌旁肝组织,YTHDF2在肝癌组织中蛋白和mRNA水平均显著高表达（P＜0.05）;GEPIA数据库分析验证：YTHDF2肝癌组织表达水平高于正常肝组织,且YTHDF2在肝癌不同分期中表达水平均高于正常肝组织（P=0.0206）,Stage III期最为明显;YTHDF2高表达患者的总体生存率（Overall Survival,OS）和无复发生存率（Disease Free Survival, DFS）均较差（P=0.00027和P=0.013）;TIMER数据库分析表明：YTHDF2在肝癌免疫微环境中与各类免疫细胞呈正相关（P＜0.05）,但肝癌患者的累积存活率不受免疫细胞的影响（P＞0.05）,YTHDF2高表达对患者的生存预后具有不良影响（P=0.007）。结论：YTHDF2在肝癌组织中显著高表达,且YTHDF2高表达水平对肝癌患者生存预后具有不良影响,YTHDF2可作为肝癌临床预后判断的分子标志物,为今后肝癌治疗靶点提供新的策略。     

实时荧光定量PCR检测人肿瘤细胞NKG2D配体基因表达

目的：建立人肿瘤细胞NKG2D配体基因（MICA、MICB、ULBP1、ULBP2、ULBP3）表达的实时荧光定量PCR（real-time fluorescence quantitativePCR）检测方法。方法：根据NCBI基因库中NKG2D配体基因序列,设计合成引物。用Trizo1法从培养的肿瘤细胞（BEC-7402、HeLa、MDA-MB-435、XWLC-05）中提取总RNA,逆转录成eDNA,建立实时荧光定量PCR检测NKG2D配体基因表达的方法,并检测NKG2D配体在肿瘤细胞株中的表达。结果：经过琼脂糖凝胶电泳、熔解曲线和标准曲线分析,用所设计的引物和SYBR GreenI能够特异扩增和定量检测NKG2D配体基因的表达。该方法成功检测4种肿瘤细胞NKG2D配体基因的表达。结论：建立了人NKG2D配体基因表达的实时荧光定量PCR检测方法,为进一步研究人NKG2D配体在肿瘤免疫中的作用提供了有效手段。     

实时定量PCR芯片——功能基因组研究的有用工具

结合实时定量PCR（real-time quantitative polymerase chain reaction,qPCR）和基因芯片技术,实时定量PCR芯片（qPCR array）为功能基因组研究提供了一种高度灵敏、可靠的方法。在基因功能分类基础上,结合qPCR准确定量的特性,qPCR array可同时定量分析上百个基因的mRNA表达水平,是研究一组特定功能基因表达的理想方法。     

EBV gene expression not altered in rheumatoid synovia despite the presence of EBV antigen-specific T cell clones

T cells infiltrating the rheumatoid arthritis (RA) joint are oligoclonal, implicating an Ag-driven process, but the putative joint-specific Ags remain elusive. Here we examine expression of selected EBV genes in RA synovia and find no abnormal expression in RA. DNA of CMV and EBV was detectable by PCR in the synovial tissue of RA. RNA of several latent and lytic EBV genes was also detectable. However, there were no differences in EBV gene expression in synovial tissues or peripheral blood when comparing RA with osteoarthritis, Gulf War syndrome, and other disease controls. RA synovia with highly expanded CD8 T cell clones reactive with defined EBV peptide Ags presented by HLA class I alleles lacked evidence of abnormal mRNA expression for the relevant EBV Ag (BZLF1) or lacked amplifiable mRNA (BMLF1). Thus, local production of EBV Ags in synovial tissues may not be the cause of the accumulation of T cell clones specific for these Ags. Instead, APCs loaded with processed EBV peptides may migrate to the synovium. Alternatively, EBV-specific T cells clones may be generated in other tissues and then migrate to synovia, perhaps due to cross-reactive joint-specific Ags or because of expression of homing receptors.     

Characterization of histopathology and gene-expression profiles of synovitis in early rheumatoid arthritis using targeted biopsy specimens

