To construct a cDNA subtractive library of genes transactivated by hepatitis C virus core protein with suppression subtractive hybridization technique. mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-)-core and pcDNA3 1(-) empty vector,respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNA were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, and then it was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain JM109. The cDNA were sequenced and analyzed in GenBank with Blast search after PCR. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR product showed that 213 clones contained 100~ 1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes before. The full length sequences were obtained with bioinformatics method,which had been accepted by GenBank. It suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein.

To screen anti idiotypic single chain variable fragments(anti Id scFv)against hepatitis C virus NS4A(HCV NS4A)so as to lay a foundation for developing anti Id scFv vaccine againist the hepatitis C virus. The recombinant phage antibody library was panned by hepatitis C virus NS4A monoclonal antibody which was coated in a microwell plate. After five rounds of biopanning, 82 clones specific to HCV NS4A antibody were determined with the enzyme linked immunoadsorbent assay(ELISA). The specificity of anti idiotypic scFv was identified by ELISA and competitive inhibition assay. The DNA sequence of the positive clone was determined. The result showed that HCV NS4A anti Id scFv had a specific combination character with hepatitis C virus NS4A monoclonal antibody. The DNA sequence data showed that the anti Id scFv coding gene included 789 bp. The results suggested that the anti Id scFv fragments to HCV NS4A monoclonal antibody could be successfully selected by recombinant phage antibody library screening technique, which might pave a way for the study of new preventive and therapeutic strategy of hepatitis C using anti Id scFv.

To screen HCV core mimotopes,HCV core monoclonal antibody was used as immobilized molecule,and a 12 mer phage peptide library was biopanned and positive clones were selected by enzyme linked immunoadsorbent assay(ELISA), competitive inhibition assay, and DNA sequencing. 10 positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results,one epitope was confirmed as a mimotope of HCV core protein. The study might provide a new approach for HCV therapy and vaccine development.

Objective To observe the effect of electroacupuncture (EA) on dopaminergic neurons in the mesencephalic substantia nigra(MSN) of rats with Parkinson′s disease (PD). Methods A model of PD was induced in rats by injecting 6-hydroxydopamine (6-OHDA) into the MSN. Acupuncture was then used to treat the model rats. To examine the apoptosis of dopaminergic neurons, the number of tyrosine hydroxylase (TH) positive neurons and the concentration of dopamine (DA) were measured in a normal control group、a sham-operation group、the EA group and the PD group which received no EA. Results In the EA group, markedly less apoptosis of dopaminergic neurons was observed, and the number of TH positive neurons and the concentration of DA were significantly higher. Conclusion Electroacupuncture may mitigate injury to the substantial nigra in Parkinson′s disease.

Objective To observe the effects of electroacupuncture (EA) on c-Jun amino terminal kinase (JNK) signaling pathway and inflammatory cytokines interferon-γ (IFN-γ) in substantia nigra (SN) cells of rotenone-induced rats model of Parkinson's disease (PD),and explore the underlying mechanism of EA on PD.Methods A total of 32 male Sprague-Dawley mice were randomly and evenly divided into a normal group,a shamoperation group,a model group and an EA group.Model group and EA group were injected intradermally with rotenone (1 mg/kg,dissolved in DMSO and saline,concentration:O.25 mg/ml) on the nape of neck.Sham-operation group was injected the same dose of DMSO and saline.Normal group had no intervention.EA group was applied to Fengfu (DU16) and Taichong (LR3) acupoints after the establishment of PD model in rats.Behavioral assessment was conducted after the treatments,the rats were sacrificed for sampling substantia nigra tissue to detect the expressions of tyrosine hydroxylase (TH),p-c-Jun amino terminal kinase (p-c-Jun) and interferon-γ(IFN-γ) protein with Western blotting (WB).Results Model rats showed significant PD syndrome characteristics,comparing with normal group and sham group,the difference was not statistically significant (P ＞ 0.05).The results of open box test showed that the scores of model group rats decreased significantly in terms of the horizontal movement [(19.12 ±2.34) points] and vertical locomotor activity [(5.27 ± 1.04) points] when compared with normal group and sham group,the difference was statistically significant (P ＜ 0.05).After EA treatment,locomotor activity of rats increased significantly when compared with model group (P ＜ 0.05),however,the normal group and sham group was not statistically and significantly different in locomotor activity (P ＞ 0.05).Compared with normal group,the expression of TH protein in (0.183 ± 0.0213) reduced significantly and the expressions of p-c-Jun (0.388 ± 0.0283) and IFN-γ protein(0.453 ± 0.0332) increased significantly in model group,the difference was statistically significant (P ＜0.05).Compared with normal group,the expression of TH protein(0.324 ± 0.0538) reduced and the expressions of p-c-Jun(0.207 ± 0.0592) and IFN-γ protein (0.239 ± 0.0215) increased in EA group,the difference was not statistically significant (P ＞ 0.05).Compared with model group,the expression of TH protein increased significantly in EA group(P ＜ 0.05),the expressions of p-c-Jun and IFN-γ protein reduced significantly in EA group(P ＜ 0.05).Conclusion EA therapy may reduce the expression of IFN-γ protein in SN of PD rats model by regulating the expression of JNK/mitogen-activated protein kinase (MAPK) pathway,which may delay the process of PD.

