乙型肝炎病毒多聚酶末端蛋白重链可变区抗体体外抑制病毒的复制

目的用蛋白片段互补法(PCA)选择HBV多聚酶末端蛋白(TP)重链可变区(VH)抗体,研究特异性VH抗体体外抑制HBV的复制与分泌。方法以HBV多聚酶TP区为抗原,用PCA法进行特异性VH抗体筛选。特异性VH抗体基因定向亚克隆人真核表达载体pZeoSV2( ),构建pZeoSV2( )-VH重组质粒,用脂质体介导的转染技术,将其导入HepG 2.2.15细胞,观察特异性抗体的生物学活性。结果用PCA法从抗体库中选择出3个特异性抗体:VH1、VH2和VH3,其中与抗原亲和力最高的是VH1。转染pZeoSV2( )-VH1的HepG 2.2.15细胞比未转染pZeoSV2 ( )-VH1或只转染空载体pZeoSV2( )的HepG 2.2.15细胞病毒颗粒分泌明显减少,上清液内为2.978 7±0.276 1比5.150 4±0.276 1和5.295 1±0.241 0(P<0.05);细胞内为5.283 9±0.162 9比8.300 4±0.323 2和8.532 1±0.138 3(P<0.05)。结论用PCA法可从抗体库中选择出HBV多聚酶TP区特异性VH抗体,该抗体在体外可抑制HBV的复制与分泌。     

中晚期食管癌支架置入术后再狭窄的危险因素分析

目的探讨中晚期食管癌支架置入术后再狭窄发生的危险因素。方法对2009年8月1日至2011年12月31日长治医学院附属和平医院65例中晚期食管癌患者行支架置入术后临床资料进行了回顾性分析。对中晚期食管癌支架置入术后再狭窄的相关因素进行单因素和多因素统计分析。结果单因素显示与临床分期(Ⅲ期和Ⅳ期,P=0.024)、组织学分级(Ⅰ级、Ⅱ级和Ⅲ级,P=0.001)、是否合并食管瘘(有食管瘘和没有食管瘘,P=0.000)、支架置入术后治疗(是否接受放化疗,P=0.004)相关。多因素Logistic回归分析显示,临床分期增加(Ⅲ期和Ⅳ期,P=0.044)、组织学分级提高(Ⅰ级、Ⅱ级和Ⅲ级,P=0.002)、合并食管瘘(有食管瘘和没有食管瘘,P=0.001)是影响中晚期食管癌患者支架置入术后再狭窄的独立风险因素,其OR值(优势比)分别为5.448、14.533、66.221。而支架置入术后接受同步放化疗(P=0.002)呈负相关,OR值为0.020成为独立的保护因素。结论临床Ⅳ期、组织学分级提高、合并食管瘘成为支架置入术后再狭窄的的高危风险因素,对于支架置入术后接受同步放化疗是减少支架再狭窄的重要因素。对于具有高危再狭窄的患者,应采取更为积极的治疗措施以预防再狭窄发生。     

临床教师在传染病学教学中应注意的几个问题

针对近几年医学的迅速发展、医学知识获得的快捷便利、医学教育模式的改变和医疗市场的变化等新的临床医学教育背景，初步探讨了新形势下如何作好临床医学教育工作的对应措施。     

