RESUMO
EGR-1 gene promoter and CDglyTK gene were used to construct the pcDNA3-EGR-CDglyTK recombinant vector, in which CDglyTK gene expression was under the control of EGR-1 gene promoter. Cationic liposome LipofectAMINE was used to transfect plasmids into human hepatoma 7402 cell line. Subsequently, the transfected cells were treated with different doses of gamma-ray. Northern blot and Western blot showed that ionizing radiation can induce CDglyTK gene expression drived by EGR-1 promoter,in a dose-dependant manner. There was no ionizing radiation-inducible effect in pcDNA3-CMV-CDglyTK control group. Ionizing radiation also markedly enhanced the sensitivity of tumor cells transfected with pcDNA3-EGR-CDglyTK to prodrugs (GCV/5-FC) in tumor cell killing. The data indicated that EGR-1 promoter was inducible by ionizing radiation, whereas the CMV promoter was not. There was a synergetic effect between GCV and 5-FC. The cytotoxic effect of the suicide gene-ionizing radiation combination was stronger than suicide gene alone or ionizing radiation alone.
Assuntos
Proteínas de Ligação a DNA/genética , Raios gama , Proteínas Imediatamente Precoces , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Northern Blotting , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Citomegalovirus/genética , Citosina Desaminase , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Proteína 1 de Resposta de Crescimento Precoce , Flucitosina/farmacologia , Ganciclovir/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Lipossomos , Nucleosídeo Desaminases/genética , Nucleosídeo Desaminases/metabolismo , Plasmídeos/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
BACKGROUND & OBJECTIVE: Interleukin-12 gene and MHC I gene have been used in gene therapy for cancer individually. To explore the synergistic antitumor effects of these two genes, the therapeutic effects of mIL-12 gene combined with MHC I (mouse H-2K) gene mediated by cationic liposome for experimental murine hepatoma were investigated. METHODS: Balb/C mouse liver cancer MM45T. Li cells were transfected with pcDNA3/mIL-12, pcDNA3/H-2Kb(H-2K of C57BL/6 mouse), and pcDNA3/GFP (reporter gene) mediated by lipofectAMINE 2000(LF 2000). The expressions of foreign genes in transfected cells were detected. Balb/C mice were inoculated subcutaneously with pcDNA3/mIL-12 and pcDNA3/H-2Kb transfected MM45T. Li. The tumorigenesis of the inoculated cells was detected. After intratumoral injection with LF2000-plasmid DNA complexes, the growth of murine tumor and the survival time of the tumor bearing mice were observated. RESULTS: The optimal ratio of LF2000: DNA is 3:1(microgram: microgram). The transfection efficiency reached to 30%. RT-PCR result showed the specific amplified fragments of the mIL-12 cDNA and H-2Kb cDNA in the transfected cells. Western blot analysis showed the expression of H-2Kb protein at 57 kDa. ELISA assay showed that the secretory mIL-12 was 48 ng/ml/10(6) cells. The tumorigenesis was decreased for transfected MM45T. Li cells with pcDNA3/mIL-12 and pcDNA3/H-2Kb. FACS assay showed that the numbers of CD3+, CD4+, and CD8+ cells from murine spleen were increased more in therapeutic group than in control group. The tumors grow slowly. The mIL-12 gene combined with H-2Kb gene has stronger antitumor effect for mouse liver cancer than single gene. CONCLUSION: The combination therapy with mIL-12 gene and MHC I gene mediated by LF-2000 have the positive synergistic antitumor effect for experimental murine hepatoma.