Abstract:Objective:To construct lentiviral vector carrying activity-defective mTERT (deleting amino acid 702-712,named mTERTΔ) and to detect its expression and function. Methods:Mice mTERTΔ gene was amplified by our previous constructed plasmid pDC315-EGFP-mTERT carrying the whole gene encoding mTERT by deletion mutant PCR. Then,eukaryotic expression vector of GV287-EGFP/mTERTΔ was constructed. After DNA sequence analysis,mTERTΔ was cloned into lentiviral vector pGC-LV to construct recombinant vector pGC-LV/mTERTΔ-EGFP. pGC-LV/mTERTΔ-EGFP was transfected into 293T cells by Lipofectamine 2000 to package lentiviral particle LV-mTERTΔ-EGFP. The particles were transfected into neuronal stem cells and primary neurons. TRAP-PCR was performed to detect telomerase activity,and fluorescence microscopy was performed to observe fragment expression and cell proliferation. Results:TERTΔ gene fragment was successfully constructed,and the analysis of DNA sequence proved that the recombinant lentiviral vector pGC-LV/mTERTΔ-EGFP was successfully constructed. Package of lentiviral particle LV-mTERTΔ-EGFP were transfected into neuronal stem cells and primary neurons. The expressed mTERTΔ detected by telomerase activity test was activity defective,and inhibited neural stem cell proliferation and endogenous mTERT function. Conclusion:The lentiviral vector of LV-mTERTΔ-EGFP was constructed successfully and infected cells could express activity-defective mTERT.

Abstract:Objective:To observe whether the Nod-like receptor pyrin domain-containing protein 3 (NLRP3)-inflammasome participates in the pathologic process of herpes simplex virus-1 (HSV-1) induced viral myocarditis (VMC). Methods:Cultured neonatal rat ventricular cardiomyocytes (NRVM) of neonatal rats were infected with 0.01 and 0.1 PFU HSV-1 for 24 hours,respectively. Morphologic changes of NRVM were observed under light microscope. The gene expression of NLRP3-inflammasome and its downstream pathways were measured by quantitative real-time PCR (qRT-PCR). The expression and location of cysteinyl aspartate-specific proteases-1 (Caspase-1) were evaluated by immunofluorescent (IF) method. Moreover,creatine kinase-MB (CK-MB) content was detected by automatic biochemical analyzer and supernatant concentration of interleukin-18 (IL-18) was measured by enzyme-linked immunosorbent assay (ELISA). Results:Cytopathic effect (CPE) was observed in NRVM infected with HSV-1. The supernatant concentration of CK-MB,one of the myocardial injury biomarkers,was significantly increased (P < 0.05). Compared with the control group,the mRNA levels of NLRP3,Caspase-1,IL-1β and IL-18 were up-regulated over 5 times in HSV-1 infected NRVM. IF showed that the expression of Caspase-1 was significantly increased. The concentration of supernatant IL-18 was increased compared with that of the control group (P < 0.05). Conclusion:NLRP3-inflammasome and its downstream pathways were activated in cell model of HSV-1 infected VMC. NLRP3-inflammasome may participate in pathologic process of VMC,and became a potential target for VMC therapy.

Abstract:Objective:To study the biological characteristics of cytotoxic T lymphocyte (CTL) originated from dendritic cells (DC) loaded with the Epstein-Birr virus latent membrane proteins (LMPs),and to detect the cytotoxic effect of LMPs-CTL on the LMPs positive nasopharyngeal carcinoma cell SUNE. Methods:The mononuclear cells were isolated from human peripheral blood by lymphocyte separation medium. The adherent method was performed. Cytokines of IL-4 and GM-CSF were performed to induce mature DC. Loaded with LMPs polypeptide antigen,the mature DC presented antigen to autologous T lymphocyte for preparation of LMPs-CTL. The cytotoxic effect of LMPs-CTL on SUNE cells was detected by the CCK8 method. The secretion of interferon-γ (IFN-γ) of LMPs-CTL and cell proliferation was detected by ELISA and carboxy fluoroscein succinimidyl ester (CFSE),respectively. Results:The killing efficiency of LMPs-CTL for SUNE was (43.47 ± 1.93)% in 12h and (77.15 ± 3.18)% in 24 h,which were higher than those in the control group[(11.45 ± 3.06)% and (24.27 ± 13.2)% (P < 0.05)]. The ability of LMPs-CTL cell proliferation simulated with SUNE cells was stronger than that in the control group. The IFN-γ secretion level secreted by LMPs-CTL was (613.40 ± 121.77) pg/ml,which was significantly higher than that secreted by the control group (86.90 ± 3.70) pg/ml (P < 0.05). Conclusion:LMPs specific CTL can be successfully induced by mature DC and possess preferable specific killing activity on LMPs positive nasopharyngeal carcinoma cells.

