Synthetic genes: a tool for identifying human papillomavirus genotypes by hybridization and polymerase chain reaction-based assays

Obtaining positive polymerase chain reaction (PCR) controls for human papillomavirus (HPV) diagnostic tests has been difficult because of prevalence variation in different geographic regions of each high-risk viral type. Overlapping oligonucleotides were designed for HPV-18, HPV-31, HPV-45, and HPV-58 type-specific (TS) sequences. Synthetic HPV viral genes were constructed by 2-step assembly PCR for accurately diagnosing TS HPV infection.     

痰标本分离革兰阴性杆菌整合子分布及分型研究

目的研究临床痰标本分离革兰阴性杆菌中整合子的分布与分型。方法用Ⅰ类、Ⅱ类和Ⅲ类3种整合酶基因通用引物扩增398株痰标本分离革兰阴性杆菌的相应基因;用琼脂对倍稀释法检测临床菌株的MIC。结果Ⅰ类整合酶基因总阳性率为48.7%,Ⅱ类整合酶基因总阳性率为2.3%,未检出Ⅲ类整合酶基因阳性菌株,Ⅰ类和Ⅱ类整合子同时阳性率为1.5%。肠杆菌科细菌Ⅰ类整合酶基因阳性率为69%,Ⅱ类整合子阳性率为2.7%;非发酵菌中Ⅰ类整合子阳性率为34%,Ⅱ类整合子阳性率为1.8%。在肠杆菌科细菌中,Ⅰ类整合酶基因阳性菌株对多种抗菌药物的耐药率均明显高于阴性菌株。结论在痰标本分离的革兰阴性杆菌中,肠杆菌科细菌Ⅰ类整合子的携带率高于非发酵菌,Ⅱ类整合子则无明显区别。     

MAGE-1基因在胃癌中的表达及其意义

目的探讨MACE-1(黑色素瘤抗原-1)基因在胃癌组织中的表达及意义。方法采用逆转录-聚合酶链反应(RT-PCR)检测58例胃癌组织及30例正常组织中MAGE-1基因表达。结果58例胃癌组织标本中有34例(58.6%)表达MAGE-1基因,而30例正常组织均不表达MAGE-1基因,两组比较差弄有显著性(P<0.05)。结论MAGE-1基因在胃癌组织中有较高的表达,对胃癌病人的免疫治疗和预后有重要意义。     

巢式PCR检测庚型肝炎病毒感染

目的探讨逆转录-巢式PCR研究庚型肝炎病毒(HGV)感染在我国南方人群中的分布。方法从HGV 5-′NCR区段设计了内、外引物对,运用逆转录-巢式PCR对来自我国南方深圳、广州和桂林三个城市总共403例,且来自不同人群组的血清/血浆标本中HGV RNA感染率进行了研究。结果献血员人群中HGV RNA的平均感染率为5.8%(11/189),其中职业献血员较义务献血员高〔9.7%(10/102)及1.1%(1/87)〕;静脉药瘾者、丙型肝炎、乙型肝炎患者人群中HGV感染率依次为50.8%(33/65)、24.0%(9/37)、2.5%(1/40);在具有严重程度不同的非A-E肝脏疾病患者中HGV RNA阳性检出率为12.5%(9/22)。结论HGV与HCV合并感染极其常见,可能与其具有共同的传播途径有关。追踪临床资料,发现HGV的感染并未加重丙型肝炎患者的病情;多次静脉滴注药物、输血/输液、血液透析所致医院感染均是引起我国人群中HGV分布较高的重要危险传播因素;HGV的致病性较弱,可能不是引起非A-E肝脏疾病的主要致病因素。     

白细胞介素6基因启动子区两个突变位点多态性检测方法的建立

目的建立一种快速检测IL-6基因启动子区-597G／A、-572G／C多态性的双重实时荧光PCR方法。方法采用1对引物，2对荧光标记探针，结合荧光共振能量转移原理和熔点曲线分析技术，检测IL-6基因启动子区-572G／C，-597G／A2个位点多态性。结果用建立的双重实时荧光PCR方法对123名健康查体者进行检测，发现中国汉族人IL-6基因启动子区-572位点有3种基因型，分别为GG，GC，CC型；-597位点仅发现4名为GA型，其余均为GG型，尚未发现从型。结论双重实时荧光PCR法简便快速，与其他方法比较具有准确、经济的特点，适合临床基因快速分型。     

