Objective To explore the roles of PKCθ(protein kinase Cθ)signaling pathway on the activation,proliferation and TH1/TH2 cytokines production of the γδT lymphocytes stimulated by Mycobacterium tuberculosis antigen(Mtb-Ag) in vitro.Methods Peripheral blood mononuclear cells(PBMC)were pretreated with or without Rottlerin at 5.0 μmol/L,and stimulated with Mtb-Ag and cultured in rhIL-2 containing medium.After different time of culture,activation molecules and cytokines production of γδT cells were measured by flow cytometry.The proliferation proportion and the percentage of each generation of γδT cells were determined by carboxylfluoreseein diacetate succinmidyl ester(CFSE)labeling technique and flow cytometry.Results After 3d of stimulation with Mtb-Ag,the expression rates of CD69 and CD25 of γδT cells were 46.2%and 45.6%,respectively.Pretreatment of 5.0 μmol/L Rottlerin markedly inhibited the both expressions of CD69 and CD25 in γδT cells(P＜0.01).After stimulation and expansion in vitro for 5,10,and 15 d,the percentages of the γδT cell were 9.6%,54.6%and 82.4%,respectively.There was a few γδT cells in propagation on the 5th day of culture,and almost all γδT cell divisions were above 6 generations on the 10th and 15th day of culture.Pretreatment of the Rotflerin significantly suppressed the γδT cell proliferation,but after 10 d culture,there were still a few parts of γδT cells in propagation.Meanwhile,after 7,14,and 21d of culture,upon stimulation with PMA+Ionomycin,the IFN-γ producing-γδT cells were about 80%at all times.But only after 21d culture,IL-4-producing γδT cells was 2.6%.,The percentage of IFN-γ producing γδT cells markedly reduced in Rottlerin group(P＜0.01).IL-4 secretion of the γδT cells was almost completely blocked.Conclusion PKCO signal pathway involves in activation,proliferation and differentiation of the γδT lymphocytes stimulated by Mtb-Ag.

Abstract:Objective To explore the effects of liposome-C-erbB2 antisense phosphorothioate oligodeoxynucleotides (S-ODNs) on C-erbB2 proto-oncogene expression and cell proliferation in human ovarian cancer cells.Methods The effects of liposome-C-erbB2 S-ODNs on C-erbB2 protein expression, cell cycle and cell proliferation in human ovarian cancer cells were studied by means of flow cytometry and 3H-thymidine incorporation.Results Liposome-C-erbB2 S-ODNs can specifically reduce C-erbB2 protein expression in human ovarian cancer cells, accompanied by a 30% inhibition of cell proliferation. The effectiveness of liposome-C-erbB2 S-ODNs on the expression of C-erbB2 was about 40 times higher than that of C-erbB2 S-ODNs.Conclusions The data suggest that antisense therapy might be a useful method of gene therapy in ovarian cancer. The effectiveness of C-erbB2 S-ODNs could be greatly increased by adsorption of S-ODNs by liposomes.