Abstract:Objective:To explore the effect of miR-342-3p on chemotherapy sensitivity of breast cancer cells. Methods:The expression levels of miR-342-3p were detected in breast cancer cell lines MCF-7,SKBr3 and MDA-MB-231. By using lipofectamine,the hsa-miR-342-3p mimic was transfected into breast cancer cell lines,which were of the lowest expression of miR-342-3p cell lines (the mimic group). The mim-NC was performed as the negative control group. Furthermore,the miR-342-3p inhibitor was transfected into breast cancer cell lines of the highest expression of miR-342-3p,the inhi-NC was performed as the negative control group. Cells from four different groups (the mimic,mim-NC,inhibitor and inhi-NC groups) were treated with 2 -滋mol/L paclitaxel,2 -滋mol/L cisplatin and 4 -滋mol/L doxorubicine for 48 hours,respectively. CCK8 assay was used for detection of cell proliferation. Results:Compared with the expression level of miR-243-3p in SKBr3,the level of miR-243-3p in MCF-7 cells was significantly increased (126.000 fold change),but it was decreased (0.017 fold change) in MDA-MB-231 cell lines. The rates of cell proliferation in the mimic group after treatment with paclitaxel and cisplatin for 48 hours were significantly lower than those in the mim-NC group,respectively (P < 0.05,respectively). However,the cell proliferation rates in the mimic group and the mim-NC group after treatment with doxorubicine for 48 hours had no significant difference (P > 0.05). The cell proliferation rates in the inhibitor group were significantly higher than those in the inhi-NC groups after treatment with paclitaxel,cisplatin and doxorubicine for 48 hours,respectively (P < 0.05,respectively). Conclusion:miR-342-3p may play a key role in the regulation of chemotherapy sensitivity to paclitaxel and cisplatin in breast cancer cell lines MDA-MB-231 and MCF-7. Up-regulation of miR-342-3p expression could not increase the chemotherapy sensitivity to doxorubicine,but down-regulation of miR-342-3p expression may weaken the chemotherapy sensitivity to doxorubicine.

Abstract:Objective:To investigate the antitumor activity of deguelin in small cell lung cancer and the possible underlying mechanisms. Methods:NCI-H446 cells were treated with deguelin at 0,5,10,20 and 50 μmol/L for 24 hours. The effect of deguelin on cell activity of NCI-H446 cells was evaluated by CCK8. EdU assay and DAPI staining were performed to assess cellular proliferation and apoptosis,respectively. According to flow cytometry analysis,20 μmol/L deguelin were given NCI-H446 cells for 0,0.5,2.0 and 4.0 hours,respectively. Western blot was applied to detect the levels of proteins involved in apoptosis. Results:Deguelin significantly inhibited proliferation and induced apoptosis of NCI-H446 in a dose-dependent manner. After treating with deguelin for different time lengths,pro-apoptotic protein Bax level was increased and anti-apoptotic protein Bcl-2 level was decreased. Conclusion:Deguelin suppresses small cell lung cancer in vitro via promoting its apoptosis probably by up-regulating pro-apoptotic protein Bax and down-regulating anti-apoptotic protein Bcl-2.

