Objective: To construct an eukaryotic expression plasmid containing gene coding for the hemagglutinin neuraminidase(HN) of Newcastle disease virus (NDV), and to study its mechanism and value in antitumor therapy. Methods: The HN cDNA was abtained from NDV with RT PCR and an eukaryotic expression vector of HN gene ( pcDNA3 HN ) was constructed. The antitumor effect was evaluated after injecting pcDNA3 HN into mice bearing B16 melanoma. Results: The HN cDNA of NDV was successfully cloned and pcDNA3 HN had a good expression in COS 7 cells. Animal experiments suggested that the pcDNA3 HN could significantly increase CTL and NK activity of tumor bearing mice. Conclusion:The eukaryotic expression plasmid containing the gene coding for the (HN) has the function of increasing CTL and NK activity of tumor bearing mice.

Objective: To study the factors effecting the expression of the reporter green fluorescent protein (GFP) gene in the mouse embryo stem cell line R1. Methods: Three different kinds of GFP euko-expression vectors were constructed, and the expression efficiency was contrasted both at mRNA and protein levels after they were integrated into the chromosomes of host cells. Results: At protein level, the GFP expression level of the colonies transfected by the expression vector-pEF-GFP with the promoter of the peptide elongation factor (EF) were significantly higher than that of the colonies transfected by pCMV-GFP with CMV promoter and by pdCMV-GFP with double copies of CMV-GFP expression unit. There was no significant difference between the colonies transfected by pCMV-GFP and pdCMV-GFP. The detection results on mRNA level of GFP had the same tendency as that at protein level. Conclusion: (1) GFP gene expression efficiency controlled by EF promoter is distinctly higher than that by CMV promoter in NIH3T3 and R1 ES cell line.(2) A slight increase of the copy number of the foreign gene expression units in the host chromosome can not make obvious increase of its expression efficiency. (3) The vector express GFP in R1 ES cell line efficiently and stablely is obtained.

Objective: To investigate the effect of four kinds of inactivation methods on the activity of soybean agglutinin (SBA). Methods: Dry-heat, humid-heat, acid-alkali and metal ion were employed to evaluate their effects on the activity of SBA, which were quantified by hemagglutination. Results: Dry-heat below 120℃ did not obviously decrease the activity of SBA, while humid-heat (95℃ for 30 min,100℃ for 20 min or 105℃ for 10 min) completely inactivated the SBA. The activity of SBA significantly decreased when soybean protein was extracted under pH 1.0-4.5 or pH 9-13. Quadrivalent and trivalent metal ions markedly inhibited the activity of SBA. Conclusion: SBA can be inactivated effectively by humid-heat, acid-alkali or metal ion. Further study is needed to clarify whether there are synergistic effects between these treatments.

Objective: To screen out DNA binding proteins specifically recognizing CpG immunostimulatory sequence (ISS) for further investigating the molecular mechanisms of ISS. Methods: Yeast one hybrid system was adapted in screening a human bone marrow cDNA library using 4 copies of ISS as bait. The ISS binding activity of the positive clone was confirmed by electrophoretic mobility shift assay (EMSA). Results: Four dual positive colonies were obtained, two of them encoded proteins with unknown functions. The other 2 encoded light chains of immunoglobulin with amino sequences homology to anti DNA Ab and HBsAb respectively. EMSA showed HBsAb specifically bound to CpG ODN at pH6.4 and pH 5.8. Conclusion: HBsAb may have ISS specific DNA binding activity.

