Objective To observe the effect of theanine in vitro and in vivo, including suppression of lung tumor growth, tumor an-giogenesis and promotion of apoptosis. Methods The inhibitory effects of theanie on lung cancer A549 cells were analyzed by MTT assays. The cell cycle and the apeptotic percentage of A549 cells were detected by FCM. The angiogenesis effect of theanine was observed with CAM model. The inhibitory effects of theanine were observed with lung carcinoma nude mice model, and the immunohistochemic technique was used to investigate the expression of CD34 and VEGF (vascular endothelial growth factor). Results Treatment of A549 cells with VEGF re-sulted in significant dose-dependent and time-dependent inhibition. FCM detection indicated that administration of EGCG resulted in an in-crease in cells in the S phase of the cell cycle and a typical apoptosis peak before the GI phase with an apoptosis rate of 15.9%. There was a significant difference in tumor volume and weight in theanine group compared with control group after two-week treatment, and the tumor in-hibition rate was 43.6%. There was no significant difference in expression of VEGF in tumor tissue according to tumor MVD marked by CD34 between the theanie group and control group. Thus, theanine could obviously inhibit tumor angiogenesis. Conclusion VEGF can ar-rest lung tumor growth via the promotion of tumor cell apoptosis and the inhibition of tumor angiogenesis.

Objective To investigate the incidence of Helicobacter pylori in teenagers and analyze the drug resistance of isolated H.pylori strains.Methods Gastric mucosa samples were harvested from 108 young patients by gastroscopic biopsy.The Kirby-Baner test was performed to test the drug susceptibility of isolated H.pylori strains.Results Thirty-six strains of H.pylori were isolated from the gastric mucosa of 108 patients(33.3%).Seven of the 36 H.pylori strains were originated from the patients aged 6-10 years,the isolation rate in this age group was 16.7%;4 strains were isolated from the patients aged 10-14 years,the isolation rate in this age group was 31.3%;26 strains were from the age group of 14-18 years with the isolation rate of 48.1%.The isolation rates of H.pylori were significantly different among the there age groups(P0.05).The drug susceptibility test indicated that the drug-resistant rate of the 36 H.pylori strains to clarithromycin,amoxicillin,gentamicin,fruranzolidone,metronidazole and levofloxacin was 8.3%(3/36),33.3%(12/36),0%(0/36),16.7%(6/36),94.4%(34/36) and 0%(0/36),respectively.Conclusions The frequency of H.pylori infection may incidence with the increase in age.For the isolated H.pylori strains in present study,the drug-resistance to metronidazole and amoxicillin is higher,to clarithromycin and fruranzolidone is lower,and no drug-resistance is found to gentamicin and levofloxacin.

By combining clinical observation and animal experiments and using FASCO - 3010 hemorheological devices to examine the relevant indices, it was found that no obvious changes can be found in the group treated with hormone before and after the treatment, whereas in the group with traditional drugs added, the RBC deformation, and specific viscosity of plasma were all markedly lowered. Comparison of rheological changes in SD rat showed that traditional Chinese drugs can markedly improve the hemorheology in such patients, indicating that these TCM treating principles can improve the concentrated, viscous, coagulated, agglutinated pathological blood status in SLE patients.

Objective To investigate the effects and distribution of 4 efflux pumps and correlated mutation of regulatory gene in multi-drug resistance Pseudomonas aeruginosa (Pa) in Hunan province. Methods Forty non-duplicated clinical strains of multiple-drug-resistant Pseudomonas aeruginosa were collected in Hunan in 2008, then the phenotype was screened with efflux pumps inhibitor phenylalanine-L-β-naphthylamide and 4 antimicrobial susceptibility test discs. Genes of the efflux pump membrane fusion protein were amplified by PCR, and correlated efflux pump regulatory genes were amplified and sequenced to analyze the role of efflux pump gene expression in multi-drug resistance compared to that of 18 strains with non-multi-drug resistance. Re-suits The positivity rates of MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM were 45.0% (18/40), 30.0% (12/40), 42.5% (17/40) and 12.5% (5/40) respectively with phenotype screening in multi-drug resistance group. The positivity rates of mexA, mexC, mexE and mexX were 100% (58/58), 22.5% (9/40), 45.0% (18/40) and 22.5% (9/40) with RT-PCR. The overexpressed positivity rates of mexA and mexX were 55.0% (22/40) and 22.5% (9/40) respectively by semi-quantitative analysis with real-time PCR. However, no over-expression of mexA and mexX in non-multi-drug resistance group with real-time PCR. The positive expression rates of mexC and mexE with RT-PCR were 11.1% (2/18), 38.9% (7/18) in non-multi-drug resistance group. The difference of overexpression of mexA and mexX was significant(P<0.001, P=0.045) between two groups. The strain Pa20 with mexA overexpressian displayed gene mutations in mexR(164GTC→GAG) and amino acid substitution(126Val→Glu), the strain Pa34 with overexpression of mexX had 164GCG→GAG,55Ala→GIu. Conclusion Most of Pa with multi-drug resistance contained the resistant mechanism of efflux pumps and most-ly due to the overexpression of MexAB-OprM and MexXY-OprM in Hunan. There are mostly regulatory genes mutation in the strains with overexpression of MexAB-OprM and MexXY-OprM.