Abstract:Objective:To establish an reliable and stable method of establishing an optimal animal model of lung injury in neonate rat intraperitoneal injection of lipopolysaccharides (LPS). Methods:Pregnant Sprague-Dawley rats on gestation day 18 were randomly divided into the control group and the LPS experimental group. Rats were injected intraperitoneally with 0.5,1.0,1.5,2.0 and 2.5 mg/kg LPS, respectively, in the LPS groups and with equal amount of saline in the control group,and then the mortality and abortion ratio of pregnant rats rate were measured. According to the statistical results,we next selected low mortality doses of LPS (0.9,0.8,0.7,0.6,and 0.5 mg/kg) and intraperitoneally injected to pregnant rats. Lung tissues were collected from neonatal rat at postnatal day 1. The indexes of pathological score and wet/dry weight ratio (W/D) of lung lobes were observed. The mRNA expression of TNF-α and IL-1β,and the TNF- α protein expression in lung tissues were examined. Furthermore,to study the LPS of 0.7 mg/kg dose,the mRNA expression of TNF-α,IL-1β in placental and fetal lung tissues were measured on day 19. The same indexes of neonatal rats lung were also examined on day 1,4 and 7 after natural birth,respectively. Results: ①The mortality and abortion ratios of pregnant rats were decreased gradually with the decreasing LPS doses. When the dosage of LPS was less than 1.0 mg/kg,these two indexes were reduced to below 25%. ②When the dosage of LPS ranged from 0.5 to 1.0 mg/kg,pathological score,W/D score and the mRNA levels of TNF-α,IL-1β were significantly higher than those in the control group under the condition of LPS≥0.7 mg/kg (P < 0.05 ). When the dosage of LPS < 0.7 mg/kg,the above indexes showed no statistical significance (P > 0.05 ). ③When the dosage of LPS = 0.7 mg/kg,the expressions of TNF-α,IL-1β mRNA in placenta and fetal issues increased more obviously (P < 0.05 ) than that in the control group. Compared to the control group,the mRNA expression of TNF-α and IL-1β in lung tissues of neonatal rats on day 1,4,and 7 was significantly increased(all P < 0.05). Conclusion:Intraperitoneal LPS (0.7 mg/kg) given to pregnant SD rats on 18th day of gestation led to lung injury,and pulmonary edema and increase TNF-α and IL-1β in newborn rats,which continued into the 7th day after birth. This is a stable and reliable method to construct the model of lung injury in neonate rat with intrauterine infection.

Abstract:Objective:To produce monoclonal antibodies against Bacillus anthracis PA15,and preliminarily establish double antibody sandwich-ELISA to detect the protective antigen obtained from serum of patient infected with Bacillus anthracis. Methods:Purified PA63 proteins were performed as immunogen to immunize mice. Hybridoma technology was performed to produce monoclonal antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to detect the purity of antibodies. Indirect ELISA,Western blot,immunoprecipitation (IP) and protein profiling were performed to analyze the specificity of monoclonal antibodies. Furthermore,double antibody sandwich-ELISA method was performed. Results:Two monoclonal antibodies against PA15 were obtained,named 3D7 and 8E9. SDS-PAGE showed the heavy and light chains of antibodies. Indirect ELISA and Western blot detected that monoclonal antibodies 3D7 and 8E9 can specifically bind PA15 and PA63. IP and protein profiling analyses showed that monoclonal antibody 3D7 can specifically bind PA83. Double antibody sandwich-ELISA detected that the limited detectable concentration of protective antigen from serum infected with Bacillus anthracis was 16 ng/ml. Conclusion:We have successfully developed monoclonal antibody against PA15,and established double antibody sandwich-ELISA to detect anthrax infected serum protective antigen.

