Objective　To study the similarities and differences on in vitro replication and expression of hepatitis C virus (HCV) between human fetal hepatocytes (HFH) and 7721 cell line. Methods　Human fetal hepatocytes and a hepatoma cell line 7721 were incubated with a serum from hepatitis C patient. After incubation, the presence of HCV RNA, the expression of HCV NS3 antigens in cells and/or supernatant were examined by RT-PCR, in situ hybridization and immunohistochemistry, respectively. Results　It was found that: ①The intracellular HCV RNA was first detected on d 2～3 post-incubation and then could be intermittently detected in cells and/or supernatant subsequently (HCV RNA could be detected in 7721 cells during a period of at least 66 days. In HFH, HCV RNA could be detected up to 25 days after incubation); ②HCV-NS3 antigen could be expressed in infected cells; ③Minus-strand RNA of HCV was mainly located within cytoplasm by in situ hybridization. Conclusion　The results suggest that both the fetal hpatocytes and the hepatoma cell line 7721 are susceptible to HCV, and especially 7721 cell line can stably support HCV replication in vitro and may be used as the target cell for long-term cultures of HCV.

ObjectiveTo explore the clinical effect of levofloxacin used on patients with HAP. Methods134 cases of patients with HAP as studied object were randomly divided into observation group and control group,observation group were given therapy of levofloxacin,and control group were given therapy of azithromycin. ResultsThe efficiency cases,effective cases,vain cases in observation group were 51,13 and 3,the efficiency rate and total effective rate were 75.0％ and 93.3％, the data in control group were 38,17,12,51.7 ％ and 81.7 ％. The efficiency rate ( x2 =4. 818, P ＜ 0.05 )and total effective rate( x2 =4. 804 ,P ＜ 0.05 ) of observation group was significantly better than that of control group. ConclusionLevofloxacin used on patients with HAP, could acquire good clinical effect,and the side-reactions were light.

The epidemic of rabies has rapidly increased and expanded in Yunnan province in recent years.In order to further analyze and understand the etiological reasons for the rapid expansion of rabies in Yunnan,a strain of rabies virus CYN1009D in Yunnan was isolated,and the complete genomic sequencing was carried out,and the bioimfomative analysis on genes/encoded proteins and phylogeny with reference to sequences in GenBank was performed.The complete genome of CYN1009D was 11923 nt in length and belonged to genotype I.The genes encoding different structural proteins were all conserved in their lengths,in comparison to other strains in China.The amino acid sequence was conserved at different antigen sites of NP,but the variation was detected at the secondary phosphorylation site of position 375; variations were also detected in the phosphorylation sites at positions 63-63 and 162 of PP; the sites playing important roles in virus synthesis,budding and viral morphology in MP were conserved; two glycosylation sites were detected at Asn37 and Asn319in GP,the neutralizing antigen sites in GP were conserved; the initial amino acid of LP (ML) was different from that of most of the strains in China (MM); the variations in G-L region in the intergenic region were significant.The phylogenic tree showed that CYN1009D has a closer genetic relationship to the strains in Southeast Asia,indicating that prevention and control on rabies in borderland areas should be reinforced meanwhile efforts are made to control rabies in China.

In recent years (2007 to 2011),although the overall number of rabies cases in China has decreased,there is evidence of emerging or re-emerging cases in regions without previous rabies cases or with low incidence of rabies.To investigate the origin and the factors affecting the spread of rabies in China,specimens were collected from 2007 to 2011 from provinces with emerging and re-emerging cases and tested for the presence of the rabies virus.Positive specimens were combined with sequences from GenBank to perform comparisons of homology and functional sites,and to carry out phylogenetic analyses.Out of these regions,five provinces had 9positive specimens from canine and cattle,and 34 canine or human specimens were obtained from previously high-incidence provinces.Complete sequences of G gene were obtained for these samples.Homology of the sequences of these 43 specimens was 87％-100％ at the nucleotide level and 93.7％ -100％ at the amino acid level.These G gene sequences were combined with reference sequence from GenBank and used to construct a phylogenetic tree.The results showed that 43 specimens were all assigned to China clade I and clade Ⅱ,with all specimens from emerging and re-emerging areas placed within clade I.Specimens isolated from Shanxi and Inner Mongolia in 2011 were distinct from previously-isolated local strains and had closer homology to strains from Hebei,Beijing and Tianjin whereas new isolates from Shanghai were tightly clustered with strains isolated in the 1990s.Finally,Shaanxi isolates were clustered with strains from adjacent Sichuan.Our results suggest that the rabies cases in emerging and re-emerging areas in China in the last 5 years are a consequence of the epidemic spreading from of neighboring provinces and regions experiencing a serious epidemic of rabies.

