Changes of centrosome and related protein in malignant transformation of BEAS-2B cell induced by coal tar pitch smoke extracts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To analyze the centrosome abnormalities in the malignant transformation of human bronchial epithelial cells (BEAS-2B) induced by coal tar pitch smoke extracts and to investigate the role and action mechanism of centrosome in the lung cancer induced by coal tar pitch.</p><p><b>METHODS</b>Medium-temperature coal tar pitch smoke extracts were used to treat immortalized human bronchial epithelial cells (BEAS-2B) and establish a malignant transformation model. The treated BEAS-2B cells were used as exposure group, and solvent control group and normal control group were also set for passage culture. The changes of centrosome in BEAS-2B cells seeded on coverslips were evaluated by indirect immunofluorescence assay. The mRNA expression of p53, p21, and cyclin E in BEAS-2B cells was measured by real-time quantitative RT-PCR, and their protein levels in BEAS-2B cells seeded on coverslips were measured by semiquantitative immunohistochemical analysis.</p><p><b>RESULTS</b>The overall rate of centrosome abnormalities in BEAS-2B cells at passage 20 was 6.56±1.01% in the exposure group, significantly higher than those in the normal control group (3.40±0.86%) and solvent control group (3.14±0.59%) (P < 0.05). In addition, the exposure group had a significantly higher overall rate of centrosome abnormalities in BEAS-2B cells at passage 30 compared with the normal control group and solvent control group (22.39±9.5% vs 4.34±1.04%, P < 0.05; 22.39±9.5% vs 4.33±1.20%, P < 0.05). Compared with the normal control group and solvent control group, the exposure group had significantly decreased mRNA and protein expression of p53 and significantly increased mRNA and protein expression of cyclin E in BEAS-2B cells at passages 20 and 30 (P < 0.05).</p><p><b>CONCLUSION</b>Centrosome abnormalities occur before the malignant transformation in BEAS-2B cells treated with coal tar pitch smoke extracts, and they may be mediated by the p53/p21/cyclin E signaling pathway.</p>

Cost-utility analysis on universal childhood hepatitis A vaccination in regions with different anti-HAV prevalence rates of China / 中华流行病学杂志

Objective To explore the inputs and outputs of areas with different anti-HAY prevalence rates on universal childhood vaccination,and to provide a scientific basis for the formulation of the immunization strategy.Methods Since hepatitis A vaccination was scheduled at 12 and 18 months of age for all the healthy children,a single cohort including 1 000 000 individuals was formed in 2009,using the Chinese inactivated vaccine.Decision analysis was used to build Markov-decision tree model.The universal childhood hepatitis A vaccination was compared with nonvaccination group to evaluate the number of symptomatic infection,hospitalization,death,qualityadjusted life years (QALYs) lost,and the incremental cost-utility from the health system and the societal perspectives.Outcomes of the vaccination for the next 70 years were also predicted.The process of analysis was run separately in five regions defined by the anti-HAV prevalence rates (around 50％,50％-69％,70％-79％,80％-89％ and ＞90％ ).Sensitivity analysis was performed to test the stability or reliability of the results,and to identify sensitive variables.Results The study projected that,in the lowest,lower,and intermediate infection regions,the cost and output indicators of universal childhood hepatitis A vaccination were all lower than non-vaccinated group.Universal vaccination could gain QALYs and save both costs from the health systen or the society.In the regions with higher infection rate,the output indicators of universal childhood hepatitis A vaccination were lower than in those non-vaccinated groups,except for the number of death due to hepatitis A,which had a 20 cases of increase.The model also predicted that in the highest infected region,universal vaccination would increase 4 560 814 and 5 840 430 RMB Yuan in the total costs from both the health system and the societies,respectively,when compared to the non-vaccination groups.Universal vaccination would also decrease the numbers of symptomatic infection,hospitalization,and QALYs lost,but would increase 51 deaths due to hepatitis A,and 1507,1929 more RMB Yuan for each QALY gained from the health system and societal respectively,in the regions with highest infection rate.Sensitivity analyses discovered that the infection rate among those susceptible population and the proportion of those who initially under protection but subsequently lost their immunity every year,were the two main sensitive variables in the model.Conclusion Our research discovered that the universal vaccination strategy should be based on the protective period of the vaccine and the anti-HAV prevalence in different endemic areas.