Abstract:Objective:To prepare long-circulating lipid membrane ultrasound contrast agent suitable for the ultrasonic diagnosis of inflammatory bowel disease (IBD) and targeted mucosal addressin cell adhesion molecule-1 (MAdCAM-1),and determine the physical characteristics of the microbubbles and investigate their affinity for SVEC4-10 cells in vitro. Methods:The long-circulating lipid membrane microbubbles were prepared by sonic dispersion,and bridged the microbubbles and anti-murine MAdCAM-1 monoclonal antibody (MECA-367) by “biotin-avidin”. The physical traits of prepared targeted microbubble contrast agent were detected. Its ability to target to IBD cell model induced by tumor necrosis factor-α (TNF-α) was determined under light microscope and confocal laser scanning microscope,and isotype control antibody IgG2a microbubbles were used as control. Results:The targeted ultrasound microbubbles had a good shape and uniform particle size,with the average diameter of (464.5 ± 85.7)nm,and the surface Zeta potential was (-19.3 ± 2.5)mV. MAdCAM-1 expression on cultured SVEC4-10 cells was positively correlated with the time of TNF stimulation. In addition,when the concentration of TNF-a was 20 ng/ml,MAdCAM-1 expression reached its peak. The targeting study in vitro showed that many targeted ultrasound contrast agents adhered more firmly to the surrounding cells expressing MAdCAM-1,while the control group had no significant binding. Conclusion:Bridging MECA-367 long-circulating lipid membrane ultrasound contrast agents were successfully prepared. The targeted ultrasound contrast agents can bind to the cell model of high expression MAdCAM-1 effectively in vitro,and the method of establishing IBD cell model on TNF-stimulated SVEC4-10 cells is simple and feasible.

Abstract:Objective:To investigate the serum levels of vascular cell adhesion molecule-1 (VCAM-1),interleukin-8 (IL-8),interleukin-11 (IL-11),interferon-inducible protein-10 (IP-10),macrophage inflammatory protein-1-茁 (MIP-1-茁),the regulated upon activation normal T cell expressed and secreted (RANTES),and angiogenin in patients with coronary heart disease (CHD),then analyze the relationship between the 7 cytokines and the characteristic of coronary artery and explore the possibility to treat them as the new biomarker of CHD. Methods:A total of 216 patients were enrolled in this study. The CHD group consisted of 158 patients who were diagnosed with CHD and the control group consisted of 58 patients with normal coronary artery. Serum levels of the seven cytokines were measured and analyzed. Results:①No significant difference was detected in the levels of IL-8,IP-10,MIP-1-茁 and RANTES between the CHD group and the control group,while the levels of VCAM-1,IL-11 and angiogenin in the CHD group were significantly higher than those in the controls (P < 0.05). ②The differences of the serum levels of VCAM-1 and IL-11 were not significant among the single vessel subgroup,two vessels subgroup and the multiple vessels subgroup (P > 0.05),while the levels of angiogenin in the multiple vessels group were significantly higher than those in the single vessel subgroup and two vessels subgroup (P < 0.05). ③The differences of the serum levels of VCAM-1 and IL-11 were not significant among the subgroups with Gensini severity score > 40,21~40 and < 20,respectively. Serum levels of angiogenin in the subgroup with Gensini severity score > 40 were significantly higher than those in the subgroup with the score between 21 and 40 (P < 0.05),and the levels in the subgroup with the score between 21 and 40 were significantly higher than those in the subgroup with the score < 20 (P < 0.05). ④The level of serum angiogenin was positively related with the Gensini severity score (r = 0.27,P = 0.046). Linear correlation analysis revealed that serum level of angiogenin was not associated with risk factors of CHD (P > 0.05). Conclusion:The expressions of VCAM-1,IL-11 and angiogenin increase in the serum of patients with coronary heart disease. The serum level of angiogenin may be a potential biological indicator to evaluate the degree of coronary artery stenosis.

