Résumé
Objective: To construct a recombinant retroviral vector carrying human inducible costimulator (ICOS) gene, screen for CHO cell line stably expressing ICOS protein and to study its biological activity. Methods: ICOS cDNA was obtained from human peripheral blood mononuclear cells (PBMC) through RT-PCR and was cloned into retroviral vector to construct retroviral recombinant pMSCV-ICOS; the latter was then packed and the high-titer virus producing cells were screened. Then CHO cell was infected by this high-titer virus and the stable cell line was screened. CHO-ICOS cells were co-cultured with PBMC (the ratio of CHO-ICOS to PBMC being 1:1,1:2,1:5, and 1:10) in presence of substimulating dose of anti-human CD3 antibody. The proliferation of PBMC and the CD25 expression on T cells were examined by 3H-TdR incorporation method and flow cytometry,respectively. CHO-pMSCV cells co-cultured with PBMC (1:1) served as the negative control and PBMC served as blank control. Results: We successfully constructed the retroviral recombinant pMSCV-ICOS and obtained CHO cell line stably expressing ICOS protein. 3H-TdR incorporation method and flow cytometry showed that, compared with the negative control group and the blank control group, co-culture with CHO-ICOS cells significantly inhibited the anti-CD3 antibody-induced activation and proliferation of PBMC(P<0.05);the maximal inhibitory rates of activation and proliferation were obtained when the ratio of CHO-ICOS to PBMC was 1:1, being (68±5.9)% and (44.08±3.26)%, respectively. Conclusion: CHO cell line stably expressing ICOS protein has been successfully established, which lays a foundation for future study.
Résumé
Objective To construct the recombinant retroviruses vector PCLNRX-ICN and to explore over-activation Notch1 on the growth of human colon carcinoma cells HT-29.MethodsICN(intracellular domain of Noctch1) genes were inserted into retrovirus expression vector pCLNRX.The expression vector pCLNRX-ICN and packaging vector pCL-10A1 were co-transfected into 293 package cells.The recombinant retroviruses were used to infect HT-29 cells.After being infected,proliferation of HT-29 cells was observed by MTT assay.The expression of c-Myc and ?-catenin detected by RT-PCR and Western Blot.ResultsRecombinant retrovirus vector pCLNRX-ICN was successfully constructed.Overactivation of Notch1 by over-expressing exogenous ICN significantly inhibited the growth of HT-29 cells,and down-regulated ?-catenin,a key regulator of Wnt signaling,in protein level but not in mRNA levels.However,the mRNA or protein levels of c-Myc were not affected by overactivation of Notch1.ConclusionOver-activated Notch1 signaling could inhibit the growth of HT-29 cells partly through down-regulation of Wnt signaling independent of c-Myc inhibition.
Résumé
Objective:To construct a recombinant retroviral vector carrying human inducible costimulator (ICOS) gene,screen for CHO cell line stably expressing ICOS protein and to study its biological activity.Methods: ICOS cDNA was obtained from human peripheral blood mononuclear cells (PBMC) through RT-PCR and was cloned into retroviral vector to construct retroviral recombinant pMSCV-ICOS; the latter was then packed and the high-titer virus producing cells were screened.Then CHO cell was infected by this high-titer virus and the stable cell line was screened.CHO-ICOS cells were co-cultured with PBMC (the ratio of CHO-ICOS to PBMC being 11,12,15, and 110) in presence of substimulating dose of anti-human CD3 antibody.The proliferation of PBMC and the CD25 expression on T cells were examined by 3H-TdR incorporation method and flow cytometry,respectively.CHO-pMSCV cells co-cultured with PBMC (11) served as the negative control and PBMC served as blank control.Results: We successfully constructed the retroviral recombinant pMSCV-ICOS and obtained CHO cell line stably expressing ICOS protein.3H-TdR incorporation method and flow cytometry showed that,compared with the negative control group and the blank control group,co-culture with CHO-ICOS cells significantly inhibited the anti-CD3 antibody-induced activation and proliferation of PBMC(P