Abstract:Objective:To investigate the expression of the recombinant plasmid of HIV-1 viral protein regulatory(Vpr) in the eukaryotic cells, and evaluate the effect on Kaposi’s sarcoma-associated herpesvirus(KSHV) lytic cycle replication by Vpr protein in BCBL-1 cells. Methods:After identification with enzyme digestion and nucleotide sequences analysis,the recombinant plasmid pVpr-Flag was transiently transfected into BCBL-1 cells and NIH3T3 cells. The mRNA and protein expression of pVpr-Flag in both cells was detected by RT-PCR, IFA and Western blot,respectively. Subsequently, RNA was obtained from BCBL-1 cells transfected with pVpr-Flag plasmid. RT-PCR was carried out to evaluate the expression of ORF26 mRNA(encoding the minor capsid protein) of KSHV. Results:Nucleotide sequences analysis indicated that the cloned Vpr sequence was 100% homology with Vpr gene registered in GenBank previously. The specific band was detected at the expected place both by RT-PCR and Western blot. And the expression of Vpr protein was also detected in BCBL-1 cells by IFA. In addition,the expression of ORF26 mRNA in BCBL-1 cells transfected with plasmid pVpr-Flag was weaker than that in the control. Conclusion:Vpr gene was correctly expressed in BCBL-1 and NIH3T3 cells,and Vpr protein might inhibit KSHV lytic cycle replication

Abstract:Objective:To investigate the effect of apoptosis of oxidant-antioxidant change on anoxia-reoxygen injury to cortical neurons in vitro. Methods:Acute glucose-oxygen deprivation and subsequent reoxygenation were used to establish anoxia-reoxygenation injury model in cultured cortical neurons. They were randomly divided into 2 groups:control group,groups with different reoxygenation time(3 h,6 h,12 h and 24 h). The content of dehydrogenase(LDH), glutathione(GSH) and NO, the activity of NOS and glutathion peroxidase(GPx) were measured by assay kits. The morphology of apoptotic cells was observed by Hochest33258 staining. Results:Compared with the control group,the level of LDH,NO and cell apoptotic rate was obviously increased,while the activity of GPx was decreased in reoxygenation groups(P < 0.05);The activity of NOS was obviously increased and the GSH level was decreased at reoxygenation 3 h and 6 h(P < 0.05), but there was no significant difference at reoxygenation 24 h(P > 0.05). Conclusion:The imbalance between the system of oxidation and reduction may be one of the reasons inducing neurons apoptosis as reoxygenation time extending in vitro.

Abstract:Objective:Establishment of glucokinase activator screening method using heterologous expression of eukaryon system. Methods:The pPIC9K vector was inserted a fragment of human liver glucokinase. The plasmid of pPIC9KGK was identified by restriction enzyme and sequencing. Then the plasmid was transfected to pichia pastoris via methanol induction. They produced the human liver recombinant GK protein,which was further detected by SDS-PAGE. The recombinant GK protein had enzymatic activity and was used in GK activator screening method. Using this method, lots of compounds to find GK activators were screened and a lead-compound LGK-01 was found. Results:Human liver recombinant GK protein was expressed in pichia pastoris, and the protein had enzymatic activity. Conclusion:GK activator screening method was established successfully in vitro.

Abstract:Objective:To construct and test the immunogenicity of codon-optimized DNA vaccines expressing gp120 from key representative HIV-1 subtypes AE,BC and B′(ThB) in China. Methods:Based on HIV-1 epidemiological information and sequence analysis of HIV-1 envelope glycoprotein(Env) of subtypes AE,BC and ThB in China,we designed the consensus gp120 sequence for each subtype AE,BC or ThB. These consensus gp120 genes were codon-optimized using codons preferred for high level expression in mammalian systems and chemically synthesized. The codon optimized gp120 genes were subcloned into the DNA vaccine vector pSW3891. The expression of gp120 by the constructed DNA vaccine plasmids in 293T cells was confirmed by Western blot. New Zealand rabbits were immunized with gp120 DNA vaccines using electroporation method,and gp120-specific antibodies in rabbit sera were evaluated by ELISA. Results:In the codon optimized gp120 gene,the frequency of the mammalian cell preferred codons was higher than that of the wild type gp120 gene. Three codon optimized gp120 DNA vaccines of consensus subtypes AE,BC or ThB were constructed and the expression of gp120 antigens was confirmed in transiently transfected 293T cells. The rabbits immunized with these gp120 DNA vaccines elicited HIV-1 Env-specific antibody responses. Conclusion:Three codon optimized DNA vaccines expressing consensus gp120 of HIV-1 sybtypes AE,BC and ThB were successfully constructed with proven expression and immunogenicity in eliciting HIV-1 Env-specific humoral responses.

