Objective To investigate the mechanism of Smad ubiquitination-related factor 2 (Smurf2)neddylation. Methods The Smurf2 protein level was tested by overexpression of Nedd8,while the method of immunoprecipitation(IP) and Western blotting were used to analyz Smurf2-Nedd8 modification.The GST-pulldown experiment was conducted to prove protein interactions.The protein was obtained by grinding mouse tissue and Western blotting was used to test the protein expression level.Results Over expression of Nedd8 could lead to the down regulation of the Smurf2′s protein level.Smurf1 and Smurf2 could interact in the GST-pulldown experiment. Smurf1 could enhance Smurf2-Nedd8 modification.The Smurf2′s protein level was up-regulated in mouse tissue of Smurf1 C426A.Conclusion There is some relationship and also difference between Smurf1 and Smurf2.Smurf1 can enhance Smurf2-Nedd8 modification.

Objective To investigate the expression of recombinant IL-37b protein and removal of the endotoxin, and identify its biological activity.Methods The prokaryotic expression vector pET28/IL-37b was constructed and to transform Escherichia coli (E.coli) Rosetta.After induction with IPTG, the recombinant protein was purified through Ni2+-NTA gel column and identified by SDS-PAGE and Coomassie brilliant blue staining.Then, the endotoxin protein was removed and was treated with LPS-stimulated RAW 264.7 cells.The culture supernatant was collected.The expression of IL-6 was detected by ELISA and the biological activity of the protein was identified.Results The recombinant IL-37b with high purity was expressed and the endotoxin produced by prokaryotic expression was reduced, and it was identified to have good biological activity.Conclusions In this study a recombinant IL-37b protein with high biological activity is successfully obtained.

Objective To establish a C57BL/6 mouse model of intestinal infection induced by S.Typhimurium.Methods In order to improve the infectious sensitivity of S.Typhimurium, C57BL/6 mice were intragastrically given 5% (w/v) NaHCO3.Then mice were challenged with S.Typhimurium.The health condition, survival and body weight of mice were observed from day 0 to day 7 after the bacterial infection.The pathological changes were also examined.Results the mice challenged with S.Typhimurium showed decreased body weight and typical clinical signs, including in appetence, piloerection and low survival rate.Macroscopic dissection revealed that intestinal hyperemia and swelling were founded in the mice challenged with S.Typhimurium.Histopathology showed intestinal epithelial and mucosal damages.Conclusions We have successfully established a C57BL/6 mouse model of S.Typhimurium infection.This model may be of crucial significance for studying the biological functions of associated immunological molecules or cytokines in the process of inflammatory bowel disease induced by S.Typhimurium.

Objective To establish lentiviral expression vectors for Smurf1 silencing and assess the effects of Smurf1 silencing on cell migration.Methods HeLa and A549 cells were infected with lentiviral expression vectors for Smurf1 silencing respectively.After 7 days,the stable cell lines with Smurf1 silencing were obtained after puromycin-resistance screening,enrichment and expansion.The intracellular gene and protein levels of Smurf1 were detected by qPCR and western blot.Transwell assay was used to assess the effect of Smurf1 silencing on cell migration.Results The stable cell lines with Smurf1 silencing are constructed successfully.Silencing of Smurf1 down-regulated cell migration rate detected by Transwell assay.Conclusion Smurf1 promotes cell migration.

Objective To investigate the effect of Smad3 on cell migration of A549 and HeLa cells.Methods Primers for pCMV-Myc-Smad3 plasmid construction and siRNA targeting Smad 3 were designed and synthesized .pCMV-Myc-Smad3 plasmid was constructed with molecular cloning techniques .Overexpression of Smad 3 with Myc-tag or silencing of endogenous Smad3, and then scratch assay was used to detect the migration ability of A 549 and HeLa cells in vitro. Results pCMV-Myc-Smad3 plasmid was successfully constructed .Overexpression of Smad 3 significantly up-regulated the migration rate of A549 and HeLa cells.Conversely, in the same cells, silencing of endogenous Smad3 or treatment with Smad3 inhibitor, SIS3, down-regulated the migration rate .Conclusions Smad3 promotes cell migration of A549 and HeLa cells.

Objective To establish a patient-derived xenografts (PDX) mouse model of liver cancer (LC) and to explore its role in precision medicine.Methods PDX model was established by subcutaneous implantation of tumor tissues in NCG mice.The morphological structure of tumor tissue was exaimed using HE staining.Fifteen BALB/c nude mice were subcutaneously inoculated with tumor cell suspension from the PDX models.The xenograft mice were randomly divided into 5-fluorouracil (5-FU) group, sorafenib group and negative control group.The tumor volume and body weight of the tumor-bearing mice were measured regularly, the tumor inhibition rate was calculated and the curative effect was evaluated.Results The success rate was 33.3% (6/18) in the establishment of liver cancer PDX mouse model, and the model well retained the characteristics of the primary tumor.In one case of PDX mouse model, the tumor inhibition rates of 5-FU and sorafenib group were 63.7% and 29.6%, with a statistically significant differece between them (P< 0.05), and there was no significant difference between the sorafenib group and negative control group, consistent with clinical observation.Conclusions The PDX mouse model of liver cancer can maintain the histological structure of primary tumor, and can be applied to precision medicine for patients with liver cancer.