Objective To reveal the impact of anti-lung cancer A549 by theanine, which may regulate T lymphocytes through dendritic cells (DCs), and to observe the immune protection of theanine on SCID mice transplanted tumors. Methods SCID mice were reconstructed after immunization, then A549 cells were inoculated. The immune protection and immune treatment of theanine stimulated DCs were observed. Results In the theanine-stimulated DC group, as well as DC group, the time needed to form tumor were significantly prolonged (F = 29. 5, P ＜ 0. 01). And the transplanted tumors were smallest (F =69. 51, P ＜0. 01) and lightest (F = 190. 9, P ＜0. 01) in the theanine-stimulated DC group. 50 days after tumor-bearing, the transplanted tumors in the theanine-stimulated DC group were smaller than in the control group, and the surface were smoother with no adhesions. Under light microscope, a large thin slice necrosis was showed in the theanine-stimulated DC group, and the VEGF expression was also reduced (F = 31.4, P ＜ 0. 01). Conclusions The anti-tumor immune protection and treatment mechanism of theanine-stimulated DCs were possibly through regulation of T cells, as well as the VEGF expression reducing,thereby inhibited tumor blood vessel formation.

ObjectiveTo investigate the prognostic factors of non-small cell lung cancer (NSCLC) after resection.MethodsClinical data of 131 NSCLC patients who underwent resection were reviewed and divided into chemotherapy group(86 cases) and non-chemotherapy group(45 cases) according to the treatment method.Survival rate was calculated by Kaplan-Meier method.The prognosis was analyzed by Cox proportional hazards model.ResultsThe median survival time (MST) of squamous cell carcinoma (76 cases),bronchial alveolar cell carcinoma( 8 cases),adenocarcinoma( 35 cases ),adenosquamous carcinoma (12 cases) was 60,54,34,24 months respectively (P＜0.05).For the patients of stage Ⅰ B,the MST of chemotherapy group and non-chemotherapy group was 75 and 76 months respectively(P ＞ 0.05 ).Multivariate analysis showed that tumor size,T stage,N stage,chemotherapy were independent prognostic factors (P =0.080,0.002,0.000,0.029).Conclusions Squamous cell carcinoma and bronchial alveolar cell carcinoma have better prognosis than adenocarcinoma,adenosquamous carcinoma.For the patients of stage Ⅰ B,the survival time can't be prolonged through platinum-based chemotherapy.Tumor size,T stage,N stage,chemotherapy are independent prognostic factors.

Objective:Preparation and immune characteristic analysis of polyclonal antibody against hypervariable region protein of Taura syndrome virus major capsid protein VP 1 as a reference for studies on immunological diagnosis reagent.Methods:The recombinant vector pET-VP1 was transformed into E.coli BL21 for protein expression.Immunizing a New Zealand rabbit with purified VP1 protein,the titer of anti-VP1 serum was determined by Agar diffusion test and ELISA.Monoclonal phage specific binding to the purified VP1 protein was used for competitive inhibition test.Results: The VP1 protein was soluble and high expression in E.coli BL21.The biological activity titer of anti-VP1 serum reached 1∶26 ,1∶217 determined by Agar diffusion test and ELISA respectively.A litter binding activity of antiserum and VP 1 protein could be blocked by monoclonal phage , but would not affect the final positive result.Conclusion:High titer antibody Preparation of the VP 1 hypervariable region protein.The binding activity of the polyclonal antibody with VP1 protein was not affected by the mutations of VP 1 protein in minority areas ,so the antiserum could be used as immu-nological detection diagnosis agent.

Objective To investigate effects of antioxidant stress protein hem e oxygenase-1 (HO-1) on lipopolysaccharide (LPS)-induced endoplasm ic reticulum stress (ERS) of rat hepatocytes. Methods The BRL cells (rat hepatocyte cell line) were cultured. The hepatocytes were treated with LPS, LPS+HO-1 si RNA , HO-1 siRNA and PB S solution, respectively. The cell viability was m easured by trypan blue ex-clusion test. The apoptosis cells were detected by the fluorescent dye Hoechst 33258. E xpressions of GR P78, C HO P, caspase-12 and HO-1 were detected by Western blotting. Results LPS caused an in-crease of HO-1 protein expression of rat hepatocytes in a dose-dependent and tim e-dependent m anner, a up-regulation of GRP78, CHO P and caspase-12, a decrease in cellviability,and an increase in apopto-sis rate of hepatocytes. Pretreatm ent of HO-1 siRNA inhibited the up-regulation of LPS-induced HO-1, however, aggravated ERS and cellular injury. Conclusion HO-1 inhibites ERS-m ediated cellular injury of rat hepatocytes induced by LPS.