ObjectiveTo construct the eukaryotic coexpression plasmid CEA/IL-2, and to lay the foundation for further studying CEA nucleiotide vaccine , adjuvant and their effects of special antitumor immunity.MethodsThe eukaryotic expression plasmid (pcDNA3-CEA)containing the gene coding for CEA was obtained by RT-PCR and gene recombination techniques.Using enzymolysis,ligation and other techniques,an eukaryotic coexpression plasmid (pIRES-CEA/IL-2)containing two expression unites of CEA and IL-2 gene connected with internal ribosome site was constructed.ResultsThe coexpression plasmids were transformed into COS7 cells and expression of two proteins were demonstrated by ELISA, and flow cytometer and elecsy.CEA and IL-2 were (23.73?0.26)ng/ml,and(20.17?0.13)ng/ml respectively.ConclusionsThe eukaryotic expression plasmids pIRES-CEA/IL-2 could be successfully constructed and transformed into COS7 cells.Expression of two proteins were demonstrated with no difference on expression.

Objective To explore the risk factors of Qi deficiency syndrome of type 2 diabetes mellitus and to provide evidence for prevention and cure diabetes mellitus with TCM.Methods On the basisof the epidemiological survey,147 cases of Type 2 diabetes mellitus were collected and divided into Qi deficiency syndrome group and non-Qi deficiency syndrome group.The relationships between risk factors and Qi deficiency syndrome were analyzed by unconditional univariate and multivariate Logistic regression.Results Two hours postprandial blood sugar[2PPBS (β value is-0.764,OR (95％CI) is 0.466 (0.236 ～ 0.919)],apolipoprotein-B [APo-B (β value is-1.005,OR (95％CI)is 0.366 (0.140～0.959)],urine glucose [β value is-1.300,OR(95％CI)is 0.273 (0.127～0.584)] were inverse correlation with Qi deficiency syndrome of type 2 diabetes mellitus.Conclusion Qi deficiency syndrome of type 2 diabetes mellitus was inverse correlation with 2PPBS、APo-B and urine glucose.

Objective To establish the decision tree model of Yang deficiency syndrome and clinical conventional indexes in type 2 diabetes mellitus patients.Methods Syndrome decision and clinical indexes collection from 249 type 2 diabetes mellitus patient were observed and analyzed.Tree structure model were built to summarize the correspondence between Yang deficiency syndrome and clinical conventional indexes based on T test,nonparametric analysis,and Spearman correlation analysis.Results The Yang deficiency syndrome accounted for 31.33％ of 249 type 2 diabetes mellitus patients.The accuracy identification rate of tree structure model of Yang deficiency syndrome with four core index,such as LPa、FT3、TSH、FINS was 84.74％,the sensitivity and specificity were 74.36％ and 89.47％.Conclusion Decision tree model can identify Yang deficiency syndrome of type 2 diabetes mellitus patients clearly and more intuitive.Decision tree model can provide the chance of syndrome objective.

Objective: To investigate changes of the endothelin-1(ET-1) and its receptor (endothelin receptor subtype A, ET AR ) mRNA expression in some organs(kidney, lung and small intestinal mucous membrane) in the sepsis and septic shock rats. Methods: Twenty-four male rats randomized into sepsis group, septic shock group, control and normal group was infused with endotoxin(LPS) via indwelling right atria catheters except normal and control group. RT-PCR was used to detect kidney, lung and small intestinal mucous membrane tissue mRNA expression of the ET-1 , ET AR and glucose-6-phospho dehydrogenase(G-6-PD) in every group.Serum BUN, Cr, ALT and A were determined. The arterial oxygen tension (PaO 2) and arterial carbon dioxide partial pressure(PaCO 2)were measured in every group. Results: ET-1 mRNA and ET AR mRNA expression in the sepsis group and septic shock group were significantly higher than in normal group. There were significant differences between the normal/control group and sepsis/shock group in BUN,Cr,ALT,PaO 2 and PaCO 2. Conclusion: A higher expression of ET-1 mRNA and ET AR mRNA may be one of the startup factors on multiple organ dysfunction syndrome (MODS) and may play an important role on pathogenesis in sepsis and septic shock.

