Study On The Effect Of CD40/CD40L In The Apoptosis Of Tumor Cells Induced By CD4～+CIKs

PartⅠ: Study on the effect of CD40/CD40L in the apoptosis of tumor cells induced by CD4~+CIKsObjects:To study the effect of the CD40/CD40L in the apoptosis of tumor cells induced by CD4~+CIKs and investigate the underlying mechanism.Methods:After large scale of amplification of CIKs in vitro, the subset of CD4~+CIK cells was isolated by magnetic beads separation columns. The expression of CD40L in CD4~+CIK was detected by flowcytometry. Apoptotic rates of MDA-MB-231 cells cocultured with CD4~+CIK for 24 hours at different ratio of E/T were evaluated by AnnexinV staining and expression of Fas was compared by flowcytometry as well. Then antibodies agaist FasL, CD40L, IFN-γwere added in the coculture, apoptotic rate and expression of Fas on MDA-MB-231 cells were detected. The mRNA expression of Bax,Bcl-2,FADD, FLIP in MDA-MB-231 cells after cocultured with CD4~+CIK for 4h,6h and 24h were analyzed by real time quantitative RT-PCR method, and the antibody blocking experiment was studied.Results:The expression of CD40L on CD4~+CIK is up to (20.72±5.18)%. The apoptotic rates of MDA-MB-231 were observed and elevated with the increasing of effector cells after 24h cocultured. The Fas on MDA-MB-231 cells increased as shown by flowcytometry (P＜0.01). After anti-FasL,anti-CD40L, anti-IFN-γblocking antibodies were added, the apoptotic rates decreased obviously from (61.5±5.43) % to (16.57±5.36)%, (20.63±6.37)%, (26.00±8.50)%. Fas on MDA-MB-231 cells decrese from (15.00±3.23)% to (10.80±4.40)%, (6.93±1.56)%, (8.73±4.70)%. The result of real time quantitative RT-PCR showed that 6h after cocultured Bax,Bcl-2 decreased and FADD,FLIP increased (P＜0.05), till 24h Bax,Bcl-2 increased and FADD,FLIP decreased(P＜0.01). The antibody blocking experiment showed that the increase of Bax,Bcl-2 and the decrease of FADD correlated with FasL.The effect of FasL increasing the Bax is conspicuous,but CD40L can decrease Bax after eliminating the effect of FasL. The decreasing of Bax, Bcl-2 and the increasing of FLIP correlated with IFN-γusing Linear Regression.Conclusion:1 Treatment of the tumor cell lines MDA-MB-231 with CD4~+CIK reverse Fas-resistant type to Fas-sensitive type, which regarding as Fas typeⅡcell.2 IFN-γ,CD40L expressed by CD4~+CIK can increase the expression of Fas and induce the apoptosis of tumor cell through Fas/FasL pathway and the apoptotic rates elevated with the increasing of effector cells, and the effect of CD40L is stronger.3 CD40L can not only induce the apoptosis of tumor cells, but also inhibit the apoptosis by decreasing Bax.PartⅡ: Establishment and identification of CHO cell line expressing sCD40LObjects:To establish a cell line expressing sCD40L and investigate the effect of CD40L in apoptosis induced by CD4~+CIK.Methods:The aimed segments were obtained from vector pDC316-sCD40L by the method of restricted enzymatic resection and were inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct a recombinant expression plasmid pIRES2-EGFP-sCD40L. The recombinant plasmid, first propagated in Escherichia coli DH5α,then extracted,purified and digested with BglⅡand SalⅠ,was confirmed to contain full length of pIRES2-EGFP-sCD40L cDNA by agarose gel analysis and DNA sequence analysis.Then the CHO cells were transfected with the plasmid using electroporation and screened with antibiotic G418. Single clones expressing sCD40L were obtained by limited-dilution method. EGFP-positive cells were detected by flow cytometry and inverted fluorescence microscopy. The DNA integration and mRNA expression of sCD40L in positive clones were detected by PCR and RT-PCR.The protein of sCD40L expressed by the positive clones was evaluated by ELISA. Then they were cocultured with MDA-MB-231 cells to observe the expression of Fas and the apoptotic rates of MDA-MB-231 cell after cocultured with the anti-Fas activating antibody(CH 11).Results:A recombinant eukaryotic expression plasmid pIRES2-EGFP-sCD40L was successfully constructed. Then the mixed CHO clones expressing sCD40Lwere selected by G418 for two weeks after transfection.Through limited-dilution cloning, three single clones were obtained,and the best one named as Bll.The percentage of cells expressing EGFP was more than 90%. The integration of gene sCD40L was identified by PCR and the sCD40L mRNA transcription was detected by RT-PCR in the positive clone. The amount of sCD40L protein identified in supematant was 4.5±2.1ng/ml. After cocultured with CHO-sCD40L, the expression of Fas on MDA-MB-231 cells increased significantly(P＜0.01). The apoptotic rates of MDA-MB-231 cells increased after coculturing with CH-11 (P＜0.01).Conclusion:A cell line expressing the sCD40L protein stably was established. It provided a useful tool to investgate the role of CD40/CD40L pathway in the adjuvant immunotherapy of tumor. The interaction of CD40/CD40L can increase the expression of functional Fas and induce the apoptosis of tumor cell.