The disease category of early rheumatoid arthritis (RA) has been limited with respect to clinical criteria. Pathological manifestations of synovitis in patients whose disease is clinically classified as early RA seem to be heterogeneous, with regular variations. To clarify the relation between the molecular and histopathological features of the synovitis, we analyzed gene-expression profiles in the synovial lining tissues to correlate them with histopathological features. Synovial tissues were obtained from knee joints of 12 patients with early RA by targeted biopsy under arthroscopy. Surgical specimens of long-standing RA (from four patients) were examined as positive controls. Each histopathological parameter characteristic of rheumatoid synovitis in synovial tissues was scored under light microscopy. Total RNAs from synovial lining tissues were obtained from the specimens selected by laser capture microdissection and the mRNAs were amplified by bacteriophage T7 RNA polymerase. Their cDNAs were analyzed in a cDNA microarray with 23,040 cDNAs, and the levels of gene expression in multilayered lining tissues, compared with those of normal-like lining tissues in specimens from the same person, were determined to estimate gene-expression profiles characteristic of the synovial proliferative lesions in each case. Based on cluster analysis of all cases, gene-expression profiles in the lesions in early RA fell into two groups. The groups had different expression levels of genes critical for proliferative inflammation, including those encoding cytokines, adhesion molecules, and extracellular matrices. One group resembled synovitis in long-standing RA and had high scores for some histopathological features – involving accumulations of lymphocytes and plasma cells – but not for other features. Possible differences in the histopathogenesis and prognosis of synovitis between the two groups are discussed in relation to the candidate genes and histopathology.     

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

Introduction

TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro.

Methods

TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry.

Results

TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38⁺ plasma cells and with CD22⁺ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22⁺ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1⁺ osteoblasts.

Conclusions

The expression of TWEAK by CD22⁺ B cells and CD38⁺ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.     

Differential Gene Expression in Synovium of Rheumatoid Arthritis and Osteoarthritis

Rheumatoid arthritis (RA) and osteoarthritis (OA) are the major types of arthritis. Although both diseases are characterized by joint destruction, their etiologies are different. To get insights into pathophysiological pathways, we used the suppression subtractive hybridization (SSH) method to identify differentially expressed genes in RA. DNA sequencing identified 12 gene products including cytoskeletal γ-actin and extracellular matrix components such as fibronectin, collagen IIIα₁, and superficial zone protein. Interferon γ-inducible genes such as a novel thiol reductase, two genes of unknown function (HSIFNIN4, RING3), and annexin II were also found. Two genes encoded proteins involved in proliferation such as elongation factor 1α and the granulin precursor. Furthermore, the protease cathepsin B and synovial phospholipase A2 group IIA were detected by SSH. To confirm the differential expression of the genes, we performed RT-PCR analyses of RA and OA synovial tissues. Compared to OA patients, 9 of the 12 genes were overexpressed in RA, suggesting that SSH is a powerful tool for the detection of differential gene expression in synovial tissues. Further characterization of the gene products may help to identify pathophysiological mechanisms in arthritic diseases.     

Dendritic cells provide a potential link between smoking and inflammation in rheumatoid arthritis

Introduction

Smoking increases the risk of developing rheumatoid arthritis (RA) and affects the severity of established RA. Smoking can impact on Th17 lymphocyte differentiation and function through activation of the aryl hydrocarbon receptor (AHR), a process with implications for the pathogenic mechanisms in RA that involve the cytokine, interleukin (IL)-17A. The objective of this study was to establish any effect of smoking on the inflammatory tissue lesions of rheumatoid arthritis via the AHR and IL-17A.

Methods

Twenty synovial and eighteen subcutaneous nodule tissue samples from 31 patients with RA were studied. Patient smoking status at the time of tissue collection was established. Expression of AHR, CYP1A1, AHRR, IL6, IL17A, IL17F, IL22, IL23, IL23R, IFNG, TBX21, IDO1 and FOXP3 genes were assessed in tissues and cultured cells using real-time PCR. Two-colour immunofluorescence was used to co-localise AHR and CYP1A1 protein in synovial tissues. The response of monocytes and monocyte-derived dendritic cells (mo-DCs) to the AHR agonist, benzo(a)pyrene (BaP) was compared in vitro.

Results

AHR gene expression was demonstrated in rheumatoid synovial tissues and nodules with significantly greater expression in synovia. Expression was not influenced by smoking in either tissue. Evidence of AHR activation, indicated by CYP1A1 and AHRR gene expression, was found only in synovia from patients who smoked. However, IL17A gene expression was lower in synovia from smokers. TBX21 and FOXP3 expression was not affected by smoking. Within the synovial tissues of smokers the principal cell type with evidence of AHR activation was a subset of synovial DCs. This observation was consistent with the sensitivity of human mo-DCs to BaP stimulation demonstrated in vitro. Exposure to BaP affected mo-DC function as demonstrated by decreased IL6 expression induced by PolyI:C, without affecting indoleamine 2,3 dioxygenase (IDO)1 expression.

Conclusion

Our findings show that one effect of smoking on inflamed rheumatoid synovial tissue involves activation of the AHR pathway. A subset of synovial DCs is important in the response to cigarette smoke. The potential for smoking to affect DC behaviour in joint tissues has relevance to both early and late phases of RA pathogenesis and warrants further investigation.     

Stromal Transcriptional Profiles Reveal Hierarchies of Anatomical Site,Serum Response and Disease and Identify Disease Specific Pathways

Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through _3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts.