Mina53在胃癌组织中的表达及临床意义

目的:探讨mina53基因在胃癌组织中的表达及其与临床分期的相关性.方法:分别应用RT-PCR和免疫组化技术,检测71例胃癌患者组织标本以及20例癌旁组织中mina53基因和蛋白的表达水平,并分析其与胃癌临床分期的相关性.结果:(1)RT-PCR结果显示20例癌旁组织中仅2例mina53基因呈弱阳性表达,阳性率为10.00％;71例胃癌组织标本中有43例mina53基因表达阳性,阳性率为60.56％ (P＜0.05);其中Ⅰ、Ⅱ、Ⅲ、Ⅳ期胃癌组织标本中的阳性比例分别为11/23、15/29、12/14及5/5(P＜0.05).(2)免疫组化结果显示20例癌旁组织中仅3例mina53蛋白呈弱阳性表达,阳性率为15.00％;71例胃癌组织标本中有35例mina53蛋白表达阳性,阳性率为49.30％(P＜0.05);其中Ⅰ、Ⅱ、Ⅲ、Ⅳ期胃癌组织标本中的阳性比例分别为7/23、13/29、11/14及4/5 (P＜0.05).结论:与癌旁组织相比,mina53在胃癌中的基因和蛋白水平存在高表达,并且随肿瘤分期的进展而表达量增高,检测mina53表达水平有助于胃癌早期诊断和预测肿瘤的侵袭程度.     

延边地区丙型肝炎患者合并TT病毒感染的检测

检测丙型肝炎患者血清标本中的TT病毒 (transfusiontransmittedvirus,TTV) ,了解延边地区丙型肝炎患者合并TTV感染状况。采用ELISA检测抗TTVIgG和巢式PCR检测丙型肝炎病毒 (HCV)感染患者血清中TTVDNA。采用全自动生化分析仪检测患者血清谷氨酸氨基转移酶 (ALT)和谷氨酸草酰乙酸氨基转移酶 (AST)。 4 5例丙型肝炎患者抗TTVIgG阳性率为 37.8% (17/45 ) ,巢式PCR阳性率为 4 2 .2 % (19/45 )。延边地区HCV感染患者重叠感染TTV较常见。     

巢式荧光定量RT-PCR检测中枢神经系统HCV方法的构建

目的 建立丙型肝炎病毒5′NCR基因的巢式荧光定量RT-PCR检测方法 ,用于中枢神经系统感染超低浓度HCV的准确和快速定量检测.方法 选择高度保守区5′NCR基因片段,设计并合成相应的特异性引物和探针,建立巢式荧光定量RT-PCR检测中枢神经系统感染样本中的超低浓度HCV正负链RNA的方法 .并对32例血清HCV抗体检查阴性的病毒性脑炎患者脑脊液有核细胞和外周血单个核细胞(PBMC)进行检测.结果 可特异性地检出脑脊液有核细胞及PBMC中HCV正负链RNA,最低检出浓度均可达7.85 copies/μl,与其他单股正链RNA病毒如登革热病毒(DEV)无交叉反应.32例血清HCV抗体检查阴性样本脑脊液有核细胞和PBMC中 HCV 5′NCR正链片段阳性的分别为1例(1/32)和2例(2/32),负链片段阳性的分别为1例(1/32)和0例(0/32).结论 本方法 的构建适用于中枢神经系统感染超低浓度HCV正负链RNA检测,且快速有效、敏感性和特异性较高,不易出现假阳性,可有效提高检出率,进一步完善目前临床常规检测HCV的方法 .     

苏南地区1b型丙型肝炎病毒包膜区变异进化研究

本研究旨在了解本地区丙型肝炎病毒(Hepatitis Cvirus,HCV)基因型构成的前提下,分析1b型丙型肝炎病毒包膜2(second envelope glycoprotein E2)区的变异和种系进化,并研究其准种变异与临床肝病活动度的关系.对宜兴市人民医院收集的抗HCV抗体阳性患者166名,RT-PCR方法检测HCVRNA,HCVRNA阳性患者采用型特异性引物分型法确定病毒基因型;选择其中未经干扰素治疗的43例1b型慢性丙型肝炎患者的血清标本,扩增E2区,从中选取肝硬化患者4例,慢性非肝硬化患者6例的E2区PCR产物纯化测序,序列采用CLUSTALW与GENBANK上多株不同型别的HCV序列进行比对分析,结果采用Phylip软件构建遗传进化树;并观察E2区高变区1(HVR-1)的氨基酸(amino acid,aa)残基序列的变异特征;采用单链构象多态性垂直电泳检测43例患者个体内HCV E2区准种的变异情况,比较不同肝病活动度患者准种变异情况.结果表明本地区HCV以1b型为主(84.3%),对E2区基因序列和氨基酸序列变异的分析显示其变异具有一定的规律性,种系进化树提示本地区HCV病毒序列与上海、湖南、日本等地的HCV株有较近的亲缘性.43例患者中ALT高于正常的丙型肝炎患者准种复杂程度明显高于ALT正常者(P＜0.05).故本地区HCV基因变异符合中国东南部的特点,基因变异与临床肝病活动度具有相关性.     

