Study On The Expression And Phosphorylation Of Signal Protein ERK And ZAP-70 In T Cells By The Effect Of Transferring TCR Gene

Objective T cell antigen receptor (TCR) provides initial stimulating signal and plays a triggering role of mediating the recognition of T lymphocyte to tumor-associated antigens. TCRβV locus contains 24 subfamilies. If there is no antigen stimulation, the rearrangement of TCR beta in T cells of normal human should be random, and lead to multifamily distribution. Each family of T cells perform their respective functions. To identify the TCRV beta expression on T cell in normal and cancer patients, someone investigated the change of 24 subfamilies of TCR V beta gene using RT-PCR. The results showed that TCR expression displayed random distribution in PBMC of healthy people, but preference distribution in PBMC of patients with hepatocellular carcinoma. Our previous investigations displayed that TCR Vβ7.1 subfamily of HCC patients showed preferable amplification respond to the stimulation of tumor cells. We speculated that TCR Vβ7 involved in the tumor immunoreaction in patients with hepatocellular carcinoma. Our previous study found that, TCR V beta 7.1 gene-modified T cells can induce apoptosis in hepatoma cells. Base on this result, this study aim to construct TCRVβ7.1 eukaryotic expression plasmid and transfer it into human T lymphocyte for expression, to further research the signal pathway activation in TCR gene-modified T-cell, and to lay a foundation for the study of TCR gene therapy for cancer patients and tumor immune tolerance mechanisms.Method To sum up, this study consists of three parts : the first part is well TCR V beta 7.1 eukaryotic expression vector construction; the second part is a study of the transfection efficiency of recombinant plasmid pcDNA3.1 (+)/TCRV beta7.1 in T cell, using flow cytometry to detect TCRV beta 7.1 expression; The third part was detected by ELISA the expression of IL-4, IFN-γproduction, and western detected Erk1/2, Zap-70 both protein for the total level of change and phosphorylation.Results (1) The amplified TCR Vβ7.1 gene were inserted into the eukaryotic expression vector pcDNA3.1 (+). The recombinant eukaryotic expression vector pcDNA3.1 (+)/TCR Vβ7.1 was confirmed by restricted digestion and PCR amplification; DNA sequencing revealed that the recombinant expression vector sequence correct.(2) After transfection with the recombinant plasmid pcDNA3.1 (+) / TCRVβ7.1, the result of FACS showed that the TCRVβ7.1expression was significantly higher in tranfected T cell than non-transfected T cell. This result indicated that TCRV 7.1βgene could be effectively transfered into PBMC with liposome-mediated transfection method followed by effective expression.(3) Western blot results showed that Transfection TCRV 7.1 beta gene PBMC group ERK, p-ERK protein expression levels significantly higher than that of non-transfected group (P <0.01). p-ERK expression is related with activation of T cell, and p-Zap-70 only expresse in the early phase after antigen attack. ELISA results showed that INF-γof the transfection group is significantly higher than the non-transfected group, but IL-4 has no change between groups.Conclusion (1) The recombinant eukaryotic expression vector pcDNA3.1 (+) /TCRVβ7.1 is successfully constructed, and effectively expressed in transfected PBMC. (2) The expression and phosphorylation levels of ERK1/2 and Zap-70 in T lymphocytes transfected with TCRVβ7.1 were studied; moreover the relationship between them and the secretion of cytokines was discussed.