Anti-tumor Effectivity Of CD40L Activated B Lymphocyte After Hepatocellular Carcinoma AFPmRNA Transfection

ObjectivesFirst, to construct AFP eukaryotic expression vector, then transcribe AFPmRNA and transfect it into B lymphocytes activated by CD40L in vitro for preparation of AFP-B cell vaccine. And induce specific CTL immunity against AFP.then discuss preliminary the feasibility of immunotherapy of hepatocellular carcinoma as a target for AFP.Second, Total RNA of Hepal-6,a hepatocelluar carcinoma cell line,were introduced into mouse B lymphocytes activated by CD40L,then study the antitumor effectivity of B lymphocytes in the context of inducing cytotoxic T lymphocytes (CTLs).MethodsFirst, T and B lymphocytes were collected, isolated and purfied from mouse spleen lymphocytes by density gradient centrifugation method. B cells were initially activated by CD40L and rmIL-4. The expression of B cell markers and major histocomability complex (MHC) on cell surface were detected by FCM.Second, the goal gene AFPcDNA was cloned by RT-PCR. Insert cloned AFPcDNA into the plasmid pGEM4Z/A64 in the direction to create the plasmid pGEM4Z/AFP/A64. It was determined by enzyme digestion and electrophoretic analysis. Linearization of pGEM4Z/AFP/A64 with SpeI, then followed by in vitro transcription AFPmRNA with T7 RNA polymerase.Third, AFPmRNA was transfected into activated B lymphocytes. The expression of B cell surface markers was determined.CTL was obtained by stimulating T lymphocytes with transfected B lymphocytesd, and then the killing activity of CTL and the IFN-r secretion were quantified. Last, Total RNA was extracted from Hepal-6, and then it was transfected into B lymphocytes. The expressions of antigen presenting cell markers and major histocomability complex on cell surface were detected, and then the killing activity of CTL and the IFN-r secretion were quantified.ResultsFirst, The MHC and co-stimulating molecules(CD40,CD86,CD80,H-2Kb,I-Ab) expression of B lymphocytes activated by CD40L and rmIL-4 were higher than unactivated B cells(p<0.05). OD value of B cell activated stimulator T cell was higher than unactivated B cell group (p<0.05).Second, the vector pGEM4Z/AFP/A64 was constructed, and it was determined by enzyme digestion and electrophoretic analysis that a clear band at 1800bp position was showed, consistenting with the target gene 1818bp. A 70.0kD specific protein band was detected by Western blot; consistenting with molecular weight of the target protein.Third, the MHC and co-stimulating molecules expression of B lymphocytes transfected by AFPmRNA were higher than untransfected B cells, comparison between the two groups (p<0.01). OD value of transfected B cell stimulator T cell was higher than untransfected B cell group(p<0.05).The killing activity and IFN-y secretion of CTL after stimulation of AFPmRNA-transfected B lymphocyte was more significant than control group, comparison between the two groups (p<0.01).Last, Electrophoresis of the whole RNA extracted from Hepal-6 cells showed that 28s,18s and 5s bands. The MHC and co-stimulating molecules expression increased significantly higher than other groups after total RNA transfected, comparison with the concrol groups p<0.05. OD value of B lymphocyte transfected by total RNA stimulator T cell was higher than control groups.The killing activity and IFN-y secretion of CTL after stimulation of RNA-transfected B lymphocyte was more significant than control group (p<0.05). ConclusionFirst, B lymphocytes loaded with AFPmRNA can induce AFP-specific CTL in vitro.Second, HCC RNA-transfected B lymphocyte is capable of inducing anti-tumor effects through CTL response. It may present two potential pathways for future HCC treatment.