RNA interference inhibits hepatitis B virus gene expression and replication in HepG_2-N10 cells

目的:评估以U6启动子作为启动序列(pSilencer2.0-U6)载体介导的小RNA干扰片段在培养细胞株中抑制HBV复制的效应.方法:将针对HBV基因组不同区域(S,X及C区)的核苷酸序列插入至pSilencer载体中,并将重组后的pSilencer质粒(分别为pS,pX及pC)载体转染入HepG2-N10细胞株(可稳定表达HBsAg、HBeAg及adw2亚型Dane颗粒)中.以ELISA法检测病毒抗原,以逆转录-聚合酶链反应法(reversetranscription-polymerasechainreaction,RT-PCR)检测病毒mRNA,并以荧光定量PCR法检测分泌入培养基的共价闭合环状DNA(covalentclosedcircularDNA,cccDNA).结果:质粒载体介导的RNA干扰能明显抑制培养基中HBsAg及HBeAg的表达.并且RT-PCR法检测显示病毒mRNA也被降解了,从而减少了蛋白表达及病毒逆转录复制的模板.荧光定量PCR法检测显示分泌入培养基的cccDNAs明显下降(HBVDNAlog10:pS:4.00±0.13;pC:4.08±0.10;pX:4.28±0.06;pN:5.05±0.07;HepG2-N10:4.74±0.06;HepG2:<2.70).结论:RNA干扰能抑制HBV基因表达及病毒复制,并且RNA干扰可能为HBV的治疗带来巨大的变化.AIM: To evaluate the effect of vector-based small interfering RNA promoted by U6 (pSilencer2.0-U6) on the inhibition of hepatitis B virus (HBV) replication in HepG2-N10 cells. METHODS: Several sequences targeting on dif- ferent regions of HBV genome were inserted into pSilencer vectors. The expression plasmids were then transfected into HepG2-N10 cells, a cell line which stably expressed HBsAg, HBeAg and adw2 subtype Dane Particles. Viral antigens were measured by enzyme linked immunosor- bent assay (ELISA). Viral mRNA was analyzedby reverse transcription-polymerase chain re- action (RT-PCR). The covalent closed circular DNA (cccDNA) secreted into the culture media were measured by quantitative real-time PCR. RESULTS: Vector-based RNA interference po- tently reduced HBsAg and HBeAg expression in cell culture. Furthermore, RT-PCR analysis showed that viral mRNAs were effectively de- graded, thus eliminating the messengers for pro- tein expression as well as templates for reverse transcription. Quantitative Real-time PCR analy- sis of cccDNAs revealed that vector-based RNA interference inhibited HBV replication efficiently (HBV DNA log10: pS: 4.00 ± 0.13; pC: 4.08 ± 0.10; pX: 4.28 ± 0.06; pN: 5.05 ± 0.07; HepG2-N10: 4.74 ± 0.06; HepG2: <2.70). CONCLUSION: RNA interference can inhibit HBV gene expression and virus replication

The regulation of trefoil factor 2 expression by the transcription factor Sp3

National Natural Science Foundation of China [81101817, 30971362]; China Postdoctoral Science Foundation [20100480724]Trefoil factor family 2 (TFF2) participates in mucus stabilization and repair, apoptosis, and inflammatory responses. Previously published reports have indicated that several growth factors and basal transcription factors are associated with the expression of TFF2. However, the detailed mechanisms that regulate TFF2 expression are not fully understood. The present study was designed to assess the essential role of the transcription factor SP3 with respect to TFF2 expression. We first demonstrated that there was a negative correlation between the expression levels of SP3 and TFF2. Thus, in the examined cells, the overexpression of SP3 decreased the expression level of TFF2, whereas the inhibition of SP3 increased the expression level of TFF2. Moreover, we discovered two GC boxes in the TFF2 promoter and confirmed the specific binding of SP3 to this promoter. On the whole, this study indicated that Sp3 was a major regulator of TFF2 expression. This knowledge should contribute to our understanding of the role that is played by SP3 in the regulation of TFF2 expression. (C) 2012 Elsevier Inc. All rights reserved

