中国人群16号染色体q12区存在系统性红斑狼疮易感基因

目的：明确16号染色体与中国人系统性红斑狼疮（systemic lupus erythematosus,SLE)相关性，并对其进行易感基因定位以期发现疾病侯选基因。方法：以16号染色体遗传距离57．79－65．1cM的5对微卫星标记，对157个SLE患者家系DNA进行扩增，产物经377DNA测序仪电泳，所得数据由Genescan软件收集，并以ETDT及Genehunter软件进行统计处理。在阳性位点处寻找疾病侯选基因以实时PCR进行基因表达研究。结果：D16S409及D16S517与SLE存在传递不平衡，其中尤以D16S517最为显著（P＜0．0001）。D16S517中，271bp等位基因优先传递给患病子代，277bp等位基因则优先传递给正常子代。对侯选基因的研究显示OAZ（OLF1／EBF－associated zinc finger protein)基因表达显著高于正常对照。结论：中国人群16q12区（58．46cM)与SLE发病相关联，OAZ是该区域可能的疾病基因，其疾病机理有待进一步研究。     

人乳头瘤病毒16型中国株L1基因的体外真核表达

目的 构建人类乳头瘤病毒16型(HPV16)中国株晚期基因L1的真核表达系统.方法 将HPV16中国株L1基因从pCR2.1/HPV16L1定向亚克隆至pTacer-CMV载体,构建重组质粒pTaeer-CMV/HPV16L1,将重组质粒用脂质体转染N1H3T3、HeLa细胞,用透射电镜、SDS-PAGE、蛋白印迹等方法观察HPV16 L1蛋白的表达.结果 NIH3T3、HeLa细胞经pTacer-CMV/HPV16L1转染后,SDS-PAGE、蛋白印迹等实验显示可表达HPV16 L1蛋白,电镜下可见病毒样颗粒(VLP).结论 pTacer-CMV/HPV16L1构建成功,可在体外表达HPV16中国株L1蛋白.     

系统性红斑狼疮易感基因单核苷酸多态性研究进展

系统性红斑狼疮(systemic lupus erythematosus,SLE)是一种复杂的多基因自身免疫性疾病,遗传因素重要而复杂.SLE的易感基因包括人类白细胞抗原(HLA)基因、免疫球蛋白Fc受体(FcR)基因、细胞毒性T细胞相关抗原4(CTLA-4)基因、免疫球蛋白受体同源体(FcRL)基因等.对易感基因单核...     

PBX1基因剪切体表达与SLE的相关研究

了解PBX1基因各种剪切体的表达在SLE患者和正常人中是否存在差异 ,探讨PBX1的表达与SLE发病的相关性。通过PCR扩增及毛细管芯片电泳 ,确证剪切体h、k、l存在于人体 ;通过实时荧光定量PCR技术 ,对剪切体h、k、l分别进行SLE患者组和正常组的mRNA表达定量比较。结果发现这 3种剪切体在患者组中的表达较正常人明显降低 ,正常人的表达是SLE的 9～ 12倍。重度患者的k、l剪切体与轻中度的病人相比表达明显降低 ,并发狼疮性肾炎的病人k剪切体的表达较无肾累及的病人显著降低。说明PBX1基因剪切体h、k、l在SLE患者中mRNA表达水平下降 ,并与SLE活动度及肾累及有关。提示机体通过PBX1的表达量的调节可能参与SLE的发病     

蝎素组分Ⅲ对THP1细胞HMGB1表达的影响

目的:研究蝎素组分Ⅲ(Scorpion venom crudeⅢ,SVC-Ⅲ)对THP1细胞HMGB1表达的影响,探讨蝎素与HMGB1相互作用的机制。方法:不同浓度SVC-Ⅲ(0.1、1.0、10和100 ng/ml)刺激培养THP1细胞48小时后,RT-PCR检测HMGB1 RNA水平表达差异;Western blot检测HMGB1蛋白水平表达情况;激光共聚焦检测HMGB1在细胞中的分布情况。结果:1.0 ng/ml SVC-Ⅲ刺激培养THP1细胞,HMGB1 RNA及蛋白表达水平升高最显著,随着SVC-Ⅲ浓度的增加,HMGB1 RNA及蛋白的表达水平反而下降;SVC-Ⅲ刺激培养THP1细胞可引起HMGB1从胞核转移入胞浆。结论:SVC-Ⅲ可以刺激或抑制THP1细胞HMGB1的表达,其作用效果与剂量密切相关。     

