| The immune responses to tumors are mostly dependent on the cell immunity, especially on T cell-mediated immunity. Successful T cell activation requires not only signals provided when antigen/MHC complex binds to the TCR, the other signals provided by costimulatory molecules are also important. Up to now many costimulatory molecules have been found, such as B7> 4-lBBLx CD40L and ICAM-1. The absence of these costimulatory molecules has been shown to lead to T cell anergy or apoptosis. Therefore, the introduction of costimulatory molecules into tumor cells to prepare tumor vaccine become to be a hotspot in the field of tumor biotherapeutic research in recent years.4-IBB ligand (4-1 BBL) is a newly found costimulatory molecule. It is a member of the tumor necrosis factor (TNF) gene family that binds to 4-IBB, a T cell activation antigen which belongs to the TNF/nerve growth factor super family. The studies have demonstrated that the binding of 4-1 BBL with 4-IBB canregulate the functions of several immune cells. 4-1BB-4-1BBL interactions can deliver a costimulatory signal for T cell activation and growth and promote the cell function of cytolysis. The powerful effects of 4-1BBL triggering on the T cell antitumor immune responses provide a novel strategy for increasing the potency of vaccines against tumors. To explore the prospects of the application of 4-1BBL in the anti-hepatocarcinoma immune response, we cloned a cDNA gene coding for the mouse costimulatory molecule 4-1BBL and further transfered it into murine hepatocellular carcinoma cell line Hepal-6. Then we observed the effects of this specific antitumor vaccine in vitro and in vivo.Firstly, the expression of 4-1BBL in lymphocyte and several hepatocellular carcinoma cell lines were analyzed by RT-PCR. The results showed that 4-1BBL could be found in the human peripheral blood lymphocytes and murine splenocytes induced by PHA, but 4-1BBL could not be found in the quiescent and non-induced lymphocytes. Only one hepatocellular carcinoma cell line HepG2 could be detected the expression of 4-1BBL while the negative results were found in the other four hepatocellular carcinoma cell lines and one normal hepatic cell line.To obtain the full gene sequence of 4-1BBL, we designed the primers of 4-1BBL according to the murine 4-1BBL gene sequence from GENEBANK. The total RNA was isolated from the murine splenocytes of C57BL/6 and the cells were induced by PHA before the abstraction of RNA. The m4-lBBL cDNA was obtained successfully by RT-PCR from the murine splenic lymphocytes. Sequencing analysis revealed that the amino acid sequence of m4-lBBL has no variation in nucleotide sequence as compared with the published sequence. The PCR product was cloned into pcDNA3.1(+) eukaryotic expression vector. This pCDNA3.1(+)- m4-lBBL vector was used to transfer the m4-lBBL gene into COS-7 cells, and the efficient expression was achieved.We used the above vector to transfer the costimulatory molecule 4-1BBL gene into syngeneic murine hepatocellular carcinoma cell line Hepal-6. The clones with stable expression were obtained through G418 screening. TheHepal-6-m4-lBBL cells efficiently expressed m4-lBBL after G418 selecting, and the exogenous gene was confirmed to have been integrated into chromosome of target cell. The gene of m4-lBBL didn't be detected in non-transfected Hepal-6 cells. The proliferation test in vitro showed that the growth rate and the ability in cloning of Hepal-6 cells weren't affected by the transfection, and they could express the m4-lBBL gene continuously. This demonstrated that the biological characters didn't be changed in wild-type tumor transfected with exogenous gene. The results of tumorigenicity experiment in syngeneic mice revealed that, as compared to wild-type tumor, mice subcutaneously injected with tumors expressing the vector Hepal-6-m4-lBBL developed fewer tumors and the latent period of tumor development was prolonged, the cell numbers for tumor development were increased and remarkable tumor growth inhibition was observed.To...
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