蛋白转导结构域介导乙型肝炎病毒聚合酶末端蛋白重链可变区抗体体外抑制病毒的复制

Objective To study a functional variable fragment of heavy chain(VH)antibody against the terminal protein(TP)region of hepatitis B virus(HBV)polymerase introduced by human immunodeficiency virus Tat protein transduction domain(TAT)and the inhibitive activity of TAT-VH on the replication of HBV in vitro.Methods The gene encoding TAT-VH was cloned into prokaryotic expression vector pET28a(+).Recombinant plasmid was transduced into E coli BL21(DE3)LysS,then the protein was expressed and purified.The purified TAT-VH fusion protein was added into HepG2.2.15 cell culture.The transduction efficiency was evaluated by indirect fluorescence assay(IFA).The cytotoxicity of TAT-VH was detected by Methabenzthiazuron(MTT)assay.HBV DNA level in HepG2.2.15 cell culture was measured using quantitative polymerase chain reaction(PCR).The data were analyzed by one-factor analysis of variance and t test.Results TAT-VH fusion protein was successfully expressed and purified.It was confirmed by IFA and MTT assay that TAT-VH was introduced into HepG2.2.15 cells and the cell growth was not affected.The level of HBV DNA in supernatant of HeDG2.2.15 cell culture with 5 000 nmol/L TAT-VH was(1.211±0.132)lg copy/mL,which was significantly lower than control group[(5.325±0.041)lg copy/mL,t=72.91,P＜0.05].Meanwhile,the level of intracellular HBV DNA was(3.521±0.411)lg copy/mL,which was significantly lower than control group[(8.532±0.132)lg copy/mL.t=28.41,P＜0.05].Conclusion The HBV replication is inhibited by anti-TP TAT-VH antibodies in vitro,which provides valuable experimemal basis for developing therapy of HBV infection with intracellular antibody.     

蛋白转导结构域介导乙型肝炎病毒聚合酶末端蛋白重链可变区抗体体外抑制病毒的复制

目的 蛋白转导结构域(TAT)介导HBV聚合酶末端蛋白(TP)重链可变区(VH)抗体,研究特异性TAT-VH抗体体外对HBV复制的影响.方法 将TAT-VH基因克隆入原核表达载体pET28a(+),在大肠埃希菌BL21(DE3)LysS内诱导融合蛋白表达并进行纯化.纯化的TAT-VH加入培养的HepG2.2.15细胞,间接免疫荧光法检测其导入HepG2.2.15细胞的效率,四甲基偶氮唑盐(MTT)法检测其对细胞生长代谢的影响,将TAT-VH加入培养的HepG2.2.15细胞,定量PCR法检测HBV DNA水平.数据行单因素方差分析和t检验.结果 成功制备了TAT-VH融合蛋白,间接免疫荧光及MTT证实TAT-VH可以跨膜导入HepG2.2.15细胞,且对细胞生长无影响;加入5 000 nmol/L TAT-VH的HepG2.2.15细胞培养上清液内HBV DNA为(1.211±0.132)lg拷贝/mL,对照组为(5.325±0.041)lg拷贝/mL(t=72.91,P＜0.05);细胞内分别为(3.521±0.411)和(8.532±0.132)Ig拷贝/mL(t=28.41,P＜0.05).结论 HBV聚合酶TP区特异性TAT-VH抗体在体外可抑制HBV复制,为应用细胞内抗体治疗HBV感染提供了良好的实验基础.     

选择HBV DNA多聚酶TP区VH抗体的一种新方法:蛋白片段互补法

目的：用PCA（Protein fragmem complementation assay，PCA，蛋白片段互补方法）选择HBV（Hepatitis Bvirus）多聚酶TP（Terminal protein，TP，末端蛋白）区VH（Variable fragments of heavy chain，VH，重链可变区）抗体。方法：用HepG2．2．15细胞分泌的HBV的多聚酶TP区作为抗原，用PCA方法进行VH抗体选择。抗原与抗体基因分别与二氢叶酸还原酶（DHFR）的两部分相连。特异性抗原、抗体相互识别，分别与他们相连的酶基因就会接触而恢复完整的DHFR酶活性，细菌得以存活。结果：对HBV DNA多聚酶TP区进行抗体选择，用PCA方法从抗体库中选择出3个对应抗体。3个TP区抗原特异性抗体有不同的氨基酸序列。结论：对HBV DNA多聚酶TP区进行抗体选择，用PCA方法可以从抗体库中较为简便的选择出对应抗体。PCA可以作为一个从抗体库中选择特异性抗体的强有力方法。     