系统性红斑狼疮患者血清中Tim-1蛋白水平及其基因多态性

目的:检测T细胞免疫球蛋白黏蛋白域蛋白-1(Tim-1)在系统性红斑狼疮患者和健康对照组血清中的浓度,比较不同基因型间Tim-1蛋白的浓度,及其基因第4外显子插入/缺失多态性,分析其与系统性红斑狼疮的关系。方法:采用ELISA方法测定Tim-1蛋白在血清中的浓度;PCR反应检测TIM-1的第4外显子插入与缺失多态性,计算基因型与等位基因的频率。结果:系统性红斑狼疮患者和健康对照组血清中Tim-1蛋白的浓度分别是:(263.083±276.953)和(58.527±92.424)pg/ml,两组之间的差异有统计学意义;SLE患者TIM-1的第4外显子缺失/缺失纯合子与缺失/插入杂合子的Tim-1蛋白的浓度分别为(307.360±284.079)和(191.750±256.708)pg/ml,两组之间无显著性差异。健康对照组TIM-1的第4外显子缺失/缺失纯合子与缺失/插入杂合子的Tim-1蛋白的浓度分别为(70.295±109.917)和(40.468±41.739)pg/ml,两组之间无显著性差异。对照组的TIM-1的第4外显子缺失/缺失纯合子,缺失/插入杂合子,插入/插入纯合子基因型频率分别是0.617,0.321,0.062;系统性红斑狼疮患者相应的基因型频率是0.652,0.326,0.022。两组之间无显著性差异。结论:Tim-1蛋白在系统性红斑狼疮患者血清中的浓度升高,与系统性红斑狼疮的正相关。但是第4外显子插入与缺失多态性与系统性红斑狼疮无相关性,该突变不改变Tim-1蛋白在血清中的水平。     

Megsin基因转染对小鼠肾小球系膜细胞单核细胞趋化蛋白-1及细胞间黏附分子-1表达的影响

目的:观察megsin基因转染对高糖环境中肾小球系膜细胞单核细胞趋化蛋白-1(MCP-1)及细胞间黏附分子-1(ICAM-1)表达的影响。方法:高糖环境中培养小鼠肾小球系膜细胞,分别培养12、24、48 h,采用MTT法检测细胞增殖程度,免疫细胞化学和Western blot法检测系膜细胞megsin、MCP-1、ICAM-1蛋白表达水平,ELISA检测细胞培养上清Ⅳ型胶原浓度。结果:高糖环境中肾小球系膜细胞megsin、MCP-1及ICAM-1表达增强,细胞增殖明显,细胞上清液中Ⅳ型胶原浓度升高,megsin基因转染后上述变化趋势更加显著,而megsin shRNA质粒转染可明显减弱上述变化。结论:Megsin可上调MCP-1及ICAM-1表达,促进系膜细胞增殖及系膜外基质积聚。     

HPV16L1晚期基因诱导表达的实验研究

目的：可为ＨＰＶ１６Ｌ１重组蛋白疫苗及诊断试剂盒的研制打下基础。方法：含有ＨＰＶ１６Ｌ１ｐＰＩＣ３．５重组质粒的ＧＳ１１５阳性菌株，经甲醇诱导在甲醇营养型酵母菌中表达ＨＰＶ１６Ｌ１蛋白，经ＳＤＳ－ＰＡＧＥ电泳，对所表达蛋白进行分析、鉴定。结果：ＨＰＶ１６Ｌ１－ｐＰＩＣ３．５重组质粒经甲醇诱导产生５５ＫＤ大小的Ｌ１靶蛋白，经ＳＤＳ－ＰＡＧＥ电泳表明，诱导第四天Ｌ１蛋白表达量最大。结论：甲醇可诱导ＨＰＶ１６Ｌ１靶蛋白的表达，并且在第四天表达量最大。     

HPV16L_1晚期基因诱导表达的实验研究

目的 :可为HPV16L1重组蛋白疫苗及诊断试剂盒的研制打下基础。方法 :含有HPV16L1pPIC3.5重组质粒的GS115阳性菌株 ,经甲醇诱导在甲醇营养型酵母菌中表达HPV16L1蛋白 ,经SDS PAGE电泳 ,对所表达蛋白进行分析、鉴定。结果 :HPV16L1 pPIC3.5重组质粒经甲醇诱导产生 5 5KD大小的L1靶蛋白 ,经SDS PAGE电泳表明 ,诱导第四天L1蛋白表达量最大。结论 :甲醇可诱导HPV16L1靶蛋白的表达 ,并且在第四天表达量最大。     