A series of phthalazine ketone compounds were synthesized and the structures were confirmed by H NMR and HR-MS spectrum. All target compounds were obtained through 7 steps, including selective reduction, nitration, bromination, ring enlargement, reduction, Knoevenagel and acylated reaction. The compounds were evaluated for their immunosuppressive effects of T-cell proliferation and inhibitory activity of IMPDH type II in vitro, as well as their structure-activity relationship were assessed. Several compounds exhibited strong immunosuppressive properties, especially compounds 7f and 7h, with IC50 values of 0.093 micromol x L(-1) and 0.14 micromol x L(-1) respectively, which were superior to mycophenolic acid. The information obtained from the studies may be useful for further research on the immunosuppressive agents.

Objective To investigate the killing effect of polymethylmethacrylate (PMMA) on spinal metastasis of transplanted VX2 carcinoma in experimental rabbit models. Methods Spinal metastasis of transplanted VX2 carcinoma model was successfully established in 18 rabbits. The experimental rabbits were randomly and equally divided into three groups with 6 rabbits in each group. Under CT guidance , PMMA or saline was injected into the center of VX2 tumor; in group A 0.3 ml of PMMA was used, in group B 0.1 ml of PMMA was used and in group C (control group) 0.3 ml saline was used. Twenty-four hours after the injection, the animals were sacrificed. Four tissue samples were obtained from the sites at 1 mm , 5 mm, 10 mm and 15 mm away from the PMMA mass in each rabbit of group A and group B , while four tissue samples were collected from different four sites from the tumor ’s center to border in each rabbit of group C. TdT-mediated dUTP nick-end labeling (TUNEL) method was used to determine the tumor cell apoptosis rate. Results After successful establishment of rabbit model, injection of PMMA was performed in sixteen among the eighteen rabbits. Technical success rates were 83.3% in both group A and B, and the success rate was 100% in group C. The difference in technical success rate was not significant. The mean tumor cell apoptosis rates of spinal VX2 carcinoma at 1 mm, 5 mm and 10 mm away from the PMMA mass in group A were (65.75±18.81)%, (50.00±14.24)% and(14.95±8.98)% respectively. The mean apoptosis rate in the control group was (9.79 ±5.24)%; the differences between the group A and the control group were statistically significant (P<0.05). The mean tumor cell apoptosis rate of spinal VX2 carcinoma at 15 mm away from the PMMA mass in group A was (10.30 ±8.13)%, which was not significantly different with that of the control group. The mean tumor cell apoptosis rates of spinal VX2 carcinoma at 1 mm and 5 mm away from the PMMA mass in group B were (49.20±15.57)% and(17.75±9.28)% respectively, which was significantly different with that of the control group(P<0.05); the mean tumor cell apoptosis rates at 10 mm and 15 mm away from the PMMA mass in group B were not significantly different with those of the control group. Statistically significant differences in the mean tumor cell apoptosis rates determined at 1 mm, 5 mm and 10 mm away from the PMMA mass existed between group A and group B(P<0.001). Conclusion PMMA can promote the apoptosis of tumor cells, properly increasing the injected amount of PMMA can enlarge the extent of tumor cell apoptosis.

AIM To determine the contents of polymethoxylated flavones in Citri Reticulatae Pericarpium from eleven cultivar origins.METHODS HPLC was applied to screening extraction solvents (methanol,anhydrous ethanol,95％ ethanol,ethyl acetate and petroleum ether).Ultraviolet-visible spectrophotometry was adopted in the content determination of polymethoxylated flavones in eighteen batches of samples,nobiletin was taken as a reference substance,and the detection wavelength was set at 330 nm.RESULTS Ethyl acetate exhibited the best extraction effect,so it was selected as extraction solvent.Nobiletin showed a good linear relationship within the range of 3.024-13.104 μg/mL (R2 =0.999 8),whose average recovery was 101.37％ with the RSD of 1.34％.The contents of polymethoxylated flavones had obvious differences among samples from different cultivar origins,which were relatively high from Citrus reticulata ‘ Chachi’,Citrus reticulata ‘ Dahongpao’,Citrus reticulata ‘ Tangerina’,Citrus reticulata ‘ Ponkan’,Citrus reticulata ‘ Kinokuni’and Citrus reticulata ‘ Shiyueju’(more than 0.4％).CONCLUSION This simple and accurate method can effectively eliminate the interference of another flavonoids,which is suitable for the content determination of polymethoxylated flavones in Citri Reticulatae Pericarpium.