脆性组氨酸三联体基因蛋白表达与炎症性肠病的临床关系

目的:探讨脆性组氨酸三联体基因(fragile histidine trail, FHIT)蛋白在38例溃疡性结肠炎(ulcerative colitis,UC)、25例Crohn’s病、11例UC相关性腺癌和13例正常对照组中的表达情况. 方法:标本常规石蜡包埋,采用免疫组化法检测FHIT在UC、Crohn’s病、UC相关性腺癌和正常对照组中的表达. 结果:38例UC中,25例FHIT蛋白表达阴性;25例Crohn’S 病中,19例表达阴性;11例UC相关性腺癌中,6例表达阴性.FHIT蛋白在UC和正常对照组(71.43%vs 7.69%, X2=18.80,P0.05). 结论:FHIT蛋白在炎症性肠病(inflammatory bowel disease, IBD)和UC相关性腺癌中呈低表达或缺失,提示该基因可能与IBD和UC相关性腺癌发生有关

Primary study on trans-activation ability of hepatitis B virus envelope proteins

目的构建系列含乙型肝炎病毒(HBV)表面抗原前-前-S区的酵母细胞表达质粒,初步探讨表达蛋白是否具有反式激活作用。方法用多聚酶链反应(PCR)扩增含前-前-S区的全S、S2和S基因,定向克隆于pDEST32载体,进行序列测定,重组质粒命名为pDEST32-wS、pDEST32-pS2和pDEST32-SHBs。用乙酸锂转化法将重组质粒转化酵母菌MaV203,经Western blot和胶体金法验证重组质粒在酵母细胞中的表达。以酵母双杂交法将重组质粒和载体pDEST22共同转入酵母菌MaV203,并在SC/-leu/-trp/-his三缺培养基及不同浓度梯度3AT培养基中验证自激活。结果重组质粒pDEST32-wS经序列测定含有HBV自前-前-S至主蛋白基因序列。经Western blot和胶体金法证实转染的酵母细胞可表达表面抗原蛋白。pDEST32-wS与pDEST22共转染实验证实被转染酵母细胞不能在浓度30 mmol/L以上的3AT培养基生存。结论构建了pDEST32-wS、pDEST32-pS2和pDEST32-SHBs表达载体,pDEST32-wS和pDEST32-SHBs在酵母细胞中可表达HBsAg... 【英文摘要】 Objective To construct yeast expression vector of HBV whole-S gene including pre-pre-S region and explore the trans-activation function of whole-S protein.Methods Whole-S gene,pre-pre-S to pre-S2 region and major HBsAg coding region containing Not I and Sal I endoenzyme sites were obtained by PCR method.After enzyme digestion,PCR products were cloned into yeast expression vector pDEST32.Recombinant plasmids were sequenced and named as pDEST32-wS,pDEST32-pS2 and pDEST32-SHBs,respectively.Recombinant plasmids...厦门市首批重大疾病科研攻关项目(WKZ0501);; 厦门市卫生局医学科研立项项目(WSK0506);; 厦门大学引进人才科研启动基金(Z03109);; 福建省青年科技人才创新项目(2006F3127);; 福建省青年科技人才创新项目(2006F3127

三叶因子1表达与胃黏膜损伤及胃癌的关系

目的　测定三叶因子 1(TFF1)在正常及病理条件下胃黏膜中的表达情况 ,探讨TFF1在胃黏膜损伤修复及胃癌抑制中的作用及意义。方法　应用免疫组化方法测定正常及不同病理条件下胃黏膜中TFF1的表达情况 ,通过图像分析软件分析阳性信号平均吸光度值以了解其表达情况。结果　胃炎、胃溃疡及十二指肠球部溃疡患者TFF1表达明显高于正常胃黏膜 (0 .5 1± 0 .0 5 ,0 .5 1± 0 .0 6 ,0 .5 0± 0 .0 6比 0 .4 4± 0 .0 6 ;P值均 0 .0 5 )。结论　TFF1表达随黏膜损伤程度的加重而表达增强 ,提示其在胃黏膜保护及促进上皮重建机制中具有一定的作用。TFF1在癌旁组织中表达增强提示其可能与肿瘤抑制及分化机制有关 ,而在癌组织中表达减弱可能与其分泌减少有关

中国式学科评估:问题与出路

今年四月份,教育部学位与研究生教育发展中心(以下简称"教育部学位中心")邀请全国学位授予单位参加全国第四轮一级学科整体水平评估。随之,各个高校展开了一场大规模、高级别的学科评估申报及材料提交总动员。第四轮学科评估自发布起也引发了学界的广泛关注和热烈讨论。高等教育是中国崛起的思想发动机,关涉民族复兴的未来,而学科评估是近年来中国高等教育学科建设成就的集中展示,其意义和影响可谓深远。为了更好推进学科评估科学进行,特别是促进高等教育健康发展,《探索与争鸣》编辑部邀请全国

