Objective To establish a druggability evaluation method for new targets of anti-tumor drugs by analyzing the mutation genes of common tumors in the digestive system. Methods We collected the mutant gene data of the five common tumors of the digestive system (esophageal cancer, gastric cancer, colorectal cancer, liver cancer and pancreatic cancer) in the Integrative Onco Genomics database, and screened out the genes with higher mutation rates in each tumor. We evaluated the druggability of these genes or their encoded proteins, and discovered the potential targets for the new anti-tumor drugs. Results A total of five tumors, 35 cohorts and 5445 tumor samples were collected in this study. The top 10 mutation genes were selected for further analysis. The canSAR database was used to analyze the druggability of unpublished mutant genes or their encoded proteins, and a total of 17 potential therapeutic drug targets were screened out. Conclusion A method for evaluating druggability of targets based on mutant genes or their encoded protein is established in this study. The application of this method can provide a reference for discovering new anti-tumor therapeutic target, saving the cost and time of target screening in new drug development.

Objective: To investigate the reverse effect and mechanism of IL-12 on chemotherapeutic medicine suppressing the immune function of NK cells. Methods: Purified NK cells were stimulated with PMAplus Ionomycin in the presence or absent of Cisplatin (DDP) and IL-12. The levels of IFN-γ and TNF-α in culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA); The content of IFN-γ and TNF-α, TRAIL (TNF-related apoptosis inducing ligand) and transcription factors including T-bet and p-STAT-4 in NK cells were analyzed by Flow cytometry. The cytotoxicity of purified NK cells (pretreated with/without chemotherapeutics and IL-12 for 48 h) to Jurkat cells was measured by Flow cytometry. Results: Chemotherapeutics significantly inhibited the production of IFN-γ, TNF-α and the expression of TRAILin NK cells, which were significantly reversed by IL-12 (P<0.05 or P<0.01). Further study revealed that chemotherapeutics down-regulated while IL-12 reversed the expression of p-STAT4 to restore cytokine production. In addition, DDP also inhibited but IL-12 recovered the cytotoxicity of NK cells against tumor cells by inducing the expression of TRAIL (P<0.05 or P<0.01). Conclusion: Chemotherapeutics inhibited the cytotoxicity of NK cells and its secretion of cytokines (IFNγ and TNF-α), which were reversed by IL-12 via up-regulating TRAIL and p-STAT-4; this might provide experimental evidence for the clinical application of IL-12 for rebuild the immune function of tumor patients receiving chemotherapy.

Objective To investigate the expression change of RACK1 ,Src and Bcl‐2 in gastric carcinoma tissue and adjacent carcinomatous tissue .Methods Eighty specimens of gastric carcinoma and adjacent carcinomatous tissues in our hospital from Au‐gust 1 ,2011 to February 1 ,2014 were collected .The immunohistochemistry staining and Western blotting methods were adopted to detect the expression of RACK1 ,Src and Bcl‐2 protein in gastric carcinoma and adjacent carcinomatous tissues ,and their correlation was analyzed and performed the statistical analysis by combining with the clinicopathological data .Results The immunohistochem‐istry staining and Western blotting detection displayed that the expression positive rate and expression level of RACK 1 in gastric carcinoma tissue were obviously lower than those in the adjacent carcinomatous tissue ,while the expression positive rate and ex‐pression level of Src and Bcl‐2 in gastric carcinoma tissue were obviously higher than those in the adjacent carcinomatous tissue ,the differences were statistically significant (P<0 .05) .The RACK1 expression in gastric carcinoma tissue was negatively correlated with the Src and Bcl‐2 expression(r= -0 .632 ,-0 .754 ,P<0 .01) ,while Src had no obvious correlation with Bcl‐2 protein(r=0 .217 ,P>0 .05) .Conclusion The expression of RACK1 in gastric carcinoma tissue is significantly decreased ,while the expres‐sions of Src and Bcl‐2 are increased .

Objective To assess the number of peripheral blood Th17 cells and mRNA expressions of IL-17A and IL-23R and their correlations with disease severity in patients with psoriasis vulgaris (PV).Methods Tissue specimens were resected from the lesions of 25 patients with PV and normal skin of 10 human controls,and venous blood samples were obtained from 20 of the patients and all of the normal human controls.Reverse transcription PCR and flow cytometry were carried out to measure the mRNA levels of IL-17A and IL-23R in these tissue specimens and quantify the number of peripheral blood CD4+IL-17+ T lymphocytes.Psoriasis area and severity index (PASI) was calculated for these patients.Results A significant increase was observed in the IL-17A and IL-23R mRNA levels in the patients compared with the controls (0.996 ± 0.231vs.0.437 ± 0.096,t =10.572,P ＜ 0.05; 1.006 ± 0.339 vs.0.491 ± 0.196,t =6.015,P ＜ 0.05).The levels of both IL-17A mRNA and IL-23R mRNA were positively correlated with PASI (r,=0.67,0.70,respectively,both P ＜ 0.05).The number of CD4+IL-1 7+ T lymphocytes in peripheral blood showed no significant differences between the patients and controls.No statistical correlation was observed between the counts of CD4+IL-17+ T lymphocytes and expression levels of IL-17A or IL-23R mRNA in psoriatic lesions.Conclusions In patients with PV,there is an increase in the expressions of IL-17A and IL-23R mRNA in lesions,which are correlated with disease severity,while no significant change is observed in the number of peripheral blood CD4+IL-17+ T cells.