OBJECTIVE To investigate the management situation of cross infection in outpatient service of stomatology in hospital and clinic in Zhanjiang city and conduct effective measure to control hospital infection.METHODS In our investigation,we selected 105 hospitals and private clinics to inspect their management situation of cross (infection) via dental(instrument).RESULTS The management in the hospitals of the city was quite qualified but the some private dental clinics were terrible.The main problems were as follows: no effective disinfection for dental instrument and the bad environment in procedure room,especially the worse sterilization situation of the dental handpieces.Most of the doctors in private clinics were short of knowledge in the sterilization for dental(instruments.) CONCLUSIONS There is no time to delay for the health administration department to normalize the management of the private dental clinics.It is essential to train the medical staffs,and implement measures for dental instrument disinfection management in private dental clinics.

Objective To identify risk factors associated with the severity of acute kidney injury (AKI) in-duced by crush syndrome and whether the patient required hemodialysis (HD). Method A retrospective study was designed. Within 19 days after the Chinese Wenchuan earthquake (May 12, 2008), 63 victims (33 men, 30women) of 2139 cases were hospitalized at Mianyang Central Hospital (Siehuau, China) because of crush syn-drome caused by crush injuries. The patients with renal dysfimcfion before the earthquake were excluded. Totally 63 patients with AKI associated with crush syndrome were included in this study and were divided into two groups: group 1, 25 patients, requiring HD (when urine volume <250 mL/d;serum potassium> 6 mmol/L) ; and group 2, 38 patients, without HD. The following data were collected retrospectively for all patients: (1) epidemiological parameters: age, gender, race, time under the rubble, liquid treatmem before being rescued; (2) clinical param-eters: blood pressure, body area crushed, amputation, fasciotomy, blood transfusion, quantity of fluid infusion, urine output in the first 24 hours; (3) initial laboratory data: complete blood count, urine analysis, serum chem-istry, arterial blood gas analysis. Comparisons between the two groups were made using SPSS 10.0. The quantita-tive data and categorical data were analyzed using t tests and χ2 tests, respectively. P -values < 0.05 were consid-ered to indicate significant differences. The significant variables were entered into logistic regression models to de-termine the risk factors for the severity of AK1 in patients with crush syndrome and whether the patient required HD. Results Four significant risk factors with P -values < 0.05 were identified: fasciotomy, cystatin C (Cys C)level, myoglobin (MB) level and lactic acidosis. The odds ratios (95% confidence intervals) were 8.641 (3.027～76.479), 6.956 (3.027～76.479), 5.379 (3.027～76.479) and 4.833 (2.569～32.764), respectively. Conclusions In addition to urine output and potassium levels, we found that four risk factors, namely faseiotomy, Cys C and MB levels, and lactic acidosis, were significanfly associated with the severity of AKI and whether the patient required hemodialysis.

Objective To study the genetic characteristrics of vaccine and varicella-zoster virus(VZV)strains isolated from patients with chickenpox or zoster by molecular analysis.Methods SNP based VZV genetic characteristrics were analyzed in 19 VZV isolates using the restriction fragment length polymorphisms analysis of DNA fragments of the open reading frames 6,38,62 and sequence alignment of the open reading frames 1,31,51,62.Results All vaccine strains were revealed Alu Ⅰ~-Pst Ⅰ~-Sma Ⅰ~+ BssH Ⅱ~+ Nae Ⅰ~+,96% clinical isolates were revealed Alu Ⅰ~+ Pst Ⅰ ~+Sma Ⅰ BssHⅡ Nae Ⅰ~-,2% clinical isolates were revealed Alu Ⅰ~-Pst Ⅰ~-Sma Ⅰ~+ BssH Ⅱ~+,2%clinical isolates were revealed Alu Ⅰ~+ Pst Ⅰ~-Sma Ⅰ~-BssHⅡ Nae Ⅰ~-by restriction fragment length polymorphisms analysis and sequence alignment revealed the mutations also presented in this four vaccine strains.Conclusion Use the restriction fragment length polymorphisms analysis of DNA fragments of the open reading,frames 6 and 62 could be distinguished VZV wild-type strains and vaccine strain in clinical isolates in China.In order to find the adverse effect caused by vaccine from certain company's,analysis on the SNPs in ORFs 1,31,51 and 62 is needed.

Objective To express human HT036 protein in Escherichia coli(E.coli.)and identify it.Methods The cDNA sequence obtained by PCR was cloned into the prokaryotic expression plasmid pET30a(+).The target protein was expressed in E.coli..induced by IPTG and analyzed by Western blotting.Results The interest gene was identified by restriction endonucleases digestion and DNA sequencing.The protein was highly expressed in E.coli..Conclusion We successfully expressed the HT036 protein.

Objective To assess the intraoperative efficacy of contrast-enhanced ultrasonography (CEUS) in the radiofrequency ablation (RFA) of renal tumors.Methods RFA was performed on 110 renal tumors (size range:1.5-8.6 cm,49 located in the left,55 right and 3 bilateral) in 107 selected patients.Conventional tumor biopsy was conducted for pathological diagnosis using 18G biopsy needle after RFA.CEUS was performed in all patients before,during and after RFA to assess the necrotic area.Once suspicious residual lession was discovered by CEUS,another round of RFA was taken immediately.Initial follow-up enhanced CT and CEUS was performed seven days after the procedure,with subsequent CEUS and enhanced CT after one month,three months,and six months to assess the necrotic area.Technical success was defined as elimination of areas that enhanced at imaging within the entire tumor.Results With CEUS performed during RFA,110 tumors (100％) were successfully ablated during one operation.Of these,five tumors was subjected to additional RFA treatment for the suspicious residual lesion detected by CEUS right after initial RFA.Elimination of areas covered the entire tumor after the second RFA.No residual or recurrence tumors was confirmed by both enhanced CT and CEUS during follow-up.All patients survived in the follow-up phase which ranged from 3 to 18 months(mean 11 months).Conclusions With the application of intraoperative CEUS,there were less residual tumors after RFA.