Construction of lentiviral mediated CyPA siRNA and its functions in non-small cell lung cancer / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer.</p><p><b>METHODS</b>First, four target sequences were selected according to CyPA mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA, and it was confirmed by PCR and sequencing. Next, it was cotransfected by Lipofectamine 2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles. At the same time, the packed virus infected non-small cell lung cancer cell (A549), the level of CyPA protein at 5 d after infection was detected by Western Blot to screen the target of CyPA. A549 were infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice. Cell cycle and apoptosis were measured by FCM.</p><p><b>RESULTS</b>It was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully. The titer of concentrated virus were 1 x 10(7) TU/ml. Flow cytometric analysis demonstrated G2-M phase (11.40% +/- 0.68%) was decreased relatively in A549/LvshCyPA compared with control groups (14.52% +/- 1.19%) (P<0.05). The apoptosis rate of A549/Lv-shCyPA (5.01% +/- 0.5%) was higher than control groups (0.35% +/- 0.17%) (P<0.05). Visible tumors were only detectable at 6th day after inoculated by A549/Lv-shCyPA. The xenograft tumors of A549/Lv-shCyPA remarkably delayed tumor growth and remained at a similarly small average size at 38th days after inoculation compared with the control group (P < 0.05).</p><p><b>CONCLUSION</b>Lentiviral-vector-mediated siRNA technique effectively inhibits the expression of CyPA, induces the NSCLC cell apoptosis, inhibits the tumor growth. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for non-small cell lung cancer.</p>

High CyPA expression in non-small cell lung cancer and its relationship with clinical pathological characteristics / 第二军医大学学报

Objective: To investigate the expression and distribution of CyPA in non-small cell lung cancer (NSCLC) and to evaluate its clinical significance in the development and progression of NSCLC. Methods: The expression of CyPA was examined in 45 NSCLC tissues, 22 paired tumor-adjacent tissues and 33 normal pulmonary tissues using tissue microarray immunohistochemistry method. The relationship between CyPA expression in NSCLC and clinic pathological characteristics of NSCLC was analyzed. Results: Expression of CyPA in NSCLC tissues and paired tumor-adjacent tissues was significantly higher than that in the normal pulmonary tissues (P<0.05). No significant correlation was found between the expression of CyPA and differentiation and histological types of NSCLC. Conclusion: CyPA is overexpressed in NSCLC tissues, suggesting that CyPA might serve as a biomarker for the development and progression of NSCLC, whose specific role remains to be further studied.

Determination of SARS-CoV by simplified nested fluorescent RT-PCR / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a simple rapid and sensitive nested RT-PCR method for detection of SARS coronavirus RNA by designing the specific primers for SARS and optimizing the parameters for PCR.</p><p><b>METHODS</b>Primers and fluorescent probes were designed according to the sequences of SARS coronavirus genes available from GenBank. The optimization of the parameters for PCR was performed in PE 7700 thermal cycle. The 36 serum samples and 40 mouthwash of SARS patients and 80 samples of healthy people were tested.</p><p><b>RESULTS</b>The positive rate of patient serum and mouthwash was 33.6%, (12/36) and 67.5%, (27/40), respectively, while the positive rate of healthy people was zero (0/160).</p><p><b>CONCLUSION</b>The simple nested RT-PCR method was a rapid, efficient and sensitive one for SARS early diagnosis.</p>

Coxsackie virus B types were discriminated by RT-PCR / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a method for detection of coxsackie B virus type 1-6 by RT-PCR.</p><p><b>METHODS</b>A pair of primers were designed to amplify all types of coxsackie B virus 1-6 efficiently. The PCR product was hybridized in micro-wells in which 6 type specific oligonucleotide probes had been coated respectively, colorimetric detection was performed to discriminate the types of coxsackie B virus.</p><p><b>RESULTS</b>This method was shown to be concordant with the IgM ELISA, 71.7% of anti-coxsackie B positive cases could be detected by RT-PCR.</p><p><b>CONCLUSION</b>The RT-PCR method can type coxsackie B virus efficiently and provides a tool for clinical diagnosis and epidemiological investigation.</p>