抗—HCV阴性献血员中丙型肝炎病毒RNA检测及序列分析

对95份抗—HCVIgG阴性献血员采用逆转录聚合酶链反应法（PCR）检测丙型肝炎病毒RNA,结果8次中有6次其检测出17份阳性标本（17／95,17．9％）,复查抗—HCVIgG仍为阴性。对其中8份阳性产物中高变区1的序列分析结果表明均为不同株HCV序列．排除了PCR污染的可能性。对其中2份阳性产物测定了全序列并与HCV各基因型代表株的相应序列比较,与HCVⅡ型相应序列的核苷酸同源性为77％～79％。而与HCVⅠ、Ⅲ、Ⅳ相应序列间的同源性为62％～69％,表明为HCVⅡ型序列。结果提示献血员抗—HCVIgG筛选不能完全排除HCV感染者,漏检不是由于HCV基因序列变异．而是检测方法本身缺陷所致。     

丙型肝炎病毒C区基因的克隆与分析

通过RT－PCR从1份来自甘肃武威地区献血员HCV阳性血清顺克隆到573bp的HCV核心基因全片段,用T－A克隆载体法将该片段接入克隆载体pUC19中并测序,结果表明武威地区分离株与Ⅰ型株HCV－Ⅰ和Ⅱ型株HCV－HeBei在该基因区段的核苷酸／氨基酸序列同源性分别为89.6%／95.4%、97.3%／97.4%,属于Ⅱ型株。     

RT套式PCR检测血浆HCV RNA及与抗HCV检测的比较

应用微量血清热变性法提取核酸,逆转录套式聚合酶链反应(RT-nest PCR)检测血浆HCV RNA,并与抗HCV ELISA检测结果比较,对HCV RNA阳性标本进行HGV RNA的筛查.结果在32例抗HCV阳性和20例抗HCV阴性血浆中,HCV RNA分别检出18例和2例,总符合率为70%,20例HCV RNA阳性者中有2例合并感染HBV,1例合并感染HGV.证明血浆样本中抗HCV与HCV RNA间存在很大的相关性.     

脂蛋白酯酶基因HindⅢ和PvuⅡ位点多态与中国人2型糖尿病的关联及meta分析

目的:探讨脂蛋白脂酶基因HindⅢ(rs320) 和PvuⅡ(rs285)位点多态与中国人2型糖尿病的相关性.方法:采用聚合酶链反应-限制性片段长度多态性方法,对919个湖北地区汉人(包括2型糖尿病患者481人,健康对照438人)脂蛋白脂酶基因内含子6PvuⅡ和内含子8HindⅢ位点多态进行基因分型和关联分析,同时对中国大陆人群的相关研究进行meta分析.结果:HindⅢ和PvuⅡ位点湖北汉族2型糖尿病人和正常人的基因型和等位基因频率均无显著差异.5个研究包括2型糖尿病患者1252例,健康对照1075例的PvuⅡ位点meta分析表明该位点与中国人2型糖尿病无显著相关性(P=0.39);9个研究包括2型糖尿病患者1515例.健康时照1022例的HindⅢ位点meta分析表明该位点与中国人2型糖尿病也无相关性(P=0.14).结论:脂蛋白脂酶基因HindⅢ和PvuⅡ位点多态与中国人2型糖尿病的发生无关.     