Abstract:Objective:To evaluate the relationships between serum high-sensitivity C-reactive protein(hs-CRP),cystatin(CysC),apolipoprotein A(ApoA),lipoprotein a[Lp(a)]and the global registry of acute coronary events(GRACE)scores in patients with non-ST segment elevation acute coronary syndrome(NSTE-ACS). Methods:A total of 155 patients with NSTE-ACS who made a definite diagnosis were selected and divided into the non-ST segment elevation myocardial infarction(NSTE-AMI)group and the unstable angina pectoris(UA) group. After admission,GRACE score was calculated and serum levels of hs-CRP,CysC,ApoA and Lp (a) were examined. Eighty-seven patients without coronary heart disease severed as control group. Results:The levels of serum hs-CRP,CysC,Lp(a) and the GRACE risk scores in NSTE-ACS patients were significantly increased compared with the control group,while the level of serum ApoA was significantly decreased and the differences were statistically significant(P < 0.05). With the increase of GRACE risk level,the levels of serum hs-CRP,CysC and Lp (a) were significantly increased,ApoA were significantly decreased and the differences were statistically significant(P < 0.05). The correlation analysis showed that GRACE scores had a positive correlation with hs-CRP,CysC,Lp(a)(r = 0.424,P < 0.01;r = 0.549,P < 0.01;r =0.134,P < 0.05),and a negative correlation with ApoA(r = -0.167,P < 0.01) in NSTE-ACS patients. Furthermore,the multiple linear regression analysis showed that CysC and hs-CRP had the greatest influence on the GRACE score. Conclusion:The levels of serum hs-CRP,CysC,ApoA and Lp(a) are closely related to coronary heart disease,which may be used as good indicators of extent of coronary atherosclerosis. For the NSTE-ACS patients,higher levels of hs-CRP,CysC,ApoA and Lp (a) indicated a higher GRACE scores. Combination of these indexes may have the important clinical value on early risk stratification,evaluation of prognosis and treatment options.

Abstract:Objective:To evaluate the long-term strategically prognostic value of optimal timely percutaneous coronary intervention (PCI) after fibrinolysis in treatment of acute myocardial infraction (AMI). Methods:The study analyzed 132 patients,who were suffered with ST-elevation myocardial infraction (STEMI) and treated with different strategies either of optimal timely PCI after fibrinolysis or primary PCI,for the disparity of medical information,including the major adverse cardiac event (MACE),left ventricular ejection fraction (LVEF) and N-terminal pro b-type natriuretic peptide (NT-Pro BNP). Results:Beyond the TIMI angiographic flow before PCI,there were no significant difference in MACE,LVEF and NT-Pro BNP levels between the two groups of patients with different strategic therapies within a six-month period. Conclusion:Contrasted with primary PCI,the strategy of optimal timely PCI after fibrinolysis has equivalent impact on long-term prognosis of patients with STEMI. This strategy is also a reliable and feasible alternative for hospitals which are unable to delivery primary PCI service for STEMI patients promptly at an urgent time.

Abstract:Objective:To observe the clinical characteristics,pathogens of inpatients with community-acquired pneumonia (CAP) and to explore the relative risk factors. Methods:The database of inpatients with CAP were retrospectively analyzed. The age,tobacco use,underlying disease,pathogens and clinical treatment were compared. The risk factors which may affect the prognosis of patients with CAP were also determined. Results:A total of 362 CAP patients(average age of 69.3 ± 17.2) were retrospectively analyzed. The top 5 underlying diseases were hypertension (164,45.3%),chronic obstructive pulmonary disease (COPD,151,41.7%),cardiac functional insufficiency (81,22.4%),diabetes (79,21.8%) and coronary heart disease (72,19.9%). A total of 177 (48.9%) patients were cured,168 patients(46.4%) improved and 17 (4.7%) patients did not improve or died. Of the 362 patients,312 patients underwent pathogen detection,and only 100 patients were detected pathogens. The top 5 pathogens were Candida albicans (31 cases),Gram-negative bacterium (11 cases),Baumanii (8 cases),Klebsiella pneumoniae (8 cases) and Pseudomonas aeruginosa (7 cases). Logistic regression analysis suggested that high age (β = 0.180,P = 0.001),high percentage of neutrophil (β = 0.127,P = 0.035),low prealbumin (β = 0.140,P = 0.024),pulmonary heart disease (β = 7.851,P = 0.046) and cerebral infarction (β = 4.861,P = 0.029) were risk factors of adverse prognosis. Conclusion:The CAP inpatients of the second affiliated hospital of NJMU were predominantly aged population. Most of them had underlying disease with hypertension,COPD,diabetes,cardiac functional insufficiency and coronary heart disease. High age,high percentage of neutrophil,low prealbumin,pulmonary heart disease and cerebral infarction were risk factors of adverse prognosis.