Objective To investigate whether R-848 could inhibit IgE production of mice immunized with OVA plus ALUM in vivo.Methods BALB/c mice were immunized s.c on the back with PBS,OVA,OVA plus R-848,OVA plus CpG,OVA plus ALUM,and OVA plus ALUM in R-848 or CpG every two weeks for three times.The blood from the mice was harvested after the last immunization,and the concentration of OVA specific or total IgE,IgG1 and IgG2a in the sera was determined;the splenocytes from the immunized mice were harvested and cultured with or without OVA for 3 days,and the concentration of IL-4 and IFNγ in the supernatants was determined.Results Firstly,we investigated that R-848 as Th1 adjuvant could enhance the production of OVA specific IgG2a in mice immunized with OVA.Secondly,further study showed that R-848 could inhibit the production of OVA specific IgE and total IgE,and promote the production of OVA specific IgG2a in the sera of mice immunized with OVA plus ALUM.Moreover,the results of ELISA showed that R-848 inhibiting IgE production was related to its reducing IL-4 production and enhancing IFNγ production.Conclusion R-848 could inhibit IgE production of mice immunized with OVA plus ALUM in vivo.

Objective To investigate the possibility of hair follicle reformation induced by hair follicle bulb cells implanted into collagen/chitosan porous scaffolds in vivo, and to observe the angiogenesis in implanted scaffolds.Methods Hair follicle bulb cells obtained by enzyme digestion from the hack skin of C57BL/6J mice were implanted into collagen/chitosan porous scaffolds followed by 2-week organotypie culture.Then,these collagen/chitosan porous scaffolds were transplanted subcutaneously into the dorsal skin of nude mice.Those nude mice transplanted with empty collagen/chitosan porous scaffolds served as the controls.The growth of hair was observed with naked eyes.Six weeks after the transplantation,skin samples were obtained from the recipient site and subjected to histological examination.Results Five weeks after the transplantation,hair growth was observed in the dorsal skin of nude mice.Six weeks later,histological examination revealed fully differentiated hair follicles and vessel-like structures in the center of collagen/chitosan porous scaffolds.However,the transplantation with empty collagen/chitosan porous scaffolds failed to elicit the same response.No hair or follicles were observed in the control mice alpng with small number of vessel-1ike structures.Con-clusions Hair follicle bulb ceils implanted into collagen/chitosan porous scaffolds in vivo could induce hair follicle reformation and promote the formation of vessel-like structure in the scafffold center.

AIM: To study the effect and mechanism of BCG on human nature killer cells. METHODS: PBMC or purified NK cells were isolated from normal human peripheral blood with negative anti-Mycobacterium tuberculosis antibody and cultured with BCG, IL-12, BCG plus IL-12 and BCG plus anti-IL-12Rβ1 mAb (2B10), respectively. The levels of IFN-γ and IL-12p40 in the culture supernatants were measured by ELISA. The frequency of IFN-γ and granzyme B producing cells were analyzed by ELISpot. The cytolytic activity was detected by MTT reduction assay. The surface expression of IL-12Rβ1 on NK cells was detected by flow cytometry. RESULTS: BCG significantly induced IFN-γ production by PBMC in a dose-dependent manner. When PBMC was stimulated with BCG, the frequency of granzyme B producing cells was higher than that in unstimulated PBMC (P<0.05). BCG enhanced the cytotoxic activity of PBMC. BCG alone didn' t induce IFN-γ production by purified NK cells, but it can augment IL-12-induced IFN-γ production by purified NK cells. The cytotoxic activities of BCG-stimulated and unstimulated purified NK cells were not significantly different (P>0.05). BCG induced IL-12 production by PBMC in a dose-dependent manner and enhanced IL-12Rβ1 expression on different subsets of NK cells. Blocking the effect of IL-12 by anti-IL-12Rβ1 mAb (2B10) inhibited BCG-induced IFN-γ production and granzyme B releasing by PBMC. CONCLUSION: BCG can indirectly promote biologic activity of NK cells and the production of endogenous IL-12 combined with up-regulation IL-12Rβ1 expression on the surface of NK cells is a part of the mechanisms of IL-12 on human NK cells.