Abstract:Objective:To investigate the role of nitric oxide(NO) on the motility function of proximal colon of diabetes mellitus mice. Methods:The distribution and expression of nNOS in the proximal colon of diabetes mellitus mice were examined by fluorescence microscopy and Western-blotting, respectively. The effect of NO precursor L-arginine(L-Arg), nNOS inhibitor 7-nitroindazole(7-NI ),iNOS Amino guanidine(AG) on the proximal colon muscle strips muscular contractions were monitored by MD-2000 system. Results:The expression of nNOS in the proximal colon was up-regulated in DM group. L-Arg decreased the proximal colon muscle strips contractile tension, 7-NI increased the muscle strips contractile tension while AG had no effect. All drugs had no influence on muscle strips contractive frequency. Conclusion:Increased expression of nNOS is demonstrated in the proximal colon of diabetes mellitus mice, and NO may play an important role in the pathogenesis of diabetic proximal colonic dysfunction.

Abstract:Objective:To investigate whether the MPZ gene contributes to the non-syndromic hearing loss of a Chinese pedigree with dominantly inherited auditory neuropathy(AN). Methods:Nine members in three generations of the family were selected for this study. Genomic DNA was isolated from the peripheral leukocytes of the patients using the Puregene DNA Isolation Kits. Firstly,the DNA fragment of the subjects was PCR amplified using oligonucleotides corresponding to exon 3 of the MPZ gene. Each fragment was purified and subsequently analyzed by direct sequencing in an Applied Biosystems 3730 automated DNA sequencer. The entire MPZ gene of one affected subject Ⅲ3 were then amplified with 6 overlapping fragments, and submitted for sequence analysis as described above. The resultant sequence data were compared with the standard sequence to identify deafness-associated mutations. Results:PCR amplifications were successfully conducted in all the subjects. We failed to detect the presence either mutations of Thr124Met and Tyr145Ser in the exon 3 that have been reported,or any other deafness-associated mutations in the whole MPZ gene,by sequence analysis. Conclusion:The MPZ gene seems not contribute to the pathogenesis of this Chinese AN family,which suggesting new gene(s) involvement.

Abstract:Objective:To investigate the incidence of chronotropic incompetence(CI) in a cohort of sinus bradycardia and the value of CI between exercise test(ET) and Holter monitoring electrocardiogram(Holter). Methods:Sixty-seven subjects of sinus bradycardia(48 male and 19 female,the mean age of 46.7 ± 13.9 years) and 47 control subjects of normal heart rate(34 male and 13 female, the mean age of 48.4 ± 7.6 years) received treadmill exercise test and Holter detection. The maximal heart rate during ET and Holter were observed. CI was defined as the failure to achieve 85% of the age predicted maximal heart rate(APMHR) during ET or failure to 100bpm during Holter. Results:①Sixteen sinus bradycardia subjects(23.88%) and 4 normal control subjects(8.51%) than 85% of APMHR(P < 0.05). ②The prevalence of CI achieved less diagnosed by ET was 23.88% and 7.46% diagnosed by Holter in the subjects of sinus bradycardia(P < 0.05). Conclusion:The population with sinus bradycardia has a higher incidence of chronotropic incompetence. ET is a more valuable diagnostic technique to CI than Holter.