内蒙古呼和浩特地区丙型肝炎病毒基因型分布

为了解内蒙古呼和浩特地区丙型肝炎病毒(HCV)基因型的分布特征,为本地区丙型肝炎的治疗、预防提供基础数据资料。收集呼和浩特地区2014年1月至2015年1月就诊的门诊和住院丙型肝炎患者血清采样标本149份,均为HCV抗体检测阳性且HCVRNA定量检测阳性。QIAGEN柱式病毒RNA提取法提取HCV RNA,反转录成cDNA,巢式PCR法扩增HCVNS5B区,对扩增片段进行测序,在NCBI BLAST上进行序列比对得到相似度最大的参考序列并确定基因型,用Megalign,Clustal W进行序列分析并建立同源关系树,分析呼和浩特地区HCV感染基因型的分布特征,以及基因型与宿主性别、年龄的关系。在测序成功的94份样本中,共检出6个基因型,1b型占73.40%(69/94),2a型占19.14%(18/94),3a、3b、6a型各占2.12%(2/94),6u型占1.06%(1/94)。性别资料明确的93例样本,男女基因型分布无显著差异(P0.05)。年龄资料明确的90例HCV样本,1+2型的患病年龄高于3+6型的患病年龄有统计学意义(P0.05)。得出内蒙古呼和浩特地区HCV感染的基因型1b为主,其次为2a型,但也有3型、6型等基因型的传入。HCV的基因型4型和5型未检测到。     

苏南地区1b型丙型肝炎病毒包膜区变异进化研究

本研究旨在了解本地区丙型肝炎病毒(Hepatitis C virus,HCV)基因型构成的前提下,分析1b型丙型肝炎病毒包膜2(second envelope glycoprotein E2)区的变异和种系进化,并研究其准种变异与临床肝病活动度的关系。对宜兴市人民医院收集的抗HCV抗体阳性患者166名,RT-PCR方法检测HCVRNA,HCVRNA阳性患者采用型特异性引物分型法确定病毒基因型;选择其中未经干扰素治疗的43例1b型慢性丙型肝炎患者的血清标本,扩增E2区,从中选取肝硬化患者4例,慢性非肝硬化患者6例的E2区PCR产物纯化测序,序列采用CLUSTALW与GENBANK上多株不同型别的HCV序列进行比对分析,结果采用Phylip软件构建遗传进化树;并观察E2区高变区1(HVR-1)的氨基酸(aminoacid,aa)残基序列的变异特征;采用单链构象多态性垂直电泳检测43例患者个体内HCVE2区准种的变异情况,比较不同肝病活动度患者准种变异情况。结果表明本地区HCV以1b型为主(84.3%),对E2区基因序列和氨基酸序列变异的分析显示其变异具有一定的规律性,种系进化树提示本地区HCV病毒序列与上海、湖南、日本等地的HCV株有较近的亲缘性。43例患者中ALT高于正常的丙型肝炎患者准种复杂程度明显高于ALT正常者(P<0.05)。故本地区HCV基因变异符合中国东南部的特点,基因变异与临床肝病活动度具有相关性。     

HCV Antibody Response and Genotype Distribution in Different Areas and Races of China

Hepatitis C virus (HCV) heterogeneity accounts for the failure of effective vaccine development and the lack of successful anti-viral therapy in some patients. Little is known about the immune response to HCV peptides and the region or race specific genotypes in China. The objective of this study was to characterize HCV antibody immune response to HCV peptides and HCV genotypes in different regions and races of China. A total of 363 serum samples were collected from HCV carriers in 6 regions in China. The immune response to HCV peptides was evaluated by ELISA. HCV genotypes were examined using nested RT-PCR. We found that the anti-HCV antibody neutralization rates were significantly different among the serum samples from different areas or from different races in the same area. For samples from Tibet and Sinkiang, the rates of neutralization by HCV peptides were only 3.2% and 30.8%, respectively. The genotypes of samples from Tibet and Sinkiang were apparently heterogeneic and included type I, II, III and multiple types (I/II/III, I/II, I/III, II/III). One specific sample with multiple-genotype (I/II/III) HCV infection was found to consist of type I, II, III, II/III and an unclassified genotype. These studies indicate that the anti-HCV antibody immune response to HCV peptides varied across regions and among races. The distribution of HCV genotypes among Tibetans in Tibet and Uighurs in Sinkiang was different from that in the inner areas of China. In addition, a “master” genotype, type II, was found to exist in HCV infection with multiple HCV genotypes.     

人原发性肝细胞肝癌中丙型肝炎病毒C_（33c）抗原及HBxAg的分布及意义

作者应用抗ＨＣＶＮＳ３区Ｃ３３ｃ抗原２Ｂ６株单克隆抗体和抗ＨＢｘＡｇ多克隆抗体,采用ＡＢＣ法对１０２例人原发性肝细胞肝癌（ＰＨＣ）组织进行了ＨＣＶ及ＨＢＶ抗原定位研究。ＨＣＶＣ３。抗原及ＨＢｘＡｇ在ＰＨＣ中的阳性检出率分别是８１．４％及７４．５％,Ｃ３３ｃ抗原或ＨＢｘＡｇ阳性占所检病例９４．１％,相同病例二者同时阳性为６１．８％。１０２例ＰＨＣ中５０例有癌旁肝组织,其Ｃ３３ｃ抗原和ＨＢｘＡｇ的阳性检出率分别是６２％和９２％。ＨＣＶＣ３３ｃ抗原定位于肝癌细胞的胞浆内,胞核未见阳性信号。Ｃ３３ｃ抗原阳性细胞在ＰＨＣ中呈散在、局灶分布为主,在癌旁肝组织呈弥漫分布为主。本文结果提示ＨＣＶ感染在ＰＨＣ的发生中可能起重要作用。     

