脂蛋白相关磷脂酶A2酶活性水平及其基因多态性与冠心病的相关性研究

目的 建立脂蛋白相关磷脂酶A2(Lp-PLA2)基因A379V点突变等位基因特异-荧光定量PCR(TaqMan-ARMS)测定方法,探讨Lp-PLA2的酶活性水平及SNPs是否是冠心病的危险因素.方法 采用等位基因特异性PCR(ARMS)技术结合荧光定量PCR技术(TaqMan探针),通过对不同基因型引物3’端正配与错配扩增效率差别的比较,建立TaqMan-ARMS检测Lp-PLA2基因(PLA2G7)A379V点突变的方法,并对方法进行评价.应用TaqMan-ARMS PCR力法,检测395例冠心病患者(CAD组)和396例非冠心病对照者(NCAD组)PLA2G7的A379V基因频率,并对Lp-PLA2的酶活性水平、年龄、CHO、GLU 、TG、HDL、LDL、Hs-CRP、Lp(a)进行测定和调查,运用独立样本t检验、卡方检验、方差分析、logistic回归对数据进行分析.结果 CAD组的Lp-PLA2酶活性水平明显高于NCAD组(31.51 nmol·ml-1·min-1＞21.31 nmol·ml-1·min-1),差异有统计学意义(F=16.40,P＜0.001)；校正传统冠心病危险因子[CHO,TG,Hs-CRP,Lp(a),GLU]后,Lp-PLA2酶活性的最高四分化数与最低四分位数相比,CAD的比值比(OR)为7.50(95％ CI:2.34～24.05);在校正年龄、性别后,A379V点突变基因型VV的OR值为2.95(95％ CI:1.22～7.15,P＜0.05).结论 成功建立了基于TaqMan-ARMS PCR技术的定性检测A379V基因突变的方法；Lp-PLA2的酶活性水平在冠心病患者中显著升高,是冠心病的危险因子；A379V点突变基因型VV是冠心病的危险因子.     

High-Efficiency Cloning of DNA Sequences Complementary to Mouse Neuroblastoma Polyadenylated RNA

A cDNA library was efficiently synthesized from mouse neuroblastoma poIy(A)⁺RNA. Several modifications of the oligo(dC)(dG) tailing procedure were used. After first strand synthesis, a dATP tail was added to the 3′-end of the cDNA. The second strand was primed for synthesis with oligo(dT). Blunt ends were produced on the cDNA by treatment with S1 nuclease. Size-enriched fractions of high molecular weight DNAs were obtained by passing the cDNA over a Sepharose CL-4B column. The optimal tailing time for each cDNA fraction was individually tested. Tailing reactions used terminal deoxynucleotidyl transferase and annealing reactions used a (G)-tailed PstI cut pBR322. E. coli K12 RR1 cells were transformed and 2.5–5 × 10⁶ transformants per μg cDNA insert were obtained for each size fraction. The transformants had an average insert size of 1200 base pairs and were 98% ampicillin sensitive. Our modifications in the method for cDNA library synthesis had 3 advantages. (1) Homopolymer-primed cDNA treated with S1 nuclease allowed the blund ends to be tailed sychronously. This allowed a higher transformation efficiency without loss of 5′-sequences. (2)Time tailing determined the most efficient tail length and optimized the transformation efficiency in each size fraction. (3) A Sephadex G-50 mini-column was used to desalt and dry nitrogen was used to concentrate the ds cDNA instead of the usual ethanol precipitation. This resulted in almost 100% recovery of synthesized products at each step of this procedure.     

Pretreatment with lipopolysaccharide ameliorates Pseudomonas exotoxin A-induced hepatotoxicity in rats

Abstract

Context: Liver injury can be induced by various hepatotoxicants, including Pseudomonas aeruginosa exotoxin A (PEA). Our previous study indicated that PEA-induced rat hepatotoxicity was T cells and Kupffer cells dependent. Several reports have demonstrated that non-toxic doses of bacterial lipopolysaccharide (LPS) can protect liver against the chemicals-induced toxicity such as acetaminophen and concanavalin-A.

Objective: This study aimed to investigate the protecting mechanisms of LPS on PEA-induced hepatotoxicity.