Abstract:Objective:To study the therapeutical effects of salvianolate on acute myocardial infarction(AMI) in swines. Methods:Twenty-one young swines were divided into high-dose,low-dose and control group randomly(n = 7,each group). AMI models were made by deligating anterior descending branch as myocardial infarction model. Salvianolate 400 mg + 5% GS 250ml or salvianolate 200 mg + 5%GS 250 ml was injected into swines through the vein once dayly immediately after coronary artery deligated for 7 days in high-dose,low-dose and control group, respectively. Four weeks after operation,coronary bypass circuit and LVEF were evaluated by coronary arteriongraphy,immunohistochemistry and gated myocardial perfusion imaging, respectively. Results:①Coronary arteriongraphy:The circulation of infarction region of therapic group was better than that of control group obviously,especially in high dose group; ②Gated myocardial perfusion imaging:LVEF in two therapic groups was higher than that in control group;③Immunohistochemistry:capillary density of therapic group was significantly higher than that of control group,but there were no significant differences between high-dose and low-dose groups. Similarly,function density of treatment groups also significantly higher than that of control group, which also was higher in the high-dose group than that in low-dose group. Conclusion:Therapeutical effect of salvianolate on acute myocardial infarction(AMI) in swines through the vein is significant. Salvianolate therapy can improve heart function and promote blood vessel regeneration,promote formn of collateral blood vessel.

Abstract:Objective:To examine the differential diagnosis between physiological and pathological left ventricular hypertrophy by using current echocardiography techniques. Methods:Twenty male SD rats were randomly divided into three groups:One group of rats performed swimming training at high intensive swimming training(HIGH);One group of rats,a 2KIC hypertension was created and maintained(2KIC);And the third rats group was sham operation(control). After 8 weeks,all rats were studied by standard and tissue Doppler echocardiography. Results:Echocardiography showed septal and posterior wall thickness increases in both the 2K1C and HIGH rats. Left ventricular increased by 36% and 41% respectively. Mitral inflow showed a decrease in late-wave velocity,thus increasing the E/A ratio in HIGH rats. However,mitral inflow showed an increase in late-wave velocity,thus decreasing the E/A ratio in 2K1C rats. There was a significant increase in Ea and Ea/Aa in HIGH rats in basal septum and lateral mitral valve annulus. And there was a significant increase in Aa,which led to significant decrease in Ea/Aa in 2K1C rats. Conclusion:Doppler echocardiography parameters of LV diastolic function can be of diagnostic importance for discrimination between pathologic and physiologic LV hypertrophy.

Abstract:Objective:Compared with the porcelain-fused metal restorations,the study is to evaluate the alteration of color on CAD/CAM alumina glass-infiltrated composite with different color Vita α veneering porcelain,helping the color-selecting and color-matching in clinical practice. Methods:Four groups of CAD/CAM alumina glass-infiltrated composite(ALC) and ten porcelain-fused Ni-Cr alloys specimens(randomized into two groups) were routinely layered with A2/A3 opaque-dentine porcelain,dentine porcelain and enamel porcelain. The resulting color differences were measured and calculated. Results:There were significant color differences in four groups of ALC,two groups of porcelain-fused Ni-Cr alloys specimens and also between the experimental and control groups(P < 0.01). Conclusion:The color space of alumina self-made glass-infiltrated composite was in the range of Chinese teeth chroma. Compared with the porcelain-fused metal restorations,the color of ALC was shifted to the lightness and yellow in b*-axis,which corresponded with the characteristics of Chinese teeth.

Abstract:Objective:To culture rat cranial osteoblastic cells in vitro by modified explants culture. Methods:Primary osteoblastic cells were obtained from the calvaria of 3-day-old new-born SD rats. The calvaria were divided into small pieces and seeded on bottom of culture bottle. When osteoblasts crawled from the explants and confluented to monolayer,they were digested and aggregated by 0.25% trypsin and explants continued culturing. MTT,ALP activity and calcific nodule were analysized in different osteoblasts. Results:The cultured cells by this method were identified according to the osteoblastic bionomics. There were no statistical differences in MTT,ALP activity and calcific nodule among the osteoblasts. Conclusion:The modified explants method to culture primary osteoblasts could obtain large amount of cells and supply osteoblasts continuously.

Abstract:Objective:To establish the method to identify protein using LC-MALDI. Methods:To investigate if the capillary liquid chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry technology is an available method to proteins sequence by comparing with the most common sequencing method of MALDI-TOF/TOF-MS. Results:17 tryptic peptides can be identified using MALDI-TOF/TOF-MS with 31.5% sequence coverage and 130 score while 35 peptides can be identified using CapLC coupled with MALDI-TOF/TOF-MS method with 59.35% sequence coverage and 2 976 score. Conclusion:The method of proteins sequence using CapLC coupled with MALDI-TOF/TOF-MS is available. Moreover,this identification method is more precise and effective than MALDI-TOF/TOF-MS in protein identification.