Objective To construct a eukaryotic expression vector pEGFP N1/IL-37b of full-length and mature IL-37b,and to detect the expression of both full-length and mature IL-37b in RAW 264.7 cells, a mouse macrophage cell line. Methods To construct the eukaryotic vectors of full-length and mature IL-37b by using plasmid pUBC/IL-37b as a template containing the coding region of IL-37b full-length gene. To detect the expression of IL-37b by western blot and confocal microscopy after transfected the recombinant plasmid into RAW 264.7 cells, and to detect the inhibition of full-length and mature IL-37b on IL-6 production by real-time PCR. Results Eukaryotic vectors pEGFP N1/IL-37b expressed full-length and mature IL-37b after transfection in cells, which inhibited LPS-induced IL-6 production. Conclusions Eukaryotic vectors of full-length and mature IL-37b can be successfully constructed,and lays a foundation for further study of anti-inflammation functions and mechanisms of IL-37b.

OBJECTIVE: To investigate the expression of costimulatory molecules CD28/B7 and CD40/CD40L in T and B lymphocytes as well as its relations with total IgE (TIgE), eosinophil cationic protein (ECP) in serum and nasal allergic symptoms in patients with allergic rhinitis (AR). The effect of specific immunotherapy (SIT) on them were also investigated. METHOD: Thirty allergic allergic rhinitis patients were chosen as observation group, and 30 healthy patients as control group. Cytofluorometric analysis was used to compare the expression level of CD28/B7-1, B7-2 and CD40/CD40L on T cells and B cells in the two groups. The relationship between the CD28/ B7-1, B7-2 and CD40/CD40L expression level and serum Total IgE, ECP level were analyzed. RESULT: The expression level of CD28/B7-2 and CD40/CD40L on T cells and B cells in allergic rhinitis patients were significantly higher than in the healthy, and serum level of TIgE has a positive relationship with the expression level of CD40L on T cells. ECP has a positive relationship with the expression level of B7-2 on B cells. The expression level of B7-1 showed no significant difference between the two groups. After specific immunotherapy for 6 months, the expression level of CD28/B7-2 and CD40/CD40L on T cells and B cells were decreased in allergic rhinitis patients but still higher than in healthy. CONCLUSIONS The upregulated level of costimulatory molecules CD28/B7-2 and CD40/ CD40L on T cells and B cells may play an important role in the pathogenesis of allergic rhinitis, specific immunotherapy can downregulate the expression level of CD28/B7-2 and CD40/CD40L, and decrease the serum level of TIgE, it may be a possible mechanism in the treatment of allergic rhinitis.
Adult ; B-Lymphocytes ; immunology ; metabolism ; B7-2 Antigen ; metabolism ; CD28 Antigens ; metabolism ; CD40 Antigens ; metabolism ; CD40 Ligand ; metabolism ; Case-Control Studies ; Desensitization, Immunologic ; Female ; Humans ; Immunoglobulin E ; blood ; Male ; Rhinitis, Allergic, Perennial ; blood ; immunology ; metabolism ; therapy ; T-Lymphocytes ; immunology ; metabolism

OBJECTIVE: To study the type and drift law of airborne pollen in Wuhan, and its relationship to pollinosis. METHOD: From November 2003 to October 2004, an airborne pollen investigation was performed in three districts of Wuhan using gravity sedimentation technique. Meanwhile, univalent shin prick tests of pollens and the study of invasion season were performed in 1200 cases with pollenosis. Among them, 352 cases underwent the airway responsiveness measurements, and the correlation between airway responsiveness and pollen concentration were analyzed. RESULT: A total of 47 pollen colonies were observed and 75,525 pollens were collected. Every year the the peak time of airborne pollen occurred in two seasons: spring (March and April) and autumn (from August to October). The incidence of pollinosis is consistent to pollen peak time; there was a negative relationship between PD20 (the provocative dose to decrease FEV1 by 20% from baseline) and airborne pollen concentration. CONCLUSION The study provides useful information for airborne pollen epidemiology in Wuhan. It provides important insights to clinical prevention, diagnosis and treatment of pollen related allergic diseases.
Allergens ; analysis ; China ; epidemiology ; Humans ; Incidence ; Pollen ; Rhinitis, Allergic, Seasonal ; epidemiology ; Skin Tests