BACKGROUNDThere is still disadvantage in animal model for lung cancer study, no matter what is xenograft nude mice or rats/mice lung neoplasm induced by carcinogenesis.The purpose of the current investigation is to explore a simple and convenient and reliable method for lung neoplasm animal model.

METHODSThe prepared 36 female Sprague Dawley (SD) rats, with the range from 180 to 220g of body weight, were randomly divided into two groups, each group included 18 rats. In the study group, the rats were given 2mg of 3, 4-benzopyrene soluting in 0.2mL corn oil every two-weekly pulmonary injection for 4 times through right middle-chest percutaneous puncture under control of anaesthesia by pentobarbital sodium i.p. The controls were only given 0.2mL corn oil injection simultaneously. The rats were sacrificed after suffering from dyspnea, and the survived rats were sacrificed at narcotism in 1 year after the first 3, 4-benzopyrene toxicosis. Lung, brain, liver, esophagus and stomach of all rates were anatomized in search of tumor.

RESULTSNo lung neoplasm was found in the control group within 1-year observation. The earliest dyspnea caused by lung malignant neoplasm was observed in 16 week after the first injection in a rat treated with 3, 4-benzopyrene oil, then 10 rats were sacrificed and found with malignant neoplasm in theirs right lung when the rats suffered from dyspnea, and 1 rat was found a huge tumour in its right lower limb in 26 weeks after treatment. The other 6 rats were sacrificed in 1 year after the first 3, 4-benzopyrene oil treatment, in which 1 rat was found with a malignant neoplasm in its right lung and 5 rats were normal. No tumor was found in brain, liver, esophagus and stomach. Out of these 18 rats, 12 (66.67%) lung malignant neoplasms and 1 limb malignant neoplasm were found within 1 year in the experimental group which yielded a total cancerogenic rate of 72.22% (13/18). The lung neoplasms included: 2 poor-differentitaed squamous cell carcinomas, 3 adenocarcinomas and 7 undifferentiated carcinomas.

CONCLUSIONSThe results suggest that 3,4-benzopyrene pulmonary injection by percutaneous puncture may provide an efficiency method for lung neoplasm model established in rats.

Objective To investigate the role and mechanism of miR-155 in invasion and metastasis of lung adenocarcinoma A549 cells.Methods Real-time PCR and fluorescence in situ hybridization were used to detect the miR-155 expression in patients′lung adenocarcinoma and adjacent tissue and lymph nodes.Scratch test and Transwell migration assay were used to assess the effect of miR-155 on the A549 cell migration and invasion capability.Bioinformatics software was used to predict the target genes of miR-155, and using luciferase to assay the target gene.Western blot and real-time PCR were performed to confirm the role of miR-155 expression in the regulation of target gene PTEN.Results The real-time quantitative PCR showed that the miR-155 expression levels in adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were 4.1±0.5, 9.6±3.1 and 7.8±2.2, respectively.The in situ hybridization showed that the expression rates of miR-155 in the adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were (23.2±15.3)%, (75.4±20.2 )% and (60.4±25.1)%,respectively.The Scratch assay showed that the wound healing rates in the miR-155 mimics group, miR-155 mimics NC group, miR-155 inhibitor group and miR-155 inhibitor NC group at 24 h were (43.2±2.2)%, (21.3±4.2)%, (24.3±5.3)%, and (35.2± 5.1)%, and that at 48 h were (75.2±4.5)%, (52.6±5.2)%, (39.4±4.2)%, and( 51.5±4.3)%, respectively.Dual luciferase reporter gene assay showed that the value of the luciferase in the miR -155 mimics group co-transfected with PTEN 3′UTR-containing wild-type and mutant plasmids were 4.7±0.5 and 7.3±0.7, and the miR-155 mimics luciferase values of the control group co-transfected with PTEN 3′UTR-containing wild-type and mutant plasmids were 7.8±0.9 and 7.5±0.8, respectively.The real-time quantitative fluorescence PCR showed that the relative expression of PTEN protein in the miR-155 mimics group, miR-155 mimics control group, miR-155 mimics inhibitor group, and miR-155 inhibitor control group were 0.5± 0.3, 1.0±0.1, 2.2±0.2 and 1.2 ±0.1, respectively.The Western blot assay detected that the relative expression of PTEN protein levels in the miR-155 mimics group, miR-155 mimics control group, miR-155 inhibitor group and miR-155 inhibitor control group were 0.4±0.1, 1.0±0.3, 2.8±0.2 and 1.4 ±0.1, respectively.The differences in PTEN mRNA and protein expressions of the four groups were statistically significant (P<0.05 for all).Conclusions miR-155 may promote the invasion and metastasis of lung adenocarcinoma through reducing the target PTEN gene expression.