逆转录-聚合酶链反应在乙型脑炎诊断中的建立

目的　运用逆转录 -半套式 -聚合酶链反应技术 (RT -semi-nested -PCR) ,建立一种新的早期快速检测乙型脑炎病毒的方法。方法　收集临床诊断的 37例乙脑和 15例非乙脑病人标本 (包括血清、脑脊液 )分别为 6 3份和 30份 ,同时用RT -semi-nested -PCR、IgM捕获法ELISA(MacELISA)进行检测。 结果　血清和脑脊液中可成功检测出JEVRNA ,经1 5 %凝胶电泳 ,出现特异性条带 ,符合预计值 4 0 0bp。结论　RT -semi-nested -PCR对检测JEV感染 ,从实验设计到实验条件 ,都是合理的 ,有较高的敏感性和特异性。可用于乙脑的早期诊断。     

HS-GGT和AFP-mRNA联合分析在肝癌诊断中的临床价值

目的　探讨肝癌特异性γ 谷氨酰转移酶 (HS GGT)和AFP mRNA联合分析在肝癌诊断中的临床价值。方法　从肝癌患者外周血有核细胞中提取总RNA ,以逆转录聚合酶链反应 (RT PCR)扩增分析AFP mMNA ,同时定量分析患者血HS GGT水平 ,比较它们在肝癌诊断中的临床价值。结果　所设计RT PCR扩增AFP基因片段大小为 15 9bp ,肝癌患者外周血AFP mRNA阳性率为60 3 % ,显著高于急性肝炎、慢性肝炎、肝硬化、肝外肿瘤及正常对照组 (P <0 0 1)。另外 ,AFP mRNA阳性率 ,在AFP浓度低于 5 0ng/ml肝癌组中为 5 7 1% ,在伴肝外转移的肝癌组中全数阳性。AFP mRNA阳性与肿瘤大小间无明显相关性 ,与HS GGT联合分析诊断肝癌的总阳性率达 93 6% 结论　HS GGT定量和外周血AFP mRNA分析有助于肝癌诊断、远处转移或术后复发的监测     

Exercise in obesity, metabolic syndrome, and diabetes

The risk of developing both metabolic syndrome and type 2 diabetes mellitus (T2DM) is inversely associated with regular exercise training (ET). Excess weight is also strongly associated with increased risk of both metabolic syndrome and T2DM. There is strong evidence that even a moderate amount of weight loss achieved through changes in diet and ET can greatly reduce the risk of developing T2DM.For the purpose of general health, exercise programs should have both aerobic and resistance training components. The 2008 federal physical activity (PA) guidelines recommend obtaining at least 150 minutes per week of moderate-intensity PA, 75 minutes per week of vigorous-intensity PA, or a combination of the 2. In addition, all individuals should strive for at least 2 days per week of resistance training activity. For the purpose of weight loss, the combination of ET and reduced energy intake has been found to be more effective than either alone.     

Detection of the nucleophosmin gene mutations in acute myelogenous leukemia through RT-PCR and polyacrylamide gel electrophoresis

Objectives: Mutations in the C‐terminal region of the nucleophosmin (NPM1) gene occur in approximately 60% of acute myeloid leukemia (AML) cases with normal karyotype and represent the most common genetic lesion presently known in this disease. Because of their frequency and favorable impact on prognostic outcome, screening for this aberration is currently recommended in routine diagnostic characterization of AML. Several techniques enabling to detect NPM1 mutation have been reported, but all require sophisticated equipment, which represent an obstacle particularly in countries with limited resources. Methods: We designed an RT‐PCR strategy to amplify NPM1 exon 12 followed by electrophoresis and fragment visualization on polyacrylamide gels to discriminate a 4–5 bp size difference resulting from mutations in this gene. A hemi‐nested method was designed to increase sensitivity for the study of minimal residual disease (MRD). Results: The assay enabled specific detection of NPM1 mutations in 12/36 patients. A 10^?2 sensitivity level was obtained using one amplification round, while the hemi‐nested PCR approach yielded a 10^?5 sensitivity level, therefore proving useful to assess MRD in patients carrying the mutation. The results were independently validated in 24 AML cases by sequencing analysis. Conclusions: This simple and low‐cost assay may integrate diagnostic work‐up of AML and could be used for assessment of response to therapy in patients with NPM1 mutations.     