Abstract:Objective:To explore the expression of tumor metastasis suppressor KAI1 in membrane and cytoplasm of papillary thyroid cancer(PTC) cell; and to explore the clinicopathological significance of total expression of KAI1 protein in PTC. Methods:we used western blot to investigate:the KAI1 protein expression in membrane and cytoplasm in 30 primary PTC cases,13 cases of tissue near the cancer,7 cases of lymph node metastases and 13 benign cases ;total expressions of KAI1 protein in 30 PTC cases. Results:the expression of KAI1 protein in membrane was significant lower than that in cytoplasm of primary PTC;while the expression were unchanged in membrane and cytoplasm of tissues near the cancer,lymph node metastases and benign cases. The positive expression of KAI1 protein was correlated with the absence of lymph node metastases and the low stage of TNM;there were no significant correlation between KAI1 expression and primary tumor size or patient age. Conclusion:Loss of location in cell membrane may be the cause of KAI1 losing its ability to suppress the tumor progression and metastases. The low expression of KAI1 protein was significantly correlated with the clinical progression and lymph node metastases of PTC and it may be used to anticipate the progression and metastases,and may be the potential target of PTC for therapy.

Abstract:Objective:To study the expressions of CTGF and VEGF and detect their clinical value and correlation in oesophageal squamous cell carcinoma tissues and paracancerous tissues. Methods:SP immunohistochemical method was used for the assayes of CTGF and VEGF on the routinely paraffin-embedded sections of surgical operated specimens of 49 cases with oesophageal squamous cell carcinoma and 41 ones with paracancerous tissues. Results:the positive rate of CTGF and the score were significantly lower in cancer tissues than those in paracancerous tissues(46.9% vs 82.9%,P < 0.05;2.21 ± 1.14 vs 4.45 ± 1.37,P < 0.01),but those of VEGF were opposite(79.6% vs 19.5%,P < 0.01;3.69 ± 1.46 vs 1.12 ± 0.74,P < 0.01). The positive rates and scores of CTGF were significantly higher in the cases without metastasis of lymphnode and in clinical stage T1 than those in the ones with metastasis of lymphnode and in clinical stage T3(P < 0.01). The positive rates and scores of VEGF were significantly lower in the cases without metastasis of lymphnode, in clinical stage T1 and histological grade Ⅰ than in those with metastasis of lymphnode,in clinical stage T3 and histological grade Ⅲ(P < 0.01). The close negative correlation was found between the score of CTGF and VEGF. Conclusion:The expressions of CTGF and VEGF migh be important biological markers in reflecting the progression,biological behaviors,metastatic potential and prognosis of oesophageal squamous cell carcinoma.

Abstract:Objective:To investigate the expression levels of MRP1,LRP and BCRPmRNA in patients with non-small cell lung cancer(NSCLC) as well as their clinical prognostic significance. Methods:RT-PCR method were used to detect the expression levels of MRP1,LRP and BCRPmRNA in tissues samples and corresponding adjacent normal lung tissues of 60 patients with NSCLC. T-Test,One-Way ANOVA,rank correlation,Kaplan-Meier survival curve,and COX multivariate regression analysis were used for statistical analysis. Results:The expression levels of MRP1and LRPmRNA were higher in NSCLC tissues than that in normal lung tissues(P = 0.02 and P = 0.000). There were significant correlations between intratumoral expressions of MRP1mRNA and LRPmRNA(rs = 0.530,P = 0.000). The expression level of BCRPmRNA was lower in NSCLC tissues than that in normal lung tissues(P = 0.042).LRPmRNA expression level was higher in lung adenocarcinomas than in squamous carcinoma(P = 0.02). None of them were related to clinical TNM stage. The patients with low expression of MRP1mRNA had significantly longer survival time than those with high expression of MRP1mRNA(P = 0.023). COX multivariate regression analysis showed that expression of MRP1mRNA was an independent prognostic factor for NSCLC. Conclusion:MRP1,LRP and BCRPmRNA were expressed in various degrees in NSCLC tissues.The expression level of MRP1mRNA was related to overall survival time of NSCLC. MRP1mRNA expressions can be used as a prognostic predictor for NSCLC.

