[Detection of UGT1A1*28 Polymorphism Using Fragment Analysis].

BACKGROUND: Uridine-diphosphoglucuronosyl transferase 1A1 (UGT1A1), UGT1A1*28 polymorphism can reduce UGT1A1 enzymatic activity, which may lead to severe toxicities in patients who receive irinotecan. This study tries to build a fragment analysis method to detect UGT1A1*28 polymorphism. METHODS: A total of 286 blood specimens from the lung cancer patients who were hospitalized in Guangdong General Hospital between April 2014 to May 2015 were detected UGT1A1*28 polymorphism by fragment analysis method. RESULTS: Comparing with Sanger sequencing, precision and accuracy of the fragment analysis method were 100%. Of the 286 patients, 236 (82.5% harbored TA6/6 genotype, 48 (16.8%) TA 6/7 genotype and 2 (0.7%) TA7/7 genotype. CONCLUSIONS: Our data suggest hat the fragment analysis method is robust for detecting UGT1A1*28 polymorphism in clinical practice. It's simple, time-saving, and easy-to-carry.

Mosaic adeno-associated virus-mediated gene transfer of calcitonin gene-related peptide for prevention of inflammatory mediator expression, macrophage infiltration and neointimal hyperplasia in an experimental rabbit vein graft model / 中国胸心血管外科临床杂志

@#Objective    To investigate the effect and mechanism of calcitonin gene-related peptide (CGRP) on the prevention and treatment of transplant vein graft disease. Methods    The 25 New Zealand white rabbits were divided into three groups: an experimental group [n=8, the rabbit jugular veins transfected with adeno-associated virus vector tipe 2/1 containing CGRP gene (AAV2/1-CGRP)], a carrier group [n=9, transfected with mosaic adeno-associated virus vector tipe 2/1 containing LacZ gene (AAV2/1-LacZ)] and a control group (n=8, saline) and then the cervical veins were implantated into the ipsilateral carotid artery by reverse end-side anastomosis. At 4 weeks after surgery, the pathology of the specimens, CD68 immunohistochemistry, in situ β-galactosidase staining were obtained. The expression of CGRP gene was detected by reverse transcription-polymerase chain reaction (RT-PCR). Monocyte chemoattractant protein-1(MCP- 1), tumour necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-9 (MMP-9) were detected by real-time polymerase chain reaction (real-time PCR). Results    The CGRP and LacZ gene expression was positive at postoperative 4 weeks. The intima/media ratio was significantly inhibited in the experimental group. Macrophage infiltration and expression of inflammatory mediators including MCP-1, TNF-α, iNOS and MMP-9 were also significantly inhibited in the experimental group. Conclusion    Transfection of AAV2/1-CGRP inhibits inflammatory mediator expression, macrophage infiltration and neointimal hyperplasia in experimental vein graft disease.

[The B lymphocytes D8/17 antigen expression and characteristics of patients with rheumatic heart disease in Guangdong].

OBJECTIVE: To investigate the D8/17 antigen expression of patients with rheumatic heart disease (RHD) in Guangdong province and study the antigen's characteristics. METHODS: The level of D8/17 antigen expression on B lymphocytes was determined with flow cytometry assay in 96 RHD patients and 83 unaffected controls. The percentage of B-cells expressing the D8/17 antigen having more than 10% was considered to be positive. D8/17 antigen was extracted by immunoprecipitation, and the antigen characteristics was analyzed by tandem mass spectrometry. RESULTS: The mean percentage of B-cells expressing the D8/17 antigen was (85.36 ± 15.15)% in the RHD patients and (82.89 ± 4.55)% in the controls, with no significant difference between the two groups (P = 0.436). Moreover, the positive rate of the D8/17 expression was 100% in either the RHD patients or the controls. The molecular weight of D8/17 antigen was found to be 40 000 - 67 000, and the purified protein was most likely to match moesin or ß-actin. CONCLUSIONS: B-cell antigen D8/17 is not associated with RHD in Guangdong province of China. Moesin or ß-actin is the most likely protein to match D8/17 antigen.

