ABSTRACT
ObjectiveTo investigate the effects and mechanisms of IL-32γ-shRNA-mediated gene silencing on the proliferation and apoptosis of fibroblast-like synoviocytes in patients with rheumatoid arthritis (RA).MethodsA eukaryotic expression plasmid of shRNA targeting IL-32γ was transfected into fibroblastlike synoviocytes by liposome in patients with rheumatoid arthritis.RT-PCR was used to determine the expression level of IL-32γ.Western blotting was used to detect the levels of cyclin D1 and p-Akt.The proliferation of RA-FLS was examined by MTT.Cell cycles were analyzed by flow-cytometry.The apoptosis of cells were measured by TUNNEL.Comparisons between groups were tested by t test.Results ① The expression of IL-32γwas significantly inhibited by shRNA-IL-32γ-expressing plamid PGCsi 3.0 targeting sequence 1,2 and 3,and the inhibition rate had reached 75.6％,66.2％ and 64.1％,respectively.② The absorbance value of proliferation of RA-FLS in EASY-shRNA-IL-32γ group was significantly lower than that in the shRNA-control group and normal group at day 3 [(0.23±0.03) vs (0.35±0.03) and (0.36±0.04),P＜0.05] and 5 [(0.27±0.03) vs (0.52±0.05) and (0.53±0.04),P＜0.01 ] after transfection.③ The rate of RA-FLS at phase G1 in the EASY-shRNA-IL-32γ group was significantly higher than that in the shRNA-control group and normal group respectively [(88±6)％ vs (69±5)％ and (68±4)％,P＜0.05],while those at phase S+G2 in the EASY-shRNA-IL-32γgroup was significantly lower than that in the shRNA-control group and normal group [ ( 13.6±3.0)％ vs (30.2±4.1)％ and(32.1±4.3)％,P＜0.01].④The rate of RA-FLS apoptosis in the EASY-shRNA-IL-32γ group was significantly higher than that in the shRNA-control group and normal group[(20.50±3.21 )％ vs (9.20±0.32)％ and (8.60±0.22)％,P＜0.01].⑤ The expression of cyclin D1(0.36±0.04) and p-Akt(0.31±0.03) in the EASY-shRNA-IL-32γ group was significantly lower than that in the shRNA-control group [ (0.59±0.08) and (0.53±0.06)] and normal group [(0.61±0.07) and (0.52±0.06),P＜0.01].ConclusionEASYshRNA-IL-32γ can inhibit RA-FLS proliferation by down-regulating the expression of cyclin D1 and induce RA-FLS apoptosis by down-regulating the expression of p-Akt.
ABSTRACT
Objective The monoclone antibody against insulin was prepared and used to measure plasma level of insulin in patients with type 2 diabetes mellitus. Methods Balb/c mice were immunedwith cross-linking insulin-BSA as immunogen. Four monoelone cell strains stably secreting insulin monoclone antibody were prepared by applying hybridoma technique. The valence, subclass and affini-ty constant of the monoclone antibody were analyzed. The serum insulin content was measured with competitive inhibition ELISA in 90 patients with type 2 diabetes mellitus and 90 healthy controls. Re-suits The affinity constant of the established monoclone antibody reached to 1011. The subclass of 1 strain was Igab(k),and the other 3 strains were Ig1(k) srbclass.The mean level of insulin was significantly lower in patients with type 2 diabetes mellitus than that of healthy controls. Conclusion These results implicated that the insulin insufficient might be due to the depletion of the pancreatic be-ta cell function of patients with type 2 diabetes mellitus, and the pathogenetic condition might be alle-viated by supplying insulin. About 10% patients are with higher insulin level than healthy controls,which may be due to insulin resistance. It is inadvisable to treat these patients with insulin therapy.
ABSTRACT
Glucagon-like peptide-1 is one of the most important members of incretin, which has an unusual therapeutic potential in type 2 diabetes mellitus and obesity. Its analogues are resistant to dipeptidyl peptidase Ⅳ hydrolysis and begin to be used clinically. These preparations may have important application perspective as new therapeutic agents in the treatment of diabetes mellitus and obesity.