Abstract:Objective:To evaluate the clinical effect of ultrasonic dissector(CUSA) in hepatectomy and partial liver transplantation. Methods:The clinical data were retrospectively analyzed among group of CUSA and without CUSA method. Results:The intraoperative bleeding and postoperative drainage of the CUSA group were significantly less than the group of without CUSA method. The aminotransferase(ALT) of with CUSA group also is lower than without CUSA group. Conclusion:CUSA is a kind of safe and effective instrument in hepatectomy,which should be suggested to apply in clinic.

Abstract:Objective:To study the changes of serum blood glucose,insulin,cortisol and TNF-α in patients underwent gynecological laparoscopic operations using abdominal wall-lifting methods under different anesthesia. Methods:Thirty-eight patients scheduled for gynecological laparoscopy,ASAⅠ~Ⅱ,were randomly divided into two groups(Ⅰ,Ⅱ) with 19 cases in each group respectively. Patients of groupⅠwere operated under general anesthesia and gasless technique. Patients of groupⅡ went through the same operative procedures under epidural anesthesia and gasless technique. All patients were monitored including blood glucose,insulin,cortisol,TNF-α at four time points:before anesthesia(T1),at 30 min after the operations beginning(T2) and 10 min after the end of the surgery(T3) and at 8 AM in the next morning(T4). Results:There were no obvious differences in blood glucose,insulin,cortisol and TNF-α between the two groups at T1(P > 0.05),and these values increased after the beginning of the operations, reaching their peak values at T4. The levels of blood glucose, insulin and TNF-α were higher in groupⅠ than those in group Ⅱ at T3(P < 0.05). Levels of serum cortisol were higher in groupⅠ than those in group Ⅱat T3(P < 0.01)and T4(P < 0.05). Conclusion:Compared with general anesthesia,epidural anesthesia might reduce stress responses in patients underwent gynecological gasless laparoscopic operation.

Abstract:Objective:To investigate the clinical significance of changes of acid-base and electrolytes in different phases during the procedure of orthotopic liver transplantation(OLT). Methods:Forty-six recipients who underwent liver transplantation with the piggyback technique under general anaesthesia were analyzed retrospectively. Blood samples collected during the phase of procedure for values included arterial pH,partial carbon dioxide pressure(PaCO2),standard bicarbonate values(HCO3-) and serum potassium(K+),ionized calcium(Ca2+),sodium(Na+),blood glucose levels(BG). The measurements were performed at nine time points:20 min after the skin incision(baseline),10,20,30,60 minutes during the anhepatic phase,and 10,20,30,60 minutes after reperfusion. Results:The pH in the phase of revascularization of liver was significantly lower at 10 min,20 min and 30 min(P < 0.001,P < 0.001 and P < 0.05,respectively); PCO2 was higher in anhepatic phase at 60 min(P < 0.05) and in the phase of revascularization of liver at all time points(P < 0.001,P < 0.001,P < 0.001 and P < 0.05,respectively); HCO3- was higher during anhepatic phase at 20 min,30 min and 60 min(P < 0.001,P < 0.01 and P < 0.01,respectively) and 10 min,20 min and 30 min during the phase of revascularization(P < 0.001,P < 0.01 and P < 0.01,respectively). K+ and Na+decreased significantly in the phase of revascularization at 10 min time point(P < 0.001,P < 0.01,respectively). No difference of Ca2+ was observed among the nine time points during the phases of procedure. Mean glucose levels increased in anhepatic phase at 10 min,20 min(both P < 0.05) and 10 min,20 min,30 min during the phase of revascularization(P < 0.001,P < 0.01 and P < 0.01,respectively). Conclusion:Mixed acid-base disturbances were observed in the early stage of revascularization phase during the procedure of OLT.