CD34, RAB20, PU.1 and GFI1 mRNA expression in myelodysplastic syndrome

Myelodysplastic syndrome (MDS) with hypocellular bone marrow (BM) is often difficult to distinguish from aplastic anemia (AA). Furthermore, the diagnosis of MDS with low blast counts and normal karyotype may be problematic. These issues highlight the need for a reliable marker for the diagnosis of MDS. This study was conducted to determine if changes of mRNA expression in any of the four selected genes would be useful markers for differentiation of hypoplastic MDS from AA, and MDS from benign disease, as well as to investigate whether mRNA expressions differ between MDS risk subgroups. Thirty-five patients diagnosed with MDS, 27 patients with AA and 17 patients with benign diseases were included. The CD34, RAB20, PU.1 and GFI1 mRNA levels were measured by real-time RT-PCR. The CD34 mRNA expressions in hypoplastic MDS were higher than those found in AA. PU.1 and GFI1 mRNA expressions were significantly lower in MDS with low blast counts and normal karyotype than those of benign disease. High-risk MDS showed higher CD34 expressions than those of low-risk MDS. This study suggests that measurement of CD34 and GFI1 mRNA expressions could be useful as a diagnostic and prognostic marker for MDS.     

原位RT-PCR法检测柯萨奇B病毒RNA的实验研究

目的建立定位检测细胞或组织中柯萨奇Ｂ病毒ＲＮＡ的原位ＲＴ－ＰＣＲ方法（直接法）。方法对柯萨奇Ｂ病毒（１～６型）感染ＨｅＬａ细胞,经核酸酶（ＤＮａｓｅ和ＲＮａｓｅ）处理后的病毒感染ＨｅＬａ细胞以及非感染的ＨｅＬａ细胞进行原位ＲＴ－ＰＣＲ检测。结果经扩增后,病毒感染的ＨｅＬａ细胞呈蓝黑阳性信号;并且这种阳性信号经ＲＮａｓｅＡ作用而消失,却不受ＤＮａｓｅ作用的影响;而非感染的ＨｅＬａ细胞则不着色。结论原位ＲＴ－ＰＣＲ法可特异地检测柯萨奇Ｂ病毒感染ＨｅＬａ细胞中的病毒ＲＮＡ,是一种特异和敏感的定位检测方法,可应用于研究该病毒的持续性感染及其与克山病等相关疾病的关系     

竞争性逆转录聚合酶链反应定量检测对HCV RNA

为了解感染丙型肝炎病素（ＨＣＶ）后的（ＨＣＶＲＮＡ）含量与丙型肝炎病情轻重，预后，治疗反应的关系，采用竞争性逆转录聚合酶链反应（ＣＲＴ－ＰＣＲ）的方法，对９例感染了ＨＣＶ患者的血清进行了ＨＣＶＲＮＡ的定量检测。结果：６．６×１０３拷贝１毫升占８８．９％，６．６×１０４拷贝／毫升占１１．１％。结果提示：急慢性丙型肝炎及无症状ＨＣＶ感染者体内均含有较高的病毒滴度，且滴度低预后好，抗病毒疗效好。     

Increased sensitivity and specificity of a sandwich ELISA for measurement of IgE antibodies

We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for measurement of IgE antibodies to the bee venom allergens phospholipase A2 (PLA2) and hyaluronidase (HYAL). The assay is 10-20 times more sensitive than conventional indirect ELISA or radioallergosorbent test (RAST). Furthermore, by using affinity purified rabbit antibodies to these allergens, the specificity of the test was increased compared to RAST. The use of antibodies to link the antigen to the solid phase removes the dependence on the individual protein's ability to bind to the microtitre plate. The increased sensitivity of the sandwich assay seems to be due to better presentation and retention of antigen on the solid phase.     

Identification of mouse genes with highly specific expression patterns in differentiated intestinal epithelium

BACKGROUND & AIMS: Few genes that regulate intestinal epithelium development, homeostasis, or function are known. We reasoned that potential candidate regulators of these processes could be identified based on their activation during intestinal epithelium development and their subsequent specific and restricted expression. METHODS: Genes were identified by differential display and microarray analyses, further selected according to sequence and UniGene expression profiles, and analyzed by RNA in situ hybridization of mouse fetal and adult intestines and in intestinal polyp tissue. RESULTS: Five genes with unknown physiological function predominantly or exclusively expressed in the intestinal epithelium were identified. Their expression is activated at distinct times during intestinal development and maturation and is maintained in highly specific, spatially distinct patterns in the adult intestinal epithelium. Two of the genes were up-regulated in intestinal tumors, 1 was down-regulated, and 2 were apparently unaltered. CONCLUSIONS: Based on sequence and expression, the identified genes represent good candidates for regulators of intestinal epithelium integrity or function. Their expression patterns suggest a morphologically not obvious molecular regionalization of the intestinal epithelium along the crypt villus axis. This approach should be an efficient means to identify novel genes required for intestinal epithelium homeostasis and function.