Abstract:Objective:To detect lymphocyte subsets in the peripheral blood of patients with different types of severe drug eruption and study its clinical significance. Methods:Immunofluorescence flow cytometry was performed to analyze the phenotypes of peripheral blood cells in 21 patients with severe drug eruption. Results:CD3+,CD3+CD4+ lymphocytes of the peripheral blood in drug-induced hypersensitivity syndrome (DIHS) patients was significantly increased in comparison with the normal value,and CD3+CD4+ lymphocytes of the peripheral blood cells in DIHS patients was significantly higher than that in Stevens-Johnson Syndrome (SJS) patients and toxic epidermal necrolysis (TEN) patients. With the increase in the number of CD3+CD4+ and CD3+CD8+ lymphocytes in the peripheral blood cells,the maximum dosage of corticosteroids progressively decreased in SJS and TEN patients. With the increase in the number of CD3-CD16+CD56+ lymphocytes of the peripheral blood cells,the maximum dosage of corticosteroids progressively increased in SJS and TEN patients. Conclusion:Different types of severe drug eruption have different lymphocyte subsets in the peripheral blood,which have different effects on the maximum dosage of corticosteroids.

Abstract:Objective:To investigate the influence of intraoperative intravenous magnesium sulfate on muscle relaxation effects of vecuronium,and evaluate its safety. Methods:Sixty patients underwent elective lumbar discectomy and internal fixation were randomly divided into two groups with 30 cases in each group. After induction of anesthesia,40 mg/kg of magnesium sulfate was given in group A through intravenous infusion,group B received the same volume of saline. Propofol (3.0~3.5 μg/ml) and remifentanil (3.0~4.0 ng/ml) were injected by plasma concentrations target-controlled infusion to maintain BIS at 45~55. Vecuronium was administrated with muscle relaxants integrated closed-loop target controlled injection system,and TOF was set to 0. Serum Mg2+,Ca2+ and K+ concentration were detected at four time points:preoperative (t1),10 min (t2) and 60 min (t3) after medication of magnesium or placebo,and t4 when vecuronium was disabled. Heart rate (HR) and mean arterial pressure (MAP) were accessed 5 min after medication of magnesium or placebo. The time of T70% (TOF recover up to 70%) and the total dosage of vecuronium were recorded. Results:Serum concentration of Mg2+ reached a maximum (1.33 ± 0.19 mmol/L) at t2,and declined gradually through the passing of operation time in group A (P < 0.05),and still higher than that in group B at t3 and t4 (P < 0.05). Serum concentration of Ca2+ and K+,as well as HR and MAP showed no significant difference between the two groups (P > 0.05). The dosage of vecuronium was (3.22 ± 0.57) μg/(kg-min) in group A,significantly less than that in group B (3.81 ± 0.58) μg/(kg-min) (P = 0.0002);T70% in group A and group B were (26.3 ± 3.6) min and (28.7 ± 4.3) min,respectively,the difference was statistically significant (P = 0.0225). Conclusion:Intravenous injection of 40 mg/kg magnesium sulfate could enhance muscle relaxation effects of vecuronium,reduce the dosage and shorten neuromuscular recovery time,and has no significant effect on serum concentration of Ca2+,K+ and hemodynamics.

