Objective To investigate the effect of non-structural protein amino acid mutations(NS1-G53D,NS3-E250V)on viral proliferation and virulence of Dengue virus(DENV)type 4 Ban18HK20 strain.Methods The mutant sites in Ban18-HK20 strain were determined by sequence alignment with an attenuated strain of DENV(PDK53). Point mutation plasmids pSPTM-Ban18HK20(G53D),pSPTM-Ban18HK20(E250V)and pSPTM-Ban18HK20(G53D+E250V)were constructed by using homologous recombination technique,and identified by enzymatic cleavage and sequencing. Viral RNA was obtained by in vitro transcription and then electrotransfected to Vero cells to rescue the point mutant virus. The biological characteristics of point mutant virus were identified by plaque assay,indirect immunofluorescence assay,virus genome sequencing,growth kinetics test,CCK-8 test and mouse neurotoxicity test.Results Enzyme digestion and sequencing confirmed that the point mutation plasmids were constructed correctly. The results of immunofluorescence assay and virus sequencing showed that the virus was rescued successfully. The point mutation virus showed smaller plaque than the wildtype virus. The virus titers of Ban18HK20(G53D),Ban18HK20(E250V)and Ban18HK20(G53D+E250V)reached the peak on the 4th day,which were 7. 67,7. 84 and 7. 78 LgPFU/mL,respectively. The titer of the wild-type virus reached the peak value 7. 68 LgPFU/mL on the 5th day. The proliferation activity of BHK21 cells infected with Ban18HK20(G53D)was significantly higher than those infected with the wild-type virus(P = 0. 003 6)and the proliferative activity of C6/36 cells infected with Ban18HK20(G53D)and Ban18HK20(G53D+E250V)was significantly higher than those infected with the wild-type virus(P < 0. 000 1). Both the point mutation virus and wild-type virus had no neurotoxicity to 4-week-old BALB/c mice. The neurotoxicity of point mutation viruses Ban18HK20(E250V)(LD_(50)=8. 51 PFU)and Ban18HK20(G53D+E250V)(LD_(50)= 0. 69 PFU)on 3-day-old BALB/c neonatal mice was lower than that of the wildtype virus(LD_(50)= 0. 69 PFU). The survival rate(60%)of nude mice injected with point mutation virus Ban18HK20(G53D +E250V)was higher than those injected with the wild-type virus(0)after intracerebral injection of the same amount of virus. The point mutation virus was genetically stable,and no recurrent mutation occurred in Vero cells after passage to the10th generation.Conclusion The non-structural protein NS1-G53D amino acid mutation weakens the cytotoxicity of Ban18HK20 strain to C6/36 and BHK21 cells,and the two-point combined mutation could weaken the neurotoxicity of Ban18HK20 strain in nude mice,which laid a foundation of the study of DENV virulence sites and the development of the vaccine.

Objective To discuss the therapeutic efficacy of budesonide and compound ipratropium bromide inhalated in treatment of infant bronchiolitis.Methods From January 2005 to February 2007,150 infants with bronchiolitis were randomly divided into treated group and control group.Both the two groups were treated with comprehensive treatment and antiviral therapy;control group were treated with budesonide suspemion and compound ipratropium bromide inhalated.Clinical efficacy of both two groups was evaluated.Results Cure rate of treatment group was 91%,cure rate of control group was 71%,the clinical therapeutic efficacy were compared between the two groups,the difference of which had statistical significance.Conclusion The result of experiment indicates that the therapeutic efficacy of budesonide and compound ipratropium bromide inhalated in treatment of infant bronchiolitis is better method.

Objective To observe the therapeutic effect of Famotidine combined with Hemocoagulase treating the neonatal stress ulcer complicating upper gastrointestinal hemorrhage. Methods 72 patients of stress ulcer complicating upper gastrointestinal hemorrhage were randomly divided into two groups:study group(35 cases) were treated with Famotidine plus Hemocoagulase;control group(37 cases)were cured by Famontidine only. The different therapeutic effects were compared. Results 35 cases of study group(100%)and 28 cases of control group(75.68%) were cured successfully. Compared the excellent rate of study group and control group,the former was significantly higher than the latter(P＜0.01). Conclusion Famotidine combined with Hemocoagulase treating the neonatal stress ulcer complicating upper gastrointestinal hemorrhage has notable therapeutic effect.

Vitamin D is a steroid hormone that exerts its action by binding to the vitamin D receptor.In addition to its actions on calcium and phosphate metabolism,it exhibits a plethora of regulatory effects on proliferation,dfferentation and function of the cells of the immune system,thus play an important role in the regulation of the immune response.Systemic lupus erythematosus (SLE) is a multisystemic auto-Immune disease that is characterized by a number of autoantibodies.VitD and VDR can inhibit the activation of autoreactive lymphocyte and the production of auto-antibodies,thus play an important role in the development and mechanism of SLE.Supplementation of VitD may improve the progrosis of SLE.

Objective:To develop an HPLC method for the simultaneous determination of oleanic acid and ursolic acid in Salvia plebeia R. Br. from different origins. Methods:The column was Agilent-C18 (250 mm × 4. 6 mm,5μm) with the temperature of 25℃, the mobile phase was methanol-water-glacial acetic acid-triethylamine (90 ∶10 ∶0. 03∶0. 06) at the flow rate of 1. 0 ml·min-1 ,and the detection wavelength was 210 nm. Results: The calibration curves were linear within the range of 7. 50-75. 00 μg · ml-1 for oleanic acid(r=0. 999 7)and 5. 00-50. 00 μg · ml-1 for ursolic acid(r=0. 999 7). The average recovery of oleanic acid and ursolic acid was 100. 15%(RSD=1. 14%,n=6)and 99. 40%(RSD=1. 67%,n=6), respectively. Conclusion: The method is simple and accurate, which is suitable for the determination of oleanic acid and ursolic acid in Salvia plebeia R. Br. from different origins.

