Objective To explore a new simpler method for the preparation of recombinant alpha virus as a novel vaccine at the DNA level. Methods Plasmids expressing ? gal protein and helper plasmids were transfected into BHK cells. Virus in culture supernatant of the transfected BHK cells were collected and purified and used to infect BHK cells in vitro to identify the expression of target gene and the titre of the recombinant virus. Results Recombinant virus with high titre, prepared by this method, could be expressed well in mammalian cells in vitro . Conclusion High titre recombinant alpha virus can be produced at the DNA level and this method can be applied for vaccine preparation and gene therapy.

Objective To investigate the biofilm formation,the alginate biosynthetic gene ex- pression and analyze the mucA gene sequence of mucoid Pseudomonas aeruginosa PA17 and nonmu- cold Pseudomonas aeruginosa PA01.Methods The modified plate culture method was used to estab lish the biofilm model in vitro.Semi-quantitative RT-PCR was used to determine the expression level of algD in planktonic condition and during the formation of biofilm.The mucA gene of PA17 and PA01 was amplified and the products were sequenced.Results PA17 biofilm was mature at 6th day, and PA01 biofilm was mature at 3rd day.The structures of the biofilms were both like pellicle.In planktonic condition,the algD expression of PAl7 was higher than PA01;in biofilm formation,the algD expression was maximal when the biofilm was mature.There was a 166～333 deletion mutation and 342A→G in mueA gene of PA17,the mucA gene of PA01 was the same with the sequence of Genbank.Conclusions The mucA gene mutation of PA17 was a new type,which maybe the reason for the little expression difference of algD between PA17 and PA01 during the biofilm formation than it in planctonic condition and the same structure of PA17 and PA01 biofilm.

Objective To investigate the effect of fluoride on expression of Runx2 mRNA and protein in bone tissue of rats. Methods Fourteen SD rats were randomly divided into two groups: control group(tap water with fluoride ＜ 0.06 mg/L), and fluorosis group(fluoride 50 mg/L in water). After 4 moths, expressions of both mRNA and protein of Runx2 in rat bone tissue were determined by RT-PCR and Western blotting. Results The results showed that the expression of Runx2 mRNA and protein in fluoride-treated bone tissue were 2.287 ± 0.261 and 0.929 ± 0.229, respectively, both of which were significantly higher than those of control group(0.995 ± 0.123,0.317 ± 0.068, t = 11.85,6.78, P ＜ 0.05). Conclusions Fluoride can increase the expression of Runx2 mRNA and protein in bone tissue of rats, and Runx2 may be involved in the pathogenesis of bone injury caused by fluoride.

Objective To study the inhibitory effects of stachyose on the hyperacute rejection in pig-to-human heart xenotransplantation.Methods A pig-to-human xenogeneic heart transplantation model was established based on an in vitro free heart blood perfusion system.The pig hearts were di- vided into two groups:group A(pig hearts treated with human blood perfusion as control)and group B(pig hearts treated with human blood plus stachyose perfusion).After perfusion for 1h,the heart xenografts were examined for deposit of lgG and IgM by immunohistochemistry and pathological analy sis.Results The mean survival time of perfusion hearts in groups A and B was(9.5?2.5)min and (46.8?8.1)min respectively(P

Objective To study the effect of cyclophosphamide pulse therapy combined with leflunomide (LEF) on refractory lupus nephropathy (RLN).Methods Thirty-six patients diagnosed as RLN were selected.Several observation parameters were compared before and after cyclophosphamide pulse therapy combined with LEF.The observation parameters included serum albumin,serum complement C3 and C4,systemic lupus erythematosus (SLE) activity index and urinary protein change.The side effects of cyclophosphamide pulse therapy combined with LEF were observed.Results The serum albumin was (20.17 ± 4.09) g/L,complement C3 was (0.40 ± 0.19) g/L,complement C4 was (0.08 ± 0.03) g/L,SLE activity index was 16.06 ± 4.17,and urinary protein was ( 9.79 ± 3.42 ) g/24 h before treatment and (38.10 ± 5.16) g/L,(0.78 ± 0.11 ) g/L,(0.16 ± 0.13)g/L,4.01 ± 1.24,( 1.14 ± 0.59) g/24 h after treatment,and there were significant differences between before treatment and after treatment (P ＜ 0.01 ).During the therapy,side effects were reported in 22 patients.However,these side effects had no impact on the therapy.After further treatment,these side effects gradually decreased and eventually disappeared.Conclusions Cyclophosphamide pulse therapy combined with LEF is effective in treating RLN.Patients' tolerance to the therapy is generally very good.

