| Part IStudy on Construction of PCI-neo-B7.1, PCI-neo-B7.2, PCI-neo-4-lBBL, pcDNA3.1-B7.1-IRES-B7.2 and Their Stable Expressions in Mouse HCC Cell LineObjectiveTo study construction of PCI-neo-B7.1, PCI-neo-B7.2, PCI-neo-4-lBBL, pcDNA3.1-B7.1-IRES-B7.2 and their stable expressions in mouse HCC cell line H22.MethodsThe murine full-length B7.1, B7.2 and 4-1BBL cDNA were obtained and subcloned into PCI-neo mammalian expression vector. The recombinants were named PCI-neo-B7.1, PCI-neo-B7.2 and PCI-neo-4-lBBL, and identificated with restriction enzyme digestion and sequencing. The recombinant named pcDNA3.1-B7.1-IRES-B7.2 was constructed and identificated with restriction enzyme digestion.Subsequently, four recombinants were transfected into H22 with Lipofectamine Reagent, then G418-resistence colonies were acquired, and named H22-CD80+ ?H22-CD86+?H22-CD137L+ ? H22-CD80/CD86+ and H22-CD80/CD86/CD137L+, respectively. RT-PCR and FCM were applied to determine whether there were stable mRNA expressions of objective genes in variants.Results1. PCI-neo-B7.1/B7.2/4-lBBL were digested, then electrophoresis of the digested products showed fragments of 862bp(mB7.1), 984bp(mB7.2) , 980bp(m4-lBBL) and 5.4kbp(PCI-neo). DNA sequenceanalysis was identical to the sequence of mB7.1/B7.2/4-1 BBL in the Genebank and did not reveal any mutation.2. pcDNA3.1-B7.1-IRES-B7.2 was digested, then electrophoresis of the digested products showed fragments of 862bp(mB7.1), 984bp(mB7.2) and 5.6kbp(pcDNA3.1), which indicated the construction was successful.3. RT-PCR showed expressions of mB7.1/B7.2/4-lBBL gene with strong intensity in H22-CD80+, H22-CD86+ and H22-CD137L+.4. RT-PCR showed dual expressions of mB7.1/B7.2 gene with strong intensity in H22-CD80/CD86+.5. FCM showed co-expression of mB7.1/B7.2/4-1 BBL gene with strong intensity in H22-CD80/CD86/CD137L+.ConclusionsThe recombinants ( PCI-neo-B7.1,PCI-neo-B7.2,PCI-neo-4-lBBL and pcDNA3.1-B7.1-IRES-B7.2) have been constructed successfully, and their stable and effective expressions also have been obtained in H22 variants, respectively.Part Impact on Immunogenicity, Anti-tumor Immunity of Wild H22 and Proliferation of Tumor with Co-expression of B7.1, B7.2 and 4-1 BBL Genes in H22-BALB/C HCC Mouse ModelObjectiveTo establish H22-BALB/C HCC mouse model and discuss the impact on immunogenicity, anti-tumor immunity of H22 variants and proliferation of tumor with co-expression of B7.1, B7.2 and 4-1 BBL genes.Methods1. The mice were divided into five groups, named A, B, C, D and E, in randomization, and group A, B were control group (CG). H22-BALB/C HCC mouse model was established by injection subcutaneously withH22-Wt, H22-neo, H22-CD80/CD86+, H22-CD137L+ and H22-CD80/CD86/CD137L+, respectively.2. The rate and incubation period of developing tumor, the rate of survival, and the proliferation of tumor in vivo were observed and recorded. The impact of gene transduction on immunogenicity and antitumor immunity were performed with re-innoculation of wild H22.3. At different time post- first innoculation of tumor cells, the mixed T lymphocyte culture (MTLCs) was performed with tumor cell and lymphocytes from spleens. To study the impact of variants on immune response(IR), Lymphocyte proliferation was tested in vitro, cytotoxic activity of CTL cells were evaluated by LDH assay, the expressions of IL-2, IFN- Y in culture solution of MTLCs were determined, RT-PCR was applied to detect the expression of IL-2, IFN- , ICAM-1, TNF- a and TNF- a receptor II mRNA.Results1. The hepatoma animal model was established successfully. The rate developing tumor in group A,B,C and D was 100%, respectively. Nevertheless, the rate was only 50% in group E, which was very lower than that of the other four groups(P< 0.01). The incubation period of developing tumor was: 6 ?1.2d in group A, 5.5 ?1.4d in group B, 13.9?.1 din group C, 14.2 ?2.9d in group D, 14.9 ?2.0d in group E, with no difference in group C, D and E, and...
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