The article points out that to improve the teaching quality and level of national medical colleges and universities,the experience of Medical College of Monpillier University,France should be introduced and learned from.Views and suggestions are also put forward to counter the existing problem in national medical teaching system and clinical practices.

Objective:To investigate the effects of adramycin on cell line of FRH-0201.Methods:Human hilar cholangiocarcinona cell line were cultured with adramycin (2?g/ml) through lasting different times,finally this cell line can survive after 72h cultured in the culture liquid with the adramycin(1?g/ml) .Then the medium was replaced with a fresh one without adramycin,and the cells were continuously cultured for 1 month.The in vitro studies included the observation of the appearance with optical microscope,MTT cell proliferation assay,analysis of the cell cycle by Flow cytometry(FACS),assays of tumor marker using en{yme-linked immunosor bent assay(ELISA),the fluorescence density of adramycin.Results:Compared to the parent FRH-0201cell line:The morphology showed typical morphological characteristics,the doubling time prolonged about 3h,the cell cycle by flow cytometry identified in G_1,G_2 and S phase of cell cycle were 40.50%,19.74%and 39.77% in trial group,and 69.83%,7.29%and 22.88% in parent group,respectively.Tumor marker has no difference between them,the fluorescence density of epirubicin descending 5.2 times.Conclusion:The adramycin can affect the cell cycle and cause the change of metabolism.

Objective To study the surgical effect of sylvian lateral fissure approach in the treatment of patients with hypertensive intracerebral hemorrhage on basal ganglia.Methods Sixty-two patients with hypertensive intracerebral hemorrhage on basal ganglia in our hospital from October 2012 to June 2015 were selected,and equally divided into control group and observation group.The control group was treated with temporal cortex approach surgery,and the observation group was treated with sylvian lateral fissure approach surgery.The hematoma clearance rates of different amount of bleeding,and nerve function indexes and ADL score before the treatment and at different time after the treatment of two groups were compared.Results The hematoma clearance rates of observation group with different amount of bleeding were higher than those of control group.The serum nerve function indexes were lower than those of control group.The ADL scores were better than those of control group.The above differences were statistically significant (P＜0.05).Conclusion The clinical effect of sylvian lateral fissure approach in the treatment of patients with hypertensive intracerebral hemorrhage on basal ganglia is better.The approach has an active role in the improvement of the hematoma clearance and nerve function and greater value for the patients with hypertensive intracerebral hemorrhage on basal ganglia.

Objective To investigate the assessment value of serum Lp-PLA2 ,hs-CRP and D-D dimer on the severity of cor-onary artery lesion .Methods A total of 136 patients with coronary heart disease acute myocardial infarction (AMI) group 46 ca-ses ,stable angina pectoris (SAP) group 38 cases and unstable angina pectoris (UAP) group 52 cases from the inpatient of cardiolo-gy department in our hospital were selected as observation group ,and 135 healthy subjects were selected as control group ,serum Lp-PLA2 was detected by enzyme kinetic method ,Serum hs-CRP and plasma D-D dimer were detected by immune turbidimetric method ,of the severity of coronary artery lesion was evaluated by the Gensini integrations .Results Serum Lp-PLA2 ,hs-CRP ,D-D dimer level and Gensini score of observation group were significantly higher than those of control group ,above indexes of AMI group were higher than those of SAP group and UAP group ,UAP group were significantly higher than SAP group ,with statistical significance between the two groups(P<0 .05) .Serum Lp-PLA2 ,hs-CRP ,D-D dimer level and Gensini score of patients with heart functional class Ⅱwere significantly lower than those of Ⅲ and Ⅳ patients ,above indexes of patients with heart functional class Ⅲwere significantly lower than Ⅳ patients ,the difference between two groups was significant(P<0 .05) .Compared with severity of coronary artery lesion ,serum Lp-PLA2 hs-CRP and D-D dimer levels of mild coronary artery lesions were significantly lower than those of moderate and high coronary artery lesions ,and moderate coronary artery lesions was obviously lower than high coronary artery lesions (P<0 .05) ,Pearson correlation analysis also found that ,Lp-PLA2 ,hs-CRP ,D-D dimer levels had positive correlation with Gensini score (r=0 .642 ,0 .651 ,0 .608 ,P<0 .05) .Conclusion The assessment of serum Lp-PLA2 ,hs-CRP and D-D dimer level in the severity of coronary artery lesion has important significance .

Objective:To investigate the injury on isolated cadaver testes induced by ischemia-reperfusion (I/R).Methods:13 isolated cadaver testes contributed by 13 persons were preserved under 0℃～4℃ hypothermia and then reperfused under 37℃.Histology and histochemical changes were observed.Results:4℃ cold ischemia in 18 hours only could induce trivial swelling and vascular degeneration of endothelial cells(ECs).Obvious pathologic changes occurred after 24 hours,including detachment of ECs,separation between basement membrane and seminiferous epitelium,malalignment of spermatogenous cell and edema of mesenchyme.Injury was worse along with the prolongation of cold preservation time.Changes of LDH and SDH activities were found by histochemical staining.Reperfusion following 6 hours ischemia induced tissue injury and unusual enzyme activity.All changes were more obvious after reperfusion following 12,18,24 or 36 hours of cold ischemia.Conclusion:HCA can preserve testes for 18 hours.Testes can't keep normal histological and histochemical structure beyond 24 hours of preservation under 4℃ hypothermia,and 37℃ reperfusion can make injury worse.