Abstract:Objective:To investigate the homology and prevalent cases of multidrug-resistant Acinetobacter baumannii (MDRAB) so as to provide reference for the prevention of nosocomial infection and clinical reasonable application of antibiotics. Methods:A total of 31 MDRAB isolates between June 2012 and December 2012 were collected,the susceptibility test of 12 antimicrobial agents was determined by K-B method,and the homology analysis of the strains was determined by pulsed-field gel electrophoresis(PFGE). Retrospective analysis of MDRAB sample cases was performed. Results:The antimicrobial susceptibility showed that the resistance rate of 31 MDRAB isolates to 9 antibiotics was 100%,including 4 kinds of antibacterial drugs. The results of PFGE demonstrated that 31 isolates were classified into 5 distinct genotypes,among those 20 isolates (accounting for 64.52%) of clone B were the predominant epidemic strains. The patients with MDRAB infection were distributed among 8 wards and mainly centralized in the intensive care unit,cardiothoracic surgery and geriatric intensive care unit. The average age of the patients was 64 years,and 97% of them had undergone invasive procedures. Conclusion:The MDRAB was seriously resistant to almost all clinically-used antibiotics. It may be closely related to a variety of factors,such as clonal spread,old age of patients,invasive treatment and patients with multiple underlying medical conditions.

Abstract:Objective:To develop a sandwich ELISA for quantitative detection enolase of Candida albicans,and apply the assay to detect enolase levels in supernatant of common fungi cultures. To investigate the potential diagnostic value of enolase in invasive candidiasis. Methods:Anti-enolase of Candida albicans monoclonal antibody was employed as coating antibody,HRP-conjugated goat polyclonal antibody against Candida albicans enolase was used as detecting antibody. The performance parameters of the sandwich ELISA including precision,linear range and limit of detection were verified by using recombinant Candida albicans enolase. Then the developed assay was applied to determine enolase levels in supernatant of pathogenic fungi cultures such as common Candida spp. Cryptococcus neoformans and Saccharomyces cerevisiae for preliminary evaluation. Results:The intra and inter-coefficient of variation was 6.61%,9.19% and 6.98%,13.81% at the concentration of 20 ng/ml and 5 ng/ml respectively. The limit of detection was 1.25 ng/ml. The linear range was 1.25~50.00 ng/ml. The level of enolase in Candida albicans culture after incubated in 37℃ for 24 h was 3.06 ng/ml and gradually increased to 33.43 ng/ml at 120 h after incubated in 37℃,consistent with growth of hyphae. The sandwich ELISA was weak cross-reactive to Candida parapsilosis and no cross-reactivity to Candida tropicalis,Candida guilliermondii,Candida glabrata,Cryptococcus neoformans and Saccharomyces cerevisiae was found. Conclusion:A sandwich ELISA for quantitative detection enolase of Candida albicans was developed,which had the potential to be applied to stndy invasive candidasis.

Abstract:Objective:To analyze the effectiveness and associated factors of highly active antiretroviral therapy (HAART) on men who have sex with men (MSM) in Jiangsu province,and to establish an evaluation system of HAART for MSM in Jiangsu province for the first time. Methods:MSM who received HAART in two cities in Jiangsu province participated in a cluster random sampling. Questionnaires were used to collect information of life history and drug use. CD4 count was acquired from AIDS therapeutic database of Jiangsu province. Epidata 3.0 and SAS 9.0 were used for data entry and statistical analysis,respectively. Results:According to AIDS therapeutic database,the total CD4 count of 157 MSM was increased,while their drug compliances were quite low. Univariable and multivariable statistical analyses showed that low condom usage,heavy smoking,and long-time interval between definite diagnosis and therapy reduced the effect of HAART,while higher CD4 count at the time of starting therapy and longer drug use were protective factors of HAART. Conclusion:Currently,HAART showed significant effect among MSM in Jiangsu province. We recommended performing early treatment and improving drug compliance of HAART among MSM who meet the therapy conditions.

Abstract:Objective:To investigate the H5N1 avian influenza antibody level in occupational exposure population in Jiangsu province during the beginning of the influenza A (H7N9) outbreak. Methods:The antibody levels of Hong Kong,Guangdong and Anhui strain were detected using serum hemagglutination inhibition(HI) tests. The results were converted into geometric mean titer (GMT) and analyzed. Statistical analysis of the influencing factors of antibody levels was performed. Results:The difference was significant between antibody levels of different gender and antigen strains of all 416 serum samples tested. Conclusion:Our study showed that the antibody level of H5N1 avian influenza is low,and the levels of male are not as high as those of female. HI antibody level of Guangdong strain is closer to currently epidemic strains of avian flu in Jiangsu Province.