Tim-3在调控IFI44表达及影响H1N1感染中的作用研究

目的探讨Tim-3信号影响免疫细胞抗感染作用的机制。方法通过基因芯片筛选及体内外验证的方法探讨Tim-3对IFI44(一种抗感染关键效应分子)表达的影响,并结合H1N1感染模型对其意义进行评价。结果在巨噬细胞上敲低Tim-3表达或通过Tim-3融合蛋白阻断Tim-3信号,均能显著提高IFI44的表达,而在Tim-3高表达的巨噬细胞中IFI44的表达受到明显抑制。在整体水平上检测Tim-3信号对IFI44表达的影响发现,给予受H1N1病毒感染小鼠腹腔注射s Tim-3蛋白能显著提高小鼠体内IFI44的表达进而降低H1N1病毒载量。结论 Tim-3在抑制抗病毒感染效应分子IFI44表达中发挥关键作用;部分解释了Tim-3调控抗感染免疫的机制,也为防治H1N1等病毒的感染提供了新的思路。     

IFI16 reduced expression is correlated with unfavorable outcome in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Its clinical course is typically indolent; however, based on a series of pathobiological, clinical, genetic, and phenotypic parameters, patient survival varies from less than 5 to more than 20 years. In this paper, we show for the first time that the expression of the interferon‐inducible DNA sensor IFI16, a member of the PYHIN protein family involved in proliferation inhibition and apoptosis regulation, is associated with the clinical outcome in CLL. We studied 99 CLLs cases by immunohistochemistry and 10 CLLs cases by gene expression profiling. We found quite variable degrees of IFI16 expression among CLLs cases. Noteworthy, we observed that a reduced IFI16 expression was associated with a very poor survival, but only in cases with ZAP70/CD38 expression. Furthermore, we found that IFI16 expression was associated with a specific gene expression signature. As IFI16 can be easily detected by immunohistochemistry or flow cytometry, it may become a part of phenotypic screening in CLL patients if its prognostic role is confirmed in independent series.     

Altered patterns of the interferon-inducible gene IFI16 expression in head and neck squamous cell carcinoma: immunohistochemical study including correlation with retinoblastoma protein, human papillomavirus infection and proliferation index

AIMS: To investigate whether the expression of interferon (IFN)-inducible gene IFI16 is inversely related to proliferative activity in vivo, we compared immunohistochemical reactivity of IFI16 in a series of head and neck squamous cell carcinomas (HNSCCs) with their proliferation index and the cell cycle regulator pRb. As human papillomavirus (HPV) infection is manifested by changes in the function or expression level of host genes such as IFN-inducible genes, we also investigated the presence of HPV DNA to determine whether head and neck cancers associated with HPV DNA can be distinguished from tumours that are presumably transformed by other mechanisms. METHODS: Thirty-six HNSCCs were evaluated for IFI16, pRb and Ki67 expression by immunohistochemistry. The presence of HPV was also detected by polymerase chain reaction. Nine tumours were located in the oropharynx (tonsillar area) and 27 in the larynx. RESULTS: HPV DNA was found in 14 of 25 (56%) laryngeal SCCs and in five of nine (56%) tonsillar SCC specimens examined; 17 out of the 19 HPV-DNA-positive cases showed high-grade IFI16 expression. Overall, proliferative activity was significantly related to tumour differentiation and histological grading. IFI16 protein expression was significantly inversely correlated with Ki67 (P = 0.039). Low-proliferating tumours positive for IFI16 staining showed a marked expression of pRb and a better prognosis than those whose tumours had low IFI16, pRb levels and a high proliferation index. CONCLUSIONS: To our knowledge, this is the first expression analysis of the IFN-inducible IFI16 gene in HNSCC. Low-proliferating tumours positive for IFI16 staining showed a marked expression of pRb and a better prognosis than those whose tumours had low IFI16, pRb levels and a high proliferation index.     