信迪利单抗联合白蛋白紫杉醇对晚期胃癌血清肿瘤标志物水平及免疫功能的影响

目的 探究信迪利单抗联合白蛋白紫杉醇对晚期胃癌血清肿瘤标志物水平及免疫功能的影响。方法 前瞻性选取2021年6月至2023年2月在长治医学院附属和平医院接受治疗的89例晚期胃癌患者为研究对象,按照随机数字表法分为对照组(n=44)和观察组(n=45)。对照组患者采取白蛋白紫杉醇治疗,观察组患者采取信迪利单抗联合白蛋白紫杉醇进行治疗。比较两组治疗效果、肿瘤标志物[糖类抗原(CA)125、CA19-9、癌胚抗原、甲胎蛋白及组织多肽特异性抗原(TPS)]水平、免疫功能指标(CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺)水平,记录患者治疗期间发生的不良反应。结果 治疗后,观察组患者的客观缓解率及疾病控制率分别为55.56%、77.78%,均高于对照组(34.09%、 47.73%),差异均有统计学意义(P<0.05)。治疗后,两组患者的各肿瘤标志物水平均较治疗前降低,且观察组CA125、CA19-9、癌胚抗原、甲胎蛋白、TPS水平分别为(16.77±2.77)U/mL、(15...     

用PCA方法选择HBV DNA多聚酶TP区VH抗体

目的:用PCA方法选择HBVDNA多聚酶TP区VH抗体.方法:从YeastDisplayscFvAntibodyLibrary中提取包含scFv抗体库的质粒pPNL6,扩增VH抗体片段.以HepG2.2.15细胞分泌的HBVDNA为模板,PCR扩增HBVDNA末端蛋白(TP).以TP区作为抗原,用PCA方法进行VH抗体选择.结果:PCR扩增的TP区共540bp.对HBVDNA多聚酶TP区进行抗体选择,用PCA方法从抗体库中选择出3个对应抗体,根据DNA序列和WernerMüller数据库可标明3个抗体.结论:对HBVDNA多聚酶TP区进行抗体选择,用PCA方法可以从抗体库中较为简便的选择出对应抗体.     

蛋白转导结构域介导乙型肝炎病毒聚合酶末端蛋白重链可变区抗体体外抑制病毒的复制

Objective To study a functional variable fragment of heavy chain(VH)antibody against the terminal protein(TP)region of hepatitis B virus(HBV)polymerase introduced by human immunodeficiency virus Tat protein transduction domain(TAT)and the inhibitive activity of TAT-VH on the replication of HBV in vitro.Methods The gene encoding TAT-VH was cloned into prokaryotic expression vector pET28a(+).Recombinant plasmid was transduced into E coli BL21(DE3)LysS,then the protein was expressed and purified.The purified TAT-VH fusion protein was added into HepG2.2.15 cell culture.The transduction efficiency was evaluated by indirect fluorescence assay(IFA).The cytotoxicity of TAT-VH was detected by Methabenzthiazuron(MTT)assay.HBV DNA level in HepG2.2.15 cell culture was measured using quantitative polymerase chain reaction(PCR).The data were analyzed by one-factor analysis of variance and t test.Results TAT-VH fusion protein was successfully expressed and purified.It was confirmed by IFA and MTT assay that TAT-VH was introduced into HepG2.2.15 cells and the cell growth was not affected.The level of HBV DNA in supernatant of HeDG2.2.15 cell culture with 5 000 nmol/L TAT-VH was(1.211±0.132)lg copy/mL,which was significantly lower than control group[(5.325±0.041)lg copy/mL,t=72.91,P＜0.05].Meanwhile,the level of intracellular HBV DNA was(3.521±0.411)lg copy/mL,which was significantly lower than control group[(8.532±0.132)lg copy/mL.t=28.41,P＜0.05].Conclusion The HBV replication is inhibited by anti-TP TAT-VH antibodies in vitro,which provides valuable experimemal basis for developing therapy of HBV infection with intracellular antibody.