BACKGROUND: Fibronectin(FN) plays the role of repair in the inflammation. There is no confirmed conclusion whether it can be applied to the refractory corneal epithelial defect.OBJECTIVE: To observe the repair effect of the human plasma FN for the refractory corneal epithelial defect.DESIGN: A controlled experimental study.SETTING: Guangzhou Red Cross Hospital, Guangzhou First People's Hospital, Guangzhou Children's Hospital, Guangzhou Hospital of Traditional Chinese Medicine and Guangzhou Second People's Hos pital.PARTICIPANTS: Totally 383 eyes with the refractory corneal epithelial defect were chosen, of which 309 were in the therapy group, and 74 in the control group.METHODS: The therapy group: Human plasma FN was administered by dropping it into the eyes once every two hours. The controlgroup: 10 g/L celacol M was administered by its dribbling into the eyes once every two hours. Weilesheng was taken orally in both groups, two pills once, three times per day. According to the state of illness, both groups received anti-bacterial or anti-viral treatment and reexamination was given every day or every other day after administration. 10 g/L fluorescein sodium was used to observe the changes of cornea.MAIN OUTCOSE MEASURES: The symptoms, results of staining using fluorescein sodium as well as the corneal epithelial healing of both groups.RESULTS: The symptoms, the results from staining using the fluorescein sodium and the corneal epithelial healing were used for the evaluation. In the therapy group, 309 eyes were followed up and the cure rate was 69.9%. The average therapeutic period was 6.5 days, while those of the control group were 58.1% and 8.7 days respectively. The difference in the curative effect between the two groups was significantly different( P＜0.01 ).CONCLUSION: The application of FN for the refractory corneal epithelial defect displays a more significant effect than conventional treatment.

Objective To investigate the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its associated mechanism during the wound healing. Methods The animal model with the full-thickness skin injury was used in the study. Fifty male mice were involved in the study and divided randomly into control group (n = 25) and GM-CSF group (n = 25). Each group had five time points (5 mice per time point). All the mice received full-thickness skin defect (1 cm × 1 cm) through the panniculus camosus on the midline of the back near the neck after anesthesia. Recombinant human granulocyte-macrophage colony stimulating factor (RhGM-CSF) gel (10 μg/cm2) were applied in the GM-CSF group and gel matrix without RhGM-CSF applied in the control group. The wound healing time and rate were observed at days 3, 5, 7, 10 and 14 after injury. The wound specimens were collected to detect the histopathological change. The microvessel density of the wound was counted based on the results of CD31 immunohistochemistry. RT-PCR was employed to detect the expression changes of GMCSF, platelet-derived growth factor (PDGF) , vascular endothelial growth factor ( VEGF) and stromal cell derived factor-1 (SDF-1). Results RT-PCR results showed that the gene expression of GM-CSF reached the peak at day 3 after injury (P＜0. 01) and kept the high level at days 3-10 after injury (P＜ 0. 05) , followed by a sharp decrease to a normal level at day 14 after wound. The wound healing time was average (2.4 ±0. 3) days earlier than the control mice after application of rhGM-CSF, with significant increase of the wound healing rate during 7-14 days after injury ( P ＜ 0. 05 ). In the GM-CSF group, the early histology of trauma wound showed a small number of neutrophils, obvious epithelial cell proliferation in the wound margin, marked hyperplasia of the granulation tissue, high cell density with quantity of spindle-shaped and oval-shaped cells and increased number of new blood vessels. The microvessel density was also increased significantly (P ＜ 0. 05) at days 7-14 after injury. The gene expressions of VEGF and SDF-1 were significantly increased at day 7 and day 10 respectively after injury (P＜0.05) and the gene expression of pro-healing factor PDGF was significantly increased in every time point (at days 5, 7 and 10,P＜0.05;at day 14,P＜0.01). Conclusion GM-CSF expresses highly in the early stage after injury and can promote the wound healing, when the mechanism may relate to the up-regulated expressions of pro-angiogenic factors and pro-healing growth factors.