Objective:To investigate the expressions and their clinical significance of vascular endothelial growth factor (VEGF) and extracellular matrix (ECM) components in non-small cell lung cancer (NSCLC). Methods:Expressions of VEGF and ECM components (fibronectin, FN and collagen Ⅳ, cⅣ) in 50 cases of NSCLC tissues and 20 cases of normal lung tissues were detected by immunohistological analysis. Their relationship with clinical features of NSCLC and the correlation of expression of VEGF and Fn and cⅣ were analyzed.Results:The positive expression rates of VEGF, Fn, and cⅣ were 96%, 78%, and 50% in NSCLC tissues. The expressions of VEGF and Fn were significantly higher than those in normal lung tissues (P<0.05). The expression of Fn and over-expression of VEGF were associated with lymph node metastasis (r=1.00, P<0.001). The survival rate of patients with over-expression of VEGF was greatly lower than that with weak expression of VEGF (P=0.022). The survival rate of Fn-negative patients was markedly higher than that of Fn-positive patients (P=0.046). Conclusion:VEGF and ECM component Fn were highly expressed in NSCLC, which correlated with lymph node metastasis and survival rate. Expression of ECM and VEGF had positive correlations, suggesting that ECM might be one of the anti-angiogenesis targets for tumor therapy.

Objective:To make a prospective study on the effectiveness and safety of toremifene (TOR) combined with novelbine/cisplatin (NP) in the treatment of patients with advanced non-small cell lung cancer (NSCLC) whose first line platinum-based chemotherapy was failure. Methods:Forty-four patients with stage ⅡB-Ⅳ NSCLC, who failed in the first line cisplatin-based chemotherapy from January 2004 to February 2006, were enrolled in this study. All the patients received TOR combined with NP second line chemotherapy for two cycles. The response rate and adverse reaction were evaluated. The survival rate was analyzed.Results:The 44 patients received average 1.8 cycles of chemotherapy (1-3 cycles). The response of 37 patients could be evaluated including 21 patients who received NP regimen before and 16 patients who received platinum-based chemotherapy. After second line therapy, 4 of the 37 patients had partial response (PR), 19 had stable disease (SD), 14 had progressive disease (PD), and no patient had complete response (CR). The total response rate (CR+PR) was 10.8% (4/37). The disease-controlling rate (CR+PR+SD) was 62.2% (23/37). The response rate and disease-controlling rate of squamous cell lung cancer (SCC) were 27.3% (3/12) and 72.7% (8/12), which were significantly higher than adenocarcinoma [0% (0/18) and 44.4% (8/18), P<0.05]. The median survival time was 8.2 months, the median time for SD was 4.0 months (1.0-10.2 months), and the 1-year survival rate was 24.4%. The median survival time and 1-year survival rate of SCC patients had no significant difference compared with adenocarcinoma patients (9.2 vs 7.1 months; 33.3% vs 27.7%, P＝0.72). There was no significant difference in survival rate between male and female patients. One patient stopped therapy for liver function injury (hyperbilirubinemia). The adverse reactions induced by chemotherapy mainly included gastrointestinal reaction, bone marrow suppression, and liver function injury. No serious adverse reaction occurred. Conclusion:The clinical efficacy of second line TOR combined with NP regimen is similar with the first line regimen for NSCLC patients, especially for SCC patients. The frequency of adverse reaction is not increased.