BACKGROUND: Membrane materials of bioreactor have exchange of substance and good physiochemical characteristics as well as good biocompatibility.OBJECTIVE: To evaluate the biocompatibility of interface of human hepatocyte/microporous polypropylene, i.e. graft modified microporous polypropylene semipermeable ultrafiltration membrane (MPP).DESIGN, TIME AND SETTING: Animal observation was performed at the Organ Transplantation Center, Ruijin Hospital of Shanghai Jiao Tong University Medical School and Polymers Institute of Zhejiang University between September 2005 and October 2007.MATERIALS: The microporous polypropylene ultrafiltration plane thin membranes, 0.2 μm diameter, M<,r> 50 000-100 000 molecular blockage, were used. Photochemical graft polymerization modification technique was adopted to graft hydrophilic acrylamide group through chemical bonds on MPP surface and succeeded in constructing an interface of human hepatocyte/microporous polypropylene, i.e. bioreactor membrane of bioartificial liver, graft modified MPP.METHODS: The biocompatibility of modified MPP was evaluated by hemolysis test, cytotoxicity test, acute systemic toxicity test, pyrogen test, skin sensitization and percutaneous stimulation test according to the requirements and biological evaluation criteria of medical device of ISO10993-1:1992.MAIN OUTCOME MEAURES: The experimental results of hemolysis, cytotoxicity, general acute toxicity, pyrogen, skin sensitization and percutaneous stimulation of modified MPP.RESULTS: The hemolytic rate of modified MPP was 1.90% (<5%), which showed that modified MPP did not lead to hemolysis. The extract solution of modified MPP exhibited no significant inhibition on the proliferative activity of L929 cells. At 24, 48 and 72 hours after MPP injection, no mice death, significant changes in body mass, or acute systemic toxicity were observed, such as ptosis, dyspnea, eyanosis, abdominal stimulation, diarrhea, decreased movement or tremor. In rabbit pyrogen test, the body temperature changed in a range from -0.2 to 0.4, which was consistent with the evaluation criteria of biomedical materials without pyrogen. Only one case was found with very slight erythema in skin sensitization test; its integral was 1 and primary stimulation index was 0.25 (<0.4), and the primary stimulation index of percutaneous stimulation test was 0.2; the average primary stimulation index was 0.068, indicating that modified MPP had no skin irritation.CONCLUSION: Modified MPP has no haemolytieus, cytotoxicity, pyrogenicity or skin sensitization, suggesting good biocompatibility by photochemical graft acrylamide on the surface of MPP.

Objective To investigate the cellular immune state and pathological mechanism of HBV chronic infection by analyzing the cytokines produced by peripheral blood mononuclear cells(PBMCs) of asymptomatic HBV carriers.Methods PBMCs were prepared from diagnosed chronic asymptomatic HBV individuals and cultured in the presence of different antigens and antibodies.The levels of IFN-?,IL-12,TNF-?,TGF-? and IL-10 in the culture supernatants were respectively detected by ELISA.Results The levels of IFN-?,IL-12 were significantly lower than in the counterparts from healthy controls;meanwhile,those of TGF-? and IL-10 were elevated markedly.Neutralization of TGF-? and IL-10 simultaneously restore the IFN-? production from PBMCs of HBV carriers,and exogenous IL-12 in low dose combined with specific HBV antigens promoted IFN-? production.Conclusion Reduction of IL-12 from PBMCs may be the fundamental reason of viral persistence in HBV chronic carriers,combined IL-12 and specific HBV antigen promoted the celluar immunity of PBMCs from HBV carriers.

Objective:Understanding of subset、memory cell generation、effector function and organ distribution of Th1 cells in vitro and in vivo.Methods:Different stages of polarized Th1 cells are generated from antigen-specific CD4 +T cells.The expression of cell surface markers and intracellular cytokines(IL-2、IFN-? and IL-4) is determined at a single cell level by FACS. Equal numbers of CFSF-labeled naive CD4 +T cells and different stages of Th1 cells are adoptively transferred into mouse.The production of IFN-? and distribution of Th1 memory cells in lymphoid and non-lymphoid organs are measured. Results:Th1 cell populations can be subdivided into several subsets based upon the production of IL-2 and IFN-?. The percentage of IFN-?-producing cells is increased and IL-2-producing cells is decreased following repeatedly stimulation under Th1 culture condition. Moreover,the expression of CD62L on Th1 cells is reduced after activation and differentiation of CD4 +T cells. 2 weeks post-transfer,the frequency of naive CD4 +T cells is comparable from lymph nodes、spleens and lungs,whereas memory Th1 cells are preferentially detected in spleens and lungs and rapidly produce IFN-? following re-exposure to the same antigen.Conclusion:Th1 cell population is composed of distinct subsets of cells. In comparson with naive CD4 +T cells,memory Th1 cells reveal the changes of surface molecule expression、biologic function and distribution in tissues.