TFF1在功能性消化不良和慢性胃炎中的表达

目的 测定三叶因子1(trefoil factor,TFF1)在正常、功能性消化不良、慢性胃炎胃粘膜中的表达情况,探讨TFF1与功能性消化不良和慢性胃炎的关系。方法 应用免疫组化方法测定正常人和功能性消化不良、慢性胃炎患者胃窦黏膜中TFF1的表达,通过图像分析软件分析其阳性信号平均光密度值。结果 慢性胃炎组TFF1的表达明显高于正常组和功能性消化不良组,而正常组和功能性消化不良组中表达均较弱,二者统计学上无差异。结论 TFF1在正常组和功能性消化不良组中表达相似,认为TFF1与功能性消化不良的发病无关:而慢性胃炎组表达较高,认为与TFF1在胃黏膜屏障功能保护和促进上皮重建的机制有关

三叶因子1在胃黏膜中的表达及存在的分子形式

目的:研究三叶因子1在人胃黏膜中的表达及存在的分子形式,探讨其胃黏膜保护作用的分子生物学机制. 方法:采用western-blot方法观察三叶因子1在人胃黏膜中的存在形式,并采用免疫组化方法了解三叶因子1在胃黏膜中的定位,比较其在胃溃疡周边及距周边5cm外胃黏膜中的表达改变. 结果:(1)三叶因子1在人胃黏膜中以三种形式存在:单聚体、二聚体及一种分子量约为21kDa的TFF1与某种分子结合而成的复合物,其中,TFF1复合物的含量最高.(2) 三叶因子1主要在人胃体及胃窦黏膜上皮表面细胞胞质中表达,核周聚集较明显,越靠近腔面的细胞表达越强.(3) 三叶因子1在胃溃疡周边黏膜的表达明显高于距溃疡周边5cm外的黏膜(P<0.001). 结论:三叶因子1主要在人胃体及胃窦黏膜上皮表面细胞胞质中表达,其与某种分子结合形成的复合物是TFF1在胃黏膜中存在的主要形式,这种TFF1复合物可能比TFF1单聚体及二聚体有更强的生物活性.TFF1在胃溃疡周边黏膜表达高于距溃疡周边5cm外的黏膜,说明其在胃黏膜保护及修护方面具有重要的作用

Upregulated Expression of hITF in Crohn's Disease and Screening of hITF Interactant by a Yeast Two-Hybrid System

Science and Technology Innovation Foundation of Fujian Province, China [WQK05022005]To study the expression of human intestinal trefoil factor (hITF) mRNA in Crohn's disease and to screen the cellular proteins that can interact with the hITF protein by a yeast two-hybrid system in order to explore the mechanism of hITF in protecting intestinal mucosa from injury. Seventy-eight patients underwent double-balloon enteroscopy (DBE). Expression of hITF mRNA was detected by quantitative real-time polymerase chain reaction analysis (qRT-PCR). The hITF gene was amplified by PCR and cloned into vector pDEST32. The yeast cells cotransformed with pDEST32-hITF and the human jejunal cDNA library were plated in a selective SC-Leu-Trp-His-Ura medium. The subsequent screen was performed with chi-gal detection, and true-positive clones were sequenced and analyzed with bioinformatics. Co-immunoprecipitation (Co-IP) was performed to confirm the binding of putative proteins to the hITF protein. Thirty-nine patients were diagnosed with Crohn's disease. We found that the expression of hITF mRNA is significantly increased in Crohn's disease compared to normal controls. A total of ten colonies were selected and sequenced. Among these, six colonies were Homo sapiens zinc finger protein 193 (ZNF193), three colonies were Homo sapiens Aldo-keto reductase family 1C 1 (AKR1C1), and one colony was of an unknown gene. A reverse two-hybrid experiment and Co-IP indicated that ZNF193 and AKR1C1 might interact with hITF. The expression of hITF mRNA is increased in Crohn's disease. ZNF193 and AKR1C1 are proteins that can interact with the hITF protein by a yeast two-hybrid system and Co-IP, hITF may contribute to the mucosal repair through this interaction