The immunoglobulin lambda-like gene cluster (14.1, 16.1 and F lambda 1) contains gene(s) selectively expressed in pre-B cells and is the human counterpart of the mouse lambda 5 gene

A human immunoglobulin (Ig)-related gene, covering approximately 8 kb, was isolated from a cosmid genomic library, by hybridization with a C lambda probe and with a lambda-like probe. This gene was identified as 14.1 It belongs to the human lambda-like cluster which is composed of three genes (14.1, 16.1 and F lambda 1) that do not rearrange. Sequence data indicate that 14.1 is organized similarly to the mouse lambda 5 gene. It contains three exons with lengths of 69, 38, and 106 codons as compared with 65, 38, and 106 for exons 1, 2, and 3 of mouse lambda 5, respectively. The corresponding homology values were 61, 66 and 75.5%. Using a 14.1 specific probe containing exon 1, we showed that this gene was selectively expressed in human pre-B cell lines. It is likely to encode a 213-amino acid lambda-like light chain that would associate with mu chains and play an important role in the early steps of B cell differentiation.     

过表达Gnb2l1基因对体外节律基因mPer1表达的影响

目的:评价过表达Gnb2l1基因对节律基因mPer1表达的影响。方法:用PMA刺激NIH3T3细胞,使节律基因稳定表达后,用脂质体介导方法将质粒pcDNA3.1/Gnb2l1转染至NIH3T3细胞,并以空载体质粒pcDNA3.1/vector为对照。利用RT-PCR技术检测不同时间点Gnb2l1 mPer1在NIH3T3细胞中的表达水平,研究过表达Gnb2l1基因对mPer1基因表达的影响。结果:采用RT-PCR技术显示成功转染,实验组比对照组Gnb2l1表达量明显增高(p0.05)。并且发现实验组的mPer1表达量比对照组明显增高,但是经过余弦分析发现两组相比mPer1的表达周期无明显改变。结论:成功过表达Gnb2l1基因使节律基因mPer1表达量增加,中值增高(p=0.038)。     

Influence of whole arm loss of chromosome 16q on gene expression patterns in oestrogen receptor-positive, invasive breast cancer

A whole chromosome arm loss of 16q belongs to the most frequent and earliest chromosomal alterations in invasive and in situ breast cancers of all common subtypes. Besides E-cadherin, several putative tumour suppressor genes residing on 16q in breast cancer have been investigated. However, the significance of these findings has remained unclear. Thus, other mechanisms leading to gene loss of function (eg haploinsufficiency, or distortion of multiple regulative subnetworks) remain to be tested as a hypothesis. To define the effect on gene expression of whole-arm loss of chromosome 16q in invasive breast cancer, we performed global gene expression analysis on a series of 18 genetically extensively characterized invasive ductal breast carcinomas and verified the results by quantitative real-time PCR (qRT-PCR). The distribution of the differential genes across the genome and their expression status was studied. A second approach by qRT-PCR in an independent series of 30 breast carcinomas helped to narrow down the observed effect. Whole-arm chromosome 16q losses, irrespective of other chromosomal changes, are associated with decreased expression of a number of candidate genes located on 16q (eg CDA08, CGI-128, SNTB2, NQO1, SF3B3, KIAA0174, ATBF1, GABARAPL2, KARS, GCSH, MBTPS1 and ZDHHC7) in breast carcinomas with a low degree of genetic instability. qRT-PCR provided evidence to suggest that the expression of these genes was reduced in a gene dosage-dependent manner. The differential expression of the candidate genes according to the chromosomal 16q-status vanished in genetically advanced breast cancer cases and changed ER status. These results corroborate previous reports about the importance of whole-arm loss of chromosome 16q in breast carcinogenesis and give evidence for the first time that haploinsufficiency, in the sense of a gene dosage effect, might be an important contributing factor in the early steps of breast carcinogenesis.     

HPV16 L1基因的原核表达及免疫反应性鉴定

目的：在大肠杆菌中表达人乳头瘤病毒16型(HPV16)主要衣壳蛋白L1，并鉴定其免疫反应性。方法：将HPV-16L1基因克隆人原核表达载体pThioHisC中，构建重组表达载体。以重组载体分别转化大肠杆菌Top10和DH5α，在IPTG诱导下表达外源基因，用SDS—PAGE和Western blot对表达产物进行鉴定和分析。结果：构建了HPV—16L1基因的原核表达质粒pThioHisC／HPV—16L1，并在大肠杆菌中表达出相对分子质量(Mr)约为70800的蛋白。表达的蛋白能与抗HPV—16L1抗体发生特异性反应。结论：在原核细胞中成功地表达HPV—16L1基因，为HPV—16L1疫苗的研制提供了必要的基础。