Objective To evaluate the diagnostic performance of Xpert MTB/RIF for the diagnosis of tuberculous meningitis (TBM).Methods This was a prospective, single center clinical trial.A total of 116 consecutive patients with suspected meningitis who were admitted to Xijing Hospital from October 2013 to June 2015 were recruited.Mycobacterium tuberculosis ( MTB) and rifampicin ( RIF) resistance mutations in 1 ml cerebrospinal fluid ( CSF) were detected with Xpert MTB/RIF and the remaining sample was tested by Ziehl-Neelsen staining , MGIT960 liquid culture and other laboratory tests .And the enrolled patients were grouped according to the 2010 South African expert consensus .The diagnostic performance of Xpert MTB/RIF was evaluated by comparing against clinical score >5 points and MGIT960 liquid culture as reference standards respectively .The comparison was performed using a χ2 test or Fisher′s exact test for categorical variables and a nonparametric rank sum test for continuous variables .Results Among the enrolled 116 subjects, 23 subjects were diagnosed as definite-TBM by MGIT960 liquid culture, 16 subjects were classified as probable TBM , 27 subjects were classified as possible TBM , and 50 subjects were classified as non-TBM.When clinical score >5 points was used as a reference standard , the sensitivity of Xpert MTB/RIF (39.4%) was comparable with that of MGIT960 liquid culture (34.8%) (χ2 =0.292, P=0.589), and significantly better than that of Ziehl-Neelsen staining (9.9%) (χ2 =16.500, 12.771, P<0.001). No significant differences were found among the specificities of Xpert MTB /RIF, MGIT960 liquid culture and Ziehl-Neelsen staining ( 98.0%, 100.0% vs 98.0%, χ2 =1.014, P=0.602 ) .When tested against MGIT960 liquid culture as a reference standard , the sensitivity of Xpert MTB/RIF was 91.3%. Conclusions Xpert MTB/RIF is a rapid and specific method to detect MTB and RIF resistance in CSF .It exhibits a good rule in value for the diagnosis of TBM and a comparable sensitivity with MGIT 960 liquid culture, thus it can be used as the initial method for the diagnosis of tuberculous meningitis .

Objective To investigate the treatment effect of proximal femoral nail antirotation (PFNA) on unstable femoral intertrochanterie fractures in the elderly. Methods A retrospective stud- y was done on 65 patients with unstable femoral intertrochanteric fractures, who were treated with PFNA from December 2005 to October 2006 and followed up for at least half a year. There were 22 males and 43 females, at age range of 67-97 (mean 78.5 years). All the operations were carried out within a week af- ter fractures. The data of operation time, operative blood loss, complications, bone healing time and hip function scores were recorded for evaluating treatment outcomes. Results All the patients were fol- lowed up for 6-18 months (average 10.5 months). The operation lasted for (29.6±7.5) minutes, with mean blood loss of (150.5±40.2) ml and bone healing time of (9.1±2.2) weeks. All patients ob- tained satisfactory treatment results, except for 3 patients with proximal thigh pain, 2 with pulmonary in- fection occurred and 1 with thrombs of lower limb vein. Harris post trauma hip function score was (88.5 ± 6.3) points. Conclusion PFNA is an effective way for femoral unstable intertrochanteric fractures in the elderly, for it is characterized by short operation time, less blood loss and stable fixation.

Objective To perform molecular cloning and sequencing, bioinformatics analysis,protein expression and function of extracellular signal regulated kinase (EgERK1) of Echinococcus granulosus in Xinjiang. Methods The specific primers of EgERK1 were designed and total RNA was extracted from Echinococcus granulosus in Xinjiang. EgERK1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and prokaryotic expression plasmid pET28a-EgERK1 was constructed and sequenced. The sequences were analyzed by DNA sequencing and bioinformatics technology. The recombinant EgERK1 protein was induced and expressed. The biological function was detected using sodium dodecyl sulfate polyacrylamide gel electropheresis and Western blot. Results The sequence of RT-PCR product was 1125 bp, encoding 374 amino acids with isoelectric point of 6.34.This gene was a new ERK-homologues gene indicated by BLAST, named EgERK1(EU701008).Homology comparisons indicated that the homology of EgERK1 and EmMPK1from Echinococcus multilocularis was 95.45%, and was 43.04%-61.88% to ERK from Caenorhabditis elegans, S. cerevisiae, D. melanogaster and human. Phylogenetic analysis showed that EgERK1 clustered with EmMPK1. Bioinformatics analysis predicted that EgERK1 contained a highly conserved T-X-Y motif and activation loop segment of ERK-like kinase.Western blot results showed the EgERK1 recombinant protein could reacted specifically with anti-human ERK monoclonal antibody. Conclusion A new EgERK1 gene of Echinococcus granulosus is successfully cloned and its recombinant protein could reacted specifically with ERK1/2 antibody, which provides the basis for further study of EgERK1 function in the host-parasite interaction.