Results: Rats pretreated with LPS (40?μg/kg, 12?h before PEA admission) significantly decreased animal mortality, serum enzyme (ALT, AST and T-bil) activities, histopathological changes and hepatocytes apoptosis following challenge with PEA. The concentrations of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-2 (IL-2) were reduced, but IL-6 and IL-10 were increased in the serum. In addition, prior treatment of these LPS-pretreated rats with gadolinium chloride (GdCl₃), a selective Kupffer cell depletion agent, markedly enhanced liver injury after PEA administration. In contrast, the pretreatment of LPS to T-cell deficient athymic nude rats still display significant attenuation of PEA-induced liver injury. This observation further confirmed our hypothesis that LPS ameliorate PEA-hepatotoxicity was through Kupffer cells but not T cells. Moreover, LPS-induced hepatoprotection ability was neutralized by co-treatment with anti-TNF-α antibodies, but not with anti-IFN-γ antibodies. Finally, replacement of LPS with RS-LPS (Rhodobacter sphaeroides LPS), a Toll like receptor-4 (TLR-4) antagonist, resulted in severe hepatotoxicity.

Conclusion: These results suggested that Kupffer cells, TNF-α and TLR-4 play central mediator roles during the hepatoprotection against PEA-induced hepatotoxicity conferred by LPS.     

CIP2A is a poor prognostic factor and can be a diagnostic marker in papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma. CIP2A has recently been described as a prognostic marker in many cancers. In this study, we assessed the value of this novel prognostic marker in PTC. A total of 178 surgical specimens of both benign and malignant thyroid tumors were collected. Immunohistochemical staining for CIP2A, HBME‐1, galectin‐3, and CK19 was performed. Western blotting for CIP2A was also performed. CIP2A was expressed in 85.3% of malignant tumors and 12.1% of benign tumors. ROC analysis showed that the AUC for CIP2A was higher than those for other tumor markers. Western blotting showed that CIP2A expression was higher in PTC than in other tumors. Poor progression‐free survival was observed in the high‐CIP2A expression group. High CIP2A expression is a poor prognostic factor and can be a diagnostic marker in PTC. The presence of any two of the three indicated makers (CIP2A, galectin‐3, and HBME‐1) is strongly correlated with the diagnosis of PTC.     

TL1A blocking ameliorates intestinal fibrosis in the T cell transfer model of chronic colitis in mice

Tumor necrosis factor like cytokine 1A (TL1A) is a member of the TNF superfamily. Accumulating evidence demonstrated the importance of TL1A in the pathogenesis of inflammatory bowel disease (IBD) and suggested a potential role of TL1A blocking in IBD therapy. Here we aimed to explore whether the anti-TL1A antibody could ameliorate intestinal inflammation and fibrosis in IBD. A T cell transfer model of chronic colitis was induced by intraperitoneal injection of CD4⁺CD45RB^high naive T cells isolated from either C57BL/6 wild type (WT) mice or LCK-CD2-Tl1a-GFP transgenic (L-Tg) mice into recombinase activating gene-1-deficient (RAG^?/?) mice. The colitis model mice were treated prophylactically or therapeutically with anti-Tl1a antibody or IgG isotype control. Haematoxylin and eosin staining (H&E staining), Masson's trichrome staining (MT staining) and sirius red staining were used to detect histopathological changes in colonic tissue; immunohistochemical staining was used to detect the expressions of collagen I, collagen III, TIMP1, vimentin, α-SMA and TGF-β1/Smad3. Results showed that anti-Tl1a antibody could reduce intestinal inflammation and fibrosis by inhibiting the activation of intestinal fibroblasts and reducing the collagen synthesis in the T cell transfer model of chronic colitis. The mechanism may be related to the inhibition of TGF-1/Smad3 signaling pathway.     

Development of a lyophilized soybean paste certified reference material for the analysis of ochratoxin A

A certified reference material (CRM) [²KRISS CRM # 108-10-018] for the analysis of ochratoxin A (OTA) in doenjang (fermented soybean paste and popular food in Korea) was produced to ensure the reliability of analytical results in testing laboratories. A home-made doenjang was chosen as a raw material after testing its OTA level. The raw material was freeze-dried, pulverized, sieved and homogenized. An isotope-dilution-liquid chromatography/tandem mass spectrometric method (ID-LC/MS/MS) which was previously developed and validated in this laboratory was used as a higher-order reference method for characterization, homogeneity studies, and short-term stability studies. The CRM had good between-bottle homogeneity with 0.56% relative standard deviation among 10 selected units. The stability of the CRM at −70 °C (the storage condition in our laboratory) and at −20 °C (the possible storage temperature at user sites) were tested for up to 8 months. No change in the OTA content was observed within the measurement uncertainty. The stability of the CRM at room temperature (for regular use and transportation) was also tested and confirmed. The certified value was (49.50 ± 1.17) μg/kg, where the expanded uncertainty was in the confidence level of 95%.