HBV前C/C基因与绿色荧光蛋白基因融合表达载体的构建及在HEK293细胞中的表达

目的 :构建含HBV前C C基因与绿色荧光蛋白基因的融合表达载体 ,并在真核细胞中表达。方法 :用PCR法扩增含 1.3倍HBVayw型全基因组真核表达载体pHBV1.3的前C C基因片断 ,应用含绿色荧光蛋白基因的真核表达质粒构建融合表达载体 ,采用脂质体介导方法将其转染到HEK2 93细胞 ,并用荧光显微镜及ELISA法检测融合蛋白的表达。结果 :经PCR及酶切鉴定 ,证实成功构建了含HBV前C C基因的真核表达重组体pEGFP -HB VC ,显微镜下观察及ELISA法证实在HEK2 93细胞中表达了相应融合蛋白。结论 :构建的重组表达载体能在真核细胞中表达目的蛋白与GFP的双功能融合蛋白 ,适合于针对HBV前C C区的相关研究。     

实验性蛛网膜下腔出血后脑血管痉挛兔海马组织中Bcl-2和Bax mRNA的表达

目的：探讨蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)造成脑损伤的机制．方法：用反转录聚合酶链式反应(RT—PCB)技术检测兔SAH后CVS时海马组织Bcl—2和BaxmRNA的表达变化．结果：Bcl—2mBNA的表达水平在SAH组1d时即开始下降，3d时降至最低，持续至7d．SAH组海马组织中BaxmRNA的表达呈上升趋势，3d时达最高，7d时仍显著高于正常组．在假手术组海马组织内的Bcl—2和BaxmRNA的表达水平保持相对恒定．结论：Bcl—2和Bax可能参与了SAH后CVS所造成的海马神经元损伤过程。     

外阴尖锐湿疣人乳头瘤病毒感染的检测与分型

目的 了解患外阴尖锐湿疣人乳头瘤病毒(HPV)6型和11型的感染情况及PCR方法对尖锐湿疣诊断与分型的临床意义。方法 采用PCR方法测定160例尖锐湿疣患病题组织或分泌物中HPV6／11型及HPVl6／18型的感染率。结果 HPV6／11型感染率为95％(152／160)，HPVl6／18型感染率为5％(8／160)，HPV6／11型 HPVl6／18型混合感染率为1．3％(2／160)。结论 外阴尖锐湿疣患以HPV6型和11型感染为主。PCR方法是一种比较适合临床HPV检测与分型的极为敏感和特异的诊断方法。     

人巨细胞病毒感染对肾移植受者血清白细胞介素－6水平的影响

应用ＰＣＲ检测ＨＣＭＶ－ＤＮＡ，ＥＬＩＳＡ检测ＨＣＭＶ－ＩｇＭ、ＩｇＧ，诊断肾移植受者ＨＣＭＶ感染，６５例受者中ＨＣＭＶ感染者３９例，非感染者２６例。应用ＭＴＴ法检测受者血清ＩＬ－６生物活性，阐明了ＨＣＭＶ感染对肾移植受者血清ＩＬ－６水平的影响。结果表明：感染与非感染组间血清ＩＬ－６水平差异无显著性（Ｐ＞０．０５）；６例原发性感染者血清ＩＬ－６水平随感染时间延长呈增高及降低双相改变，表明慢性迁延性感染者血清ＩＬ－６水平降低。临床工作中监测ＨＣＭＶ感染的肾移植受者血清ＩＬ－６水平变化具有重要意义。     

Reduced peripheral blood mitochondrial DNA content is not a risk factor for Type 2 diabetes.

AIMS: Mitochondrial depletion in pancreatic beta cells is known to reduce glucose stimulated insulin secretion. We aimed to determine whether the offspring of patients with early onset Type 2 diabetes had reduced peripheral blood mitochondrial content relative to control subjects and whether this could lead to a predisposition to type 2 diabetes in later life. METHODS: We measured the levels of mitochondria relative to a single copy genomic target by real time polymerase chain reaction in a series of peripheral blood samples taken from the offspring of Caucasian patients with Type 2 diabetes and matched controls. Measures of insulin sensitivity and beta cell function were also taken. RESULTS: In contrast with previous studies, mitochondrial DNA content was not decreased in the offspring of patients with Type 2 diabetes relative to matched controls in our cohort. Conversely, we noted a small proliferation in mitochondrial numbers in our case subjects. In agreement with these findings, no correlations with either insulin sensitivity or beta cell function were noted. CONCLUSIONS: Our results indicate that reduced mitochondrial DNA content in peripheral blood is not a risk factor for the development of Type 2 diabetes in the offspring of patients with early onset Type 2 diabetes.     