Objective To observe the efficiency of immunotherapy on allergic rhinitis and explore irs correla- tive factors.Methods 185 patients with AR were treated with immunotherapy in this department between 1993 and 2003.Specific allergens were selected through skin test.The patients were given subcutaneous injections with al- lergen extract produced by Xiehe Hospital.The doses were gradually increased to the maintenance dose.The clinical effectiveness evaluation based on the symptoms and signs before and after immunotherapy was judged as marked, moderated and poor response.The possihie factors which affected the efficacy of immunotherapy were selected.These factors included sex,age,illness duration,disease condition,chronic sinusitis,asthma,duration of immunotherapy and reaction condition of the skin test.The correlative factors with efficacy were sifted with multifactorial stepwise re- gression analysis.Results The marked,moderated and poor responses were 22.9 %,51.7 %,and 25.4 % respective- ly.The efficacy of immunotherapy correlated well with the duration of immunotherapy(P

Objective To detect the genetic transcription and protein expression level of endoplasmic reticulum aminopeptidase 1 (ERAP1) in human ovarian cancer cells and tissue and study their relationship with directional lymphatic metastasis. Methods Real-time fluorescent quantitative PCR and western blot were used to determine the expressions of ERAP1 gene and protein in the human ovarian cancer cell lines between non-directional (SKOV3) and directional highly lymphatic metastasis (SKOV3-pm2, SKOV3-pm3, SKOV3-pm4 ). Immunohistochemistry method was used to further validate the ERAP1 expressions of the transplanted ovarian tumor primarily focus and the lesions of lymph node metastasis from nude mice and the human ovarian cancer primarily focus and the lesions of lymph node metastasis. Results The expression level of ERAP1 gene and protein were down-regulated in SKOV3, SKOV3-pm2, SKOV3-pro3, SKOV3-pm4 cell sublines (0.118±0.012, 0.031±0.003,0.028±0.003, 0.016±0.005 ; 0.91± 0.33, 0.09±0.03, 0.10±0.04, 0.05+0.04; respectively), and the level of ERAP1 in SKOV3 cell was higher than those in the other three kinds of cell lines (P<0.05 ). The results showed that there were significant declining trend of expression of ERAP1 in the human ovarian cancer cell lines between non-directional and directional highly lymphatic metastasis; the transplanted ovarian tumor primarily focus and the metastasis lesions of lymph node from nude mice (143±22 vs. 97±12, P<0.05), the primarily focus (184±14) and the lesions of lymph node metastasis from human ovarian tumors (P<0.05). Conclusion The absence or down-regulated expression of ERAP1 is closely related to the metastasis and invasion of lymph node in ovarian carcinoma.

Background N-ethyl-N-nitrosourea (ENU)-induced mouse mutagenesis is a powerful approach for the study of gene function and the generation of human disease models.Objective This study was to create the corneal morphologic change and map the mutant gene of a kind of corneal opacity in ENU mutagenesis in mouse.Methods ENU was intraperitoneally injected in forty C57BL/ 6J (B6) male mice aged 8-10 weeks old.The male mice were mated with the same strain female mice.Their progenies were screened for visible eye mutation,and the mutant mice were mated with the same strain mice to confirm the heredity of mutation phenotypes.Hematoxylin & eosin staining was used to examine the histopathological change of cornea in one mouse with ENU-induced corneal opacity.To map the mutant gene,[(B6×D2)F1 ×B6] N2 mutant mice were bred,and the genome of the N2 mice was scanned by microsatellite markers distributed equally on the mouse chromosome.The microsatellite linked to the mutant gene was determined by the log odds score.This experimental procedure was approved by Ethic Committee about Experimental Animal Care and Use of Yangzhou University.Results The founder mouse,which was the progeny of an ENU-treated B6 male mouse and an untreated B6 female mouse,had a corneal opacity phenotype.After mating the mutant with B6 mice,19 of 59 descendants appeared corneal opacity phenotype.Thickening of corneal stroma,neoangiogenesis,infiltration of inflammatory cells and proliferation of fibroblasts were exhibited in cloudy cornea in ENU-induced mutated mice under the optical microscope.After linkage analysis between microsatellite markers and the mutant gene,the mutant gene was linked to D2Mi307,which was located at 63.42 cM.Three cases of 26 N2 mice underwent recombination with the LOD 3.79.The mutant gene associated with the cornea phenotype was located on chromosome 2.Conclusions This study map the mutant gene associated with the cornea phenotype on chromosome 2.The strain might be used as a mouse model for heritable human corneal opacity.