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.

This article summarizes some methods in teaching physical chemistry,such as inspiring students’ interests and improving teacher’s teaching level. Then,how to enhance the teaching quality of physical chemistry is discussed.

Objective To study morphological characteristics of placenta in severe pregnancy induced hypertension (SPIH) and its relationship to pregnant outcome. Methods Morphological changes were observed by light microscopy; Blood biochemical analyses were used to predispose pregnant outcome. Results Difference in pathological changes of placental bed between normal term pregnancy (NTP) and SPIH groups was significant: the placental weight, proliferation of cytotrophoblasts, numbers of the placental villi with syncytial knots, thickness of basal lamina, fibrinoid necrosis and deposition of matrix, stromal edema and fibrosis of villi, the vascular numbers of villi, stasis; lack of physiologic changes in decidual spiral arteries. Clinical examination showed that the rate of anemia, thrombocytopenia, blood concentration, hypoproteinemia, ascites, eye ground artery spasm in SPIH were higher in NIT. Conclusions Pathological changes of placenta play important roles in development of SPIH, and the pathological changes are paralleled with the severity of the disease.

Objective To establish the triplex Taqman probes real-time RT-PCR method for simultaneously detecting of EV71,CA16 and EV.Methods Retrospective study.Specific primers and probes were designed based on conserved regions of EV71,CA16 and EV.The sensitivity,specificity and reproducibility were assessed by the optimized reaction system.A total of 176 throat swabs as the experimental group were collected from children with suspected hand foot mouth disease (HFMD),who admitted from April 2012 to July 2012 in Nanjing Children's Hospital affiliated to Nanjing Medical University.During this time,10 cases of healthy children,10 cases of outpatients with flu-like symptoms and 90 cases of inpatients in pneumology department of our hospital were recruited as control group,whose throat swabs were also collected.All of 286 samples were tested by the triplex Taqman probes real-time RT-PCR for simultaneously detecting EV71,CA16 and EV.SPSS13.0 was used to analyze the results.Results The sensitivities of the triplex Taqman probes real-time RT-PCR was 1.0 × 103 copies per milliliter for EV71,CA16 and EV.It showed 100％ specificity for 9 enterovirus and 3 non-enterovirus.Analysis with 1.0 × 103-1.0 × 105 copies per milliliter constructed plasmids demonstrated high reproducibility with coefficient of variation of 0.44％-1.04％ for EV71,0.38％-0.73％ for CA16,and 0.46％-0.90％ for EV.More over 176 samples collected from children with suspected HFMD were detected by triplex Taqman probes real-time RTPCR and real-time RT-PCR.The results showed 97.2％ (171/176)agreement and 0.94 Kappa value with high concordance.Conclusions The triplex Taqman probes real-time RT-PCR detecting EV71,CA16 and EV simultaneously has been established successfully.The assay,with high sensitivity and specificity,provide good basis for the rapid clinical diagnosis of EV71,CA16 and EV and open up broad prospects for clinical and relevant researches.

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 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 investigate the possible existence of HBoV in children with acute respiratory infections in Nanjing area and explore its relationship with clinical characteristics.Methods A total of 397 nasopharyngeal secretion samples were collected from children with acute respiratory infection,admitted from July 2009 to June 2010 in Nanjing Children'S Hospital affiliated to Nanjing Medical University,and 50 cases of children without symptoms of respiratory infection were recruited as control group,whose nasopharyngeal secretion samples were also collected.HBoV was determined by real-time fluorescence quantitative PCR.MP and CT were detected by real-time fluorescence quantitative PCR in those HBoV-positive samples.RSV,ADV,IVA,IVB,PIV-1,PIV-2,PIV-3 and hMPV were detected by direct antigen-specific immunofluorescence assays.HBoV NP-1 fragments were amplified and sequenced in 5 HBoV positive samples randomly selected.The results were compared with the known GenBank sequence,and thereby the phylogenetic tree was established.The epidemiological characteristics,clinical presentation and the final clinical diagnosis of HBoV were analyzed according to the clinical data of the HBoV-positive patients.Results Thirty-three HBoV-positive cases were detected by real-time fluorescence quantitative PCR method with a positivity rate of 8. 3% ( 33/397 ). Among the 33 HBoV-positive cases, 19 cases (57.6%) were multiple infections with HBoV and other pathogens, the top three of which were MP (27.3% ,9/33 ),RSV (24.2% , 8/33 ) and PIV-3 ( 12. 1% ,4/33 ). Affected children aged from 7 to 36 months old accounted for 75.8% of the total ( 25/33 ). The measured HBoV NP-1 gene sequences of 5 specimens were consistent,indicating a high homology (99% to 100% ) with the stl, st2 and WHL-1. Conclusions HBoV is one of the pathogens of children's acute respiratory infections in Nanjing. HBoV NP-1 gene is highly conserved,with little variation in different seasons and in different regions and therefore can be used as a marker for real-time fluorescence quantitative PCR and other methods.

The authors have made an investigation on the conditions of the internet access,the platform for network-aided teaching,the teaching resource and the teaching effect for physical chemistry among 127 pharmacy students of 2008 enrollment.Based on the results of the investigation,an analysis on the current situation and problems of network-aided teaching for this course was performed and recommendations were put forward accordingly.

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.