Abstract:Objective:To explore the clinical Value of different-time 99Tcm-MIBI SPECT/CT fusion imaging for the diagnosis of the lung cancer. Methods:Sixty five patients with pulmonary abnormal lesions were chosen for performing different-time 99Tcm-MIBI SPECT/CT fusion imaging. They were divided into lung cancer and benign groups according to the pathologic results. Then the results were analyzed. The uptake ratio(UR) was calculated. Finally the diagnostic efficacy of different-time 99Tcm-MIBI SPECT/CT fusion imaging for the lung cancer was evaluated by the threshold of x+1s of the lung benign group. Results:The UR value of 45 cases in lung cancer group was 2.56 ± 0.53 at the time of 60 min SPECT/CT fusion imaging,while 20 cases of benign group was 1.47 ± 0.42. There was a significant UR difference in two groups(P < 0.05). Using the x + 1s of benign group UR value as a threshold,the diagnostic sensitivity,specificity and accuracy of different-time 99Tcm-MIBI SPECT/CT fusion imaging for the lung cancer was 86.67%,85.00% and 86.15% respectively. The false positive and false negative rate were 15.00% and 13.33%. The positive and negative predictive value were 92.86% and 73.91%. Conclusion:Different-time 99Tcm-MIBI SPECT/CT fusion imaging may be used in the diagnosis of lung cancer with great value and it could provide the important clinical information for clinical treatment planning.

Abstract:Objective:To explore the relationship of dose and effects between insulin glargine combined with oral antihyperglycemic drug(OAD) and insulin pump to intensive treatment in patients with type 2 diabetes mellitus. Methods:Patients with type 2 diabetes were modulated their blood glucose with insulin pump in an ideal level about 2 weeks,then insulin pump was removed to use insulin glargin at bedtime combined with oral hyperglycemia drugs. The effects in controlling blood glucose and insulin doses are compared between insulin glargine treatment group and insulin pump therapy group, and their relationships were analyzed. Results: The effects in modulating blood glucose were similar between insulin glargine greatment and insulin pump therapy. The insulin dose in insulin glargine treatment is 39.0% of the total insulin dose and 87.6% of the basal insulin dose in insulin pump therapy. The coefficient correlation is 0.76(P < 0.01) and 0.81(P < 0.01). Conclusion:The effects in controlling blood glucose were similar between insulin glargine trentment and insulin pump therapy, and there is positive correlation between the doses of insulin glargine and total insulin in insulin pump therapy,and between the doses of insulin glargine and basic insulin in insulin pump therapy.

Abstract:Objective:To investigate the mental health status,cigarette smoking,alcohol drinking and their relationship among medical staff. Methods:The 1 418 medical staff in 7 comprehensive hospitals of Jiangsu province were investigated using symptom check list 90(SCL-90) and a self-designed questionnaire. Data were input to create a database with EpiData 3.02 and analyzed with SPSS 13.0 software package. Results:Total 1 447 questionnaires were sent out and 1 418 qualified ones were collected, and the effective response rate was 98.0%. There were 30.5% male and 69.5% female,with the mean age 35.6 ± 11.1 years. This survey showed that the total smoking rate was 10.0%,with 31.2% in male and 0.8% in female. The standardized total smoking rate was 11.7% according to the national population proportion by age in 2000,with 26.6% in male and 1.3% in female. Smoking rate in male was significant higher than in female,especially in the medical technician(25.8%). The total drinking rate was 14.0%,with 40.4% in male and 2.6% in female. The standardized total drinking rate was 16.3%,with 37.0% in male and 1.4% in female. Drinking rate in male is more significant than that in female,especially in the medical technician(28.9%). Comparing the smokers and the non-smokers,there were 3 factors(hostility,paranoid ideation and psychoticism) without significance,while others(somatization,obsession-compulsion,interpersonal sensitivity,depression,anxiety,phobic anxiety) of the smokers were all significantly higher than the non-smokers. According to whether they drank or not,the medical staff were divided into two groups. The result revealed that there were significant differences in the mean scores of somatization and phobic anxiety between the two groups,while other 7 factors(obsession-compulsion,interpersonal sensitivity,depression,anxiety,hostility,paranoid ideation and psychoticism) had no significance between the two groups. Conclusion:Drinking and smoking rate of the medical staff in comprehensive hospitals were lower than that of general population,while the smokers of medical staff had lower metal health level.