Abstract:Objective:To explore the effect of microRNA-194 (miR-194) on cisplatin resistance of non-small cell lung cancer (NSCLC) A549/DDP cell lines and its related mechanisms. Methods:The real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect miR-194 expression differences between NSCLC A549/DDP cells and A549 cells. MiR-194 expression differences were detected after MiR-194-inhibitor transfected into A549/DDP cells. MMT assay,colony formation and flow-cytometric analysis were sequentially performed to detect transfected A549/DDP cells to DDP drug sensitivity,cell proliferation ability,and apoptosis on treatment with DDP. The protein expressions of Bax and Bcl-2 in transfected A549/DDP cells were detected by Western blot. Results:The level of miR-194 in A549/DDP cells was significantly higher than that in A549 cells (P < 0.05). The miR-194 level in A549/DDP cells was significantly lower than that in A549/DDP cells after transfected with miR-194 inhibitor (P < 0.05). The inhibited expression of miR-194 could produce following effects: compared to the control group,the half inhibition concentration (IC50) of DDP in A549/DDP with miR-194-inhibitor cells was decreased (P < 0.05),proliferation ability of A549/DDP with miR-194-inhibitor cells was diminished (P < 0.05),apoptosis after treatment with DDP was increased (P < 0.05). Western blotting results showed that,the expression of Bcl-2 was down-regulated and the expression of Bax was up-regulated in A549/DDP/miR-194-inhibitor cells than those in the control group. Conclusion:miR-194 increases the resistance of A549/DDP cells to DDP through inhibition of apoptosis,up-regulating Bcl-2 protein expression and down-regulating Bax. The inhibition of miR-194 expression can reverse the resistance of A549/DDP cells to DDP.

Abstract:Objective:To prepare a human scFv-Fc fusion antibody against rabies virus and to analyze its binding activity. Methods:The human scFv-Fc prokaryotic expression vector was constructed. After optimizing prokaryotic expression system,the human scFv-Fc fusion antibody against rabies virus was expressed and purified. Purified humanized scFv-Fc antibody was confirmed its binding activity by binding to purified inactivated rabies virus. Results:After sequence analysis,the human scFv-Fc-pColdⅡprokaryotic expression vector was successfully established. Through transforming E.coli.BL21 (DE3),human scFv-Fc fusion antibody was expressed by optimized induction of IPTG at concentration of 0.5 mmol/L. The expression of scFv-Fc fusion antibody was increased and its molecular weight was 57 000. By purification,the human scFv-Fc fusion antibody was purified with excellent binding activity even in 512 times diluted concentration. Conclusion:We have successfully established a whole human anti-rabies virus scFv-Fc-pColdⅡ prokaryotic expression vector,and expressed and purified scFv-Fc fusion antibody. This fusion antibody can lay a foundation for developing novel antibody-targeted drugs of rabies prophylaxis after exposure.

Abstract:Objective:To analyze the polymorphism distributions of 19 STR loci including D21S11,D6S1043,Penta E,PGA,D2S1338,D11S51,D19S433,D12S391,CSF1PO,D3S1358,Penta D,VWA,D13S317,THO1,TPOX,D8S1179,D16S539,D5S818 and D7S820 in Jiangsu population,and calculate their gene frequency(P),power of discrimination(DP),unbiased expected heterozygosity(H),polymorphism information content(PIC),and probability of paternity exclusion(PE). Methods:Capillary electrophoresis was detected using PCR via 3130 sequencing. Analysis software was used to analyze data. Results:The DP value and PE value of Penta E were the highest in the 19 STR sites. The cumulate PE of the 19 STR loci was above 0.9999. Conclusion:Objective allelic polymorphism data were obtained from a large number of sample experiments and allele frequency,which provide an objective basis for identification in disputed paternity of Jiangsu area.