The internal ribosome entry site (IRES) of hepatitis C virus visualized by electron microscopy

Translation of hepatitis C virus (HCV) RNA is initiated via the internal ribosome entry site (IRES), located within the 5' untranslated region. Although the secondary structure of this element has been predicted, little information on the tertiary structure is available. Here we report the first structural characterization of the HCV IRES using electron microscopy. In vitro transcribed RNA appeared as particles with characteristic morphology and gold labeling using a specific oligonucleotide confirmed them to be HCV IRES. Dimerization of the IRES by hybridization with tandem repeat oligonucleotides allowed the identification of domain III and an assignment of domains II and IV to distinct regions within the molecule. Using immunogold labeling, the pyrimidine tract binding protein (PTB) was shown to bind to domain III. Structure-function relationships based on the flexible hinge between domains II and III are suggested. Finally, the architecture of the HCV IRES was seen to be markedly different from that of a picornavirus, foot-and-mouth disease virus (FMDV).     

我国非肠道传播非甲非乙型肝炎(丙型)病毒NS3蛋白的部份cDNA克隆及序列分析

用15ml非肠道传播非甲非乙型肝炎病人的混合血清提取病毒RNA,经逆转录和聚合酶链反应(PCR)扩增,获得583个核苷酸的非肠道传播非甲非乙型肝炎病毒(HCV)NS3蛋白的cDNA片段.该片段与美国报道的同片段HCV cDNA原型比较,核酸序列同源性为80.9％,氨基酸序列同源性为93.2％。与日本报道的同片段J1 HCV cDNA相比较,核酸序列和氨基酸序列的同源性分别为92.6％和95.2％。用α-~(32)P同位素标记该片段,与HCV病人血清出现杂交反应。     

Aberrant expression of a fetal glycoprotein 68 in hepatocellular carcinoma: a comparative study on the expression of alpha-fetoprotein and carcinoembryonic antigen

A rat IgG2a monoclonal antibody against a stage-specific fetal glycoprotein with a molecular mass of 68 kDa (FGP68) was produced and applied to paraffin sections. This monoclonal antibody was used to compare the expression of FGP68 with that of both alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in 75 hepatocellular carcinomas (HCCs). Seventy-five primary HCCs from patients aged 36 to 77 years were examined. Formalin-fixed, paraffin-embedded tissue sections were used for immunohistochemical analyses. Histologically, 6 cases of HCC were classified as type I according to the Edmondson and Steiner criteria, 57 cases as type II, and 12 cases as type III. The cancer tissues showed positive reactions with the antibody against FGP68. Approximately one-third of the HCCs (26/75) contained tumor cells that expressed FGP68 -(21/57 for Edmondson and Steiner type II; 4/12 for type III; and 1/6 for type I) - and positive immunoreactivity was observed in the cytoplasm of the cancer cells. Twenty-five of the 75 HCCs had tumor cells that expressed AFP and there was a significant correlation between FGP68 expression and AFP expression. Twenty-three of the 75 HCCs had tumor cells that expressed CEA and there was no significant correlation between FGP68 expression and CEA expression. No positive reactions for FGP68, AFP and CEA were observed in samples of non-neoplastic liver tissues. Based on the possibility that stage-specific FGP68 plays an important role in liver embryogenesis, FGP68-expressing tumor cells might ontogenetically revert to more primitive cells.     

Domains on the hepatitis C virus internal ribosome entry site for 40s subunit binding

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA is known to interact with the 40S ribosomal subunit alone, in the absence of any additional initiation factors or Met-tRNAi. Previous work from this laboratory on the 80S and 48S ribosomal initiation complexes involving the HCV IRES showed that stem-loop III, the pseudoknot domain, and some coding sequence were protected from pancreatic RNase digestion. Stem-loop II is never protected by these complexes. Furthermore, there is no prior evidence reported showing extensive direct binding of stem-loop II to ribosomes or subunits. Using direct analysis of RNase-protected HCV IRES domains bound to 40S ribosomal subunits, we have determined that stem-loops II and III and the pseudoknot of the HCV IRES are involved in this initial binding step. The start AUG codon is only minimally protected. The HCV-40S subunit binary complex thus involves recognition and binding of stem-loop II, revealing its role in the first step of a multistep initiation process that may also involve rearrangement of the bound IRES RNA as it progresses.     