PCR-SSCP法检测结核分枝杆菌耐药性

目的：探讨耐多药结核分枝杆菌耐药基因突变与耐药性的关系以及聚合酶链反应-单链构象多态性分析(poly merase chain reaction-single strand cinfomlation polymorphism，PCR-SSCP)方法的临床应用价值。方法：用PCR-SSCP方法检测58株结核分枝杆菌临床分离株katG，rpoB，rpsL基因突变并与常规药敏试验检测结果进行对比。结果：经常规药敏试验检测，58株结核分枝杆菌临床分离株中共有41株耐药，其中，耐异烟肼(INH)为35株，高耐药株27株；耐利福平(RFP)为31株，高耐药株24株；耐链霉素(SM)有31株，高耐药株26株。同时耐3种药物的有21株(51．2％)，耐2种药物的14株(34．1％)，单耐药株6株(14．6％)．PCR-SSCP方法对58株临床分离株katG，rpoB，rpsL基因突变的检测率为40％(23／58)，45％(26／58)，38％(22／58)，其中检出3个基因同时突变的有13株(32％)，2种基因突变的12株(29％)，1种基因突变的有10株(23．8％)．常规药敏试验与PCR-SSCP法检出结核分枝杆菌同时耐3种药物的符合率为61．9％(13／21)，检出耐2种药物的符合率为85．70k，(12／14)，检出耐1种药物的符合率为50％(3／6)．高耐药株中突变率为80．5％(62／77)，低耐药株中突变率为60％(12／20)．结论：PCR-SS-CP方法对耐2种以上药物的结核杆菌检出率较高，且耐药基因突变率随着耐药浓度增高而增高。将PCR-SSCP法与药敏试验联合应用可互相弥补，已成为临床指导用药的好方法。     

抗-HCV与HCV-RNA在丙型肝炎诊断中的观察

目的 探讨早期检测抗-HCV和HCV-RNA对诊断丙型肝炎的价值。方法 通过用酶联免疫吸附法(ELISA)和逆转录PCR法，对198例丙型肝炎患者检测其抗-HCV和HCV-RNA。结果 抗-HCV总阳性率为84％，抗-HCV、HCV-RNA同时阳性为58％，抗-HCV单项阳性为26％，抗-HCV阴性而HCV-RNA阳性为16％。结论 HCV-RNA对早期HCV感染的诊断以及抗-HCV阴性的慢性丙肝的诊断有重要意义。对怀疑HCV感染者应同时检测抗-HCV和HCV-RNA，才能对不同时期的HCV感婆作出准确及时的诊断。     

多聚酶链反应检测细菌DNA对大鼠空、回肠吻合口瘘的早期诊断价值

目的:探讨PCR检测大鼠外周血及腹水中细菌DNA对空肠-空肠、回肠-回肠吻合口瘘的早期诊断价值。方法:健康Wistar雌性大鼠50只，随机分成5组，每组10只：A组为假手术组;B组为空肠-空肠吻合组;C组为空肠吻合口瘘组;D组为回肠-回肠吻合组;E组为回肠吻合口瘘组。采集手术前后外周血及术后腹水，抽提DNA, 比较lacZ基因和16SrRNA基因的PCR阳性率，并观察各组的病理学情况。结果:（1）C，E组术后外周血lacZ基因PCR阳性率与B，D组无显著性差异（P>0.05）;C，E组术后外周血16SrRNA基因PCR阳性率显著高于B，D组（P<0.05）。（2）C，E组腹水lacZ基因和16SrRNA基因PCR阳性率均显著高于B，D组（P<0.05）。（3）C，E组腹水lacZ基因阳性率显著高于外周血（P<0.05）;C，E组腹水16SrRNA基因阳性率与外周血无显著性差异（P>0.05）。结论:（1）PCR检测术后外周血16SrRNA基因对空、回肠吻合口瘘的早期诊断有一定意义;（2）检测术后腹水lacZ基因和16SrRNA基因对空肠-空肠、回肠-回肠吻合口瘘的早期诊断也有一定意义。     

Tau isoform expression in frontotemporal dementia without tau deposition

In many cases of sporadic frontotemporal dementia (FTD) and in FTD caused by tau mutations (FTDP-17) there is disruption of the normal splicing of tau leading to the aberrant expression of tau isoforms and neurodegeneration. This suggests a central role for tau in the pathogenesis of FTD. However, more than half the cases of sporadic FTD show no tau deposition. We question whether altered expression is also involved in the pathogenesis of tau-negative FTD. Real-time polymerase chain reaction was used to investigate tau isoform expression in tau-negative FTD and age-matched controls. There were no differences in total tau mRNA or 4R versus 3R isoform expression. Our study suggests that perturbed tau mRNA expression is unlikely to be involved in the pathogenesis of tau-negative FTD.     

Absence of structural alterations of the multidrug resistance genes in transitional cell carcinoma

Summary Tumor DNA from 27 patients with treated or untreated transitional cell carcinomas of the urinary tract was screened for genomic alterations of the multidrug resistance genes in order to determine whether structural changes of these genes are important in primary urothelial tumors. None of the tumors showed evidence of amplification or rearrangements of either mdr1 or mdr2. The lack of amplification or rearrangements observed in these tumors suggests that structural alterations of the mdr1 and mdr2 genes are not important mediators of drug resistance in TCC.Supported in part by grant CA-34775 from the National Institutes of Health and by a grant from the Heckscher Foundation for ChildrenDr. Klein is a fellow of the American Cancer Society