电针预处理对心肌缺血再灌注模型大鼠心肌组织FXR基因及心肌细胞线粒体功能的影响

目的探讨电针预处理改善心肌缺血再灌注损伤的可能作用机制。方法40只Wistar大鼠随机分为假手术组、缺血再灌注组、FXR抑制剂组及电针预处理组,每组10只。各组大鼠均予捆绑固定7天后,除假手组外其余各组均进行心肌缺血再灌注造模处理。FXR抑制剂组第8天尾静脉注射法尼酯X受体(FXR)抑制剂z-Guggulsterone 0.4μl/g后再造模,电针预处理组从第1天开始取"内关""足三里""关元"电针7天,第8天再造模。造模后测定大鼠血清白细胞介素8(IL-8)浓度及心肌细胞线粒体呼吸链酶活性及Na^+-K^+-ATP酶、Ca^2+-Mg^2+-ATP酶活性,检测心肌组织FXR基因表达水平。结果与假手术组相比,缺血再灌注组IL-8浓度升高,呼吸链酶Ⅰ、Ⅱ、Ⅲ、Ⅳ及Na^+-K^+-ATP酶、Ca^2+-Mg^2+-ATP酶活性均降低,FXR基因表达水平上调(P<0.01)。与缺血再灌注组相比,FXR抑制剂组与电针预处理组IL-8浓度均降低,呼吸链酶Ⅰ、Ⅱ、Ⅲ、Ⅳ及Na^+-K^+-ATP酶、Ca^2+-Mg^2+-ATP酶活性升高,FXR基因表达水平降低(P<0.01)。与FXR抑制剂组比较,电针预处理组IL-8浓度、FXR基因表达水平升高,Na^+-K^+-ATP酶、Ca^2+-Mg^2+-ATP酶活性及呼吸链酶Ⅲ、Ⅳ均降低(P<0.05)。结论电针预处理可能通过降低血清IL-8浓度,提高心肌细胞线粒体呼吸链复合酶、ATP酶活性,下调FXR基因表达,从而改善心肌缺血再灌注损伤。     

实时荧光定量端粒重复扩增法检测大肠癌患者单个核细胞端粒酶基因表达

目的 研究大肠癌患者外周血单个核细胞(PBMC)端粒酶基因表达与临床病理的关系.方法 用实时荧光定量端粒重复扩增法(RTQ-TRAP)检测71例大肠癌和20例良性大肠疾病患者,以及25名健康人PBMC端粒酶基因表达,同时检测大肠癌患者血浆癌胚抗原(CEA)含量.结果 71例大肠癌患者中,50例端粒酶基因表达阳性,阳性率70.4%;20例良性大肠疾病表达阳性1例,阳性率为5.0%,大肠癌组与良性大肠疾病组和健康对照组的PBMC端粒酶基因表达差异有统计学意义(χ2=24.521,P<0.001).大肠癌患者在不同性别(χ2=0.114,P=0.736)、年龄(χ2=0.113,P=0.735)、肿瘤部位(χ2=1.523,P=0.677)、Dukes分期(χ2=2.282,P=0.320)间的端粒酶基因表达差异无统计学意义.71例大肠癌患者PBMC端粒酶基因表达阳性率与CEA阳性率(63.4%)比较,差异无统计学意义(χ2=2.286,P=0.125).结论 RTQ-TRAP是一种快速、准确、定量检测端粒酶基因表达的方法,大肠癌患者PBMC端粒酶活性是一项有效的辅助诊断大肠癌的分子标志.     

聚合酶链反应结合探针杂交检测军团菌

利用来源于嗜肺军团菌巨噬细胞感染增强因子（ｍｉｐ）基因序列的引物，建立了快速检测军团菌的聚合酶链反应技术。扩增反应产物经与地高辛标记的特异寡核苷酸探针杂交证实。用本方法检测接种过军团菌的支气管肺泡灌洗液标本。可检出１００ＣＦＵ／ｍｌ细菌。研究表明它是军团病诊断的一种有效手段。     

轻链型多发性骨髓瘤患者免疫固定电泳检测差异的分析

目的 探讨轻链型多发性骨髓瘤患者实验室检查上的特点.方法 回顾性分析我院2003年5月至2007年9月间采用免疫固定电泳(IFE)检测的30例轻链型患者的实验室结果的特征.结果 轻链型多发性骨髓瘤患者中,κ型患者κ/λ显著升高(P＜0.05),λ型κ/λ显著降低(P＜0.05);两组κ/λ均与正常值差异有显著性(P＜0.05).结论 κ/λ比值可作为多发性骨髓瘤的诊断、鉴别诊断敏感和特异性指标.     

Characterization of angiotensin II-receptor subtypes in podocytes

Glomerular podocytes play a key role in maintaining the integrity of the glomerular filtration barrier. This function may be regulated by angiotensin II (Ang II) through activation of cell-surface receptors. Although studies suggest that podocytes express receptors for Ang II, the Ang II binding site has not been characterized with radioligand binding techniques. We therefore used iodine 125-labeled Ang II to monitor Ang II-receptor density during differentiation of a mouse podocyte cell line. Scatchard analyses of equilibrium binding data revealed a single class of high-affinity binding sites (dissociation constant approximately 3 nmol/L) in both differentiated and nondifferentiated cells. During differentiation, the density of Ang II-receptor sites increased roughly 15-fold in differentiated podocytes (maximal density of specific binding sites 881 fmol/mg protein) compared with that in nondifferentiated cells (52 fmol/mg protein; P<.005). Glomerular podocytes expressed messenger RNA for AT1A, AT1B, and AT2 receptor subtypes, and competitive binding studies found that differentiated podocytes expressed mostly AT1 receptors (approximately 75%) with lesser amounts of AT2 (approximately 25%). Up-regulation of Ang II-receptor number was associated with increased Ang II-receptor responsiveness, as evidenced by enhanced Ang II-stimulated inositol phosphate (IP) generation and incorporation of tritiated thymidine. Both [3H]thymidine incorporation and IP generation were mediated by AT1-receptor activation. These data suggest that glomerular podocytes express a high-affinity binding site for Ang II with pharmacologic characteristics of both AT1 and AT2 receptors. This receptor site is up-regulated during podocyte differentiation, and receptor activation induces both IP generation and DNA synthesis by AT1-dependent mechanisms. We speculate that activation of podocyte Ang II receptors contributes to glomerular damage in disease states.