64排螺旋CT对粗隆间骨折Evans分型的影响研究

目的:探讨64排螺旋CT对粗隆间骨折Evans分型的影响,为临床使用提供参考依据。方法:2015年3月至2017年3月,三甲医院高年资创伤骨科主任医师2名,医师1、医师2分别按照术前X线、术前64排螺旋CT平扫和三位重建结果对128例新鲜闭合单侧粗隆间骨折患者进行Evans分型,分别记为X线分型、CT分型。本院术者依据围术期X线、CT及术中所见骨折情况进行Evans分型(逆粗隆间骨折定义为Ⅴ型)作为最终分型。记录分型结果,计算并对比准确率、误诊率。结果:(1)剔除5例,90.09%(123/128)的患者完成研究。(2)分型结果:X线分型中,3例(最终分型Ⅲ型2例,Ⅳ型1例)无法定型;Ⅰ型正确1例,改为Ⅱ型1例;Ⅱ型正确18例,改为Ⅰ型2例,改为Ⅲ型3例,Ⅳ型2例;Ⅲ型正确45例,改为Ⅱ型7例,改为Ⅳ型1例;Ⅳ型正确19例,改为Ⅱ型3例,改为Ⅲ型15例。CT分型中,Ⅰ型正确3例,Ⅱ型正确29例,Ⅲ型正确64例,改为Ⅳ型1例,Ⅳ型正确22例,Ⅴ型正确3例。(3)CT分型的总准确率、总误诊率优于X线分型(99.19%vs67.48%、0.81%vs30.08%,P0.05)。(4)Ⅰ型、Ⅱ型、Ⅲ型、Ⅳ型骨折进行CT分型,准确率高于X线分型(P0.05),误诊率低于X线分型(P0.05);Ⅴ型骨折,两种分型准确率、误诊率相等。结论:64排螺旋CT平扫及三维重建是粗隆间骨折Evans分型较为可靠的辅助检查,可考虑推广运用。     

蛋白激酶底物p36在肝硬变、肝细胞肝癌中的表达及与HBV、HCV感染的关系

本文应用鼠抗蛋白激酶底物ｐ３６单克隆抗体,采用免疫组织化学法对ｐ３６在５４例肝硬变,７９例肝细胞肝癌中的表达分布进行了研究,同时结合ＨＢＶ、ＨＣＶ感染情况分析其相互关系,结果显示：ｐ３６在肝硬变及肝细胞肝癌中定位于肝细胞或癌细胞胞浆内,在胞浆内弥漫分布,阳性细胞呈灶状或弥漫分布,部分病例癌周肝细胞信号较癌组织为强,ｐ３６在肝硬变、肝细胞癌中的阳性率分别为８８．８％（４８／５４）及８２．３（６５／７９）,ＨＢｘＡｇ在两种组织的阳性率分别为７０．４％及７６％,ＨＣＶ核心抗原在两种组织的阳性率分别为８０％及７８．５％;三者同时阳性分别为５５．５％及５８．２％;ｐ３６、ＨＢｘＡｇ同时阳性分别为６８．５％及６４．５％;ｐ３６、核心抗原同时阳性分别为７４．１％及７０．８％,我们的结果提示,肝硬变、肝细胞肝癌组织中ｐ３６存在高表达,其高表达可能与ＨＢＶ、ＨＣＶ感染密切相关     

Differential localization of protein kinase C isozymes in retinal neurons

We report the immunohistochemical localization of protein kinase C isozymes (types I, II, and III) in the rabbit retina using the monospecific monoclonal antibodies MC-1a, MC-2a, and MC-3a. Using immunoblot analysis of partially purified protein kinase C preparations of rabbit retina, types II and III isozymes alone were detected. The activity of type III was the stronger. By light microscopic immunohistochemical analysis, retinal neurons were negative for type I and positive for type II and type III isozymes. Type II was more diffusely distributed through the retinal layers, but was distinctive in ganglion cells, bipolar cells, and outer segments. The immunoreactivity was stronger for type III isozyme, and it was observed in mop (rod) bipolar cells and amacrine cells. By using immunoelectron microscopy, the cytoplasm of the cell body, the axon, and dendrites of the mop bipolar cells were strongly immunoreactive for type III. The so-called rod bipolar cells were for the first time seen to form synapses with rod photoreceptor cells. These differential localizations of respective isozymes in retinal neurons suggest that each isozyme has a different site of function in each neuron.