Inhibitory effects of calcitonin gene-related peptides on experimental vein graft disease.

BACKGROUND: Vein graft disease is a chronic inflammatory disease and limits the long-term clinical outcome of coronary revascularization. Because calcitonin gene-related peptide (CGRP) inhibits macrophage infiltration and inflammatory mediators, we hypothesized that transfected CGRP gene would inhibit macrophage infiltration and expression of inflammatory mediators in vein graft disease. METHODS: Autologous rabbit jugular vein grafts were incubated ex vivo in a solution of mosaic adeno-associated virus vectors containing CGRP gene (AAV2/1.CGRP) or Escherichia coli B-galactosidase gene (LacZ) or a saline solution and then interposed in the carotid artery. Expression of CGRP gene was identified by reverse transcription-polymerase chain reaction, and E. coli LacZ gene expression was identified by X-gal staining. Intima to media ratios were evaluated at postoperative 4 weeks. Macrophages were identified with CD68 antibody by immunocytochemistry. Inflammatory mediators were measured with real-time polymerase chain reaction. RESULTS: The CGRP and LacZ gene expression were positive at postoperative 4 weeks. The intima to media ratio was significantly inhibited in the AAV2/1.CGRP group. Macrophage infiltration and expression of inflammatory mediators including monocyte chemoattractant protein-1, tumor necrosis factor-alpha, inducible nitric oxide synthase, and matrix metalloproteinase-9 were also significantly inhibited in the AAV2/1.CGRP group. CONCLUSIONS: Transfection of AAV2/1.CGRP inhibited inflammatory mediator expression, macrophage infiltration, and neointimal hyperplasia in experimental vein graft disease.

KDR expression is associated with the stage and cigarette smoking of the patients with lung cancer.

PURPOSE: Kinase insert domain-containing receptor (KDR) is one of the molecular targets used in cancer therapy. We studied the KDR expression characteristics and the relationship with the clinical parameters of the patients with lung cancer, to give the basic evidence and clue for tailoring therapy. METHODS: Reverse transcriptase and real-time PCR were used to evaluate the KDR mRNA expression levels in 222 tissue samples (106 tumor tissues, 106 matched normal tissues obtained from the same patients with lung cancer, and 10 normal lung specimens from individuals without lung cancer). The KDR mRNA expression level and clinical parameters were analyzed by paired-sample t test, ANOVA and linear regression, respectively. The Kaplan-Meier method and the log-rank test were used for survival analysis. Expression of KDR protein was also examined immunohistochemically in 15 tumor samples and 15 matched normal lung specimens. RESULTS: The KDR mRNA expression levels were significantly higher in normal tissues (mean 4.50 +/- 0.51) than that in the carcinoma tissues (mean 4.12 +/- 0.50, P < 0.0005). KDR expression in tumor tissues is associated with the histological status, tumor stage, cigarette smoking, and N stage of the patients with lung cancer (P < 0.05) analyzed by using ANOVA methods. Multivariate analysis showed that tumor stages and cigarette smoking status were the two most important independent predictors for the KDR expression levels in tumor tissues (R = 0.415, R (2) = 0.172, F = 10.694, P < 0.0005). Tumors with KDR mRNA expression levels above the mean had a shorter survival (466 +/- 313 days) than did patients with KDR expression levels below the mean (671 +/- 264 days), whereas Kaplan-Meier analysis and log-rank test showed no significant difference in the overall survival between the patients (P = 0.2055). All the 15 normal lung tissues detected showed scale 2 KDR immunostaining. The intensity of immunostaining for KDR in tumor specimens varied from negative (scale 0) to strongest (scale 3) staining. CONCLUSIONS: Locally advanced and non-cigarette smoking patients with lung cancer may be the two valuable surrogate markers for KDR mRNA higher levels. Non-squamous lung cancer, N 2 stage may be the secondary markers for that. The KDR expression level in normal lung tissue is stable, but varied in tumor tissues. Targeting KDR therapy in lung cancer might considerate these clinical and KDR expression information. Further confirmation study must be needed.