脂质体介导mIL-12基因与MHCⅠ基因联合对小鼠实验性肝癌的治疗作用

背景与目的：白细胞介素-12及MHCⅠ基因均已单独用于肿瘤基因治疗,为探索两者的抗肿瘤协同效应,本研究探讨小鼠白细胞介素-12（mIL-12)基因与同种异型的MHCⅠ（小鼠为H-2K）基因联合治疗Balb/C小鼠实验性肝癌。方法：分别应用含mIL-12基因,C57BL/6小鼠H-2K^bcDNA及绿色荧光蛋白（GFP）报告基因的真核表达质粒载体pcDNA3。体外经新型脂质体LipofectAMINE2000(LF2000)介导转染小鼠肝癌细胞株MM45T.Li,检测转染细胞外源基因的表达,将经LF2000介导pcDNA3/mIL-12与pcDNA3/H-2K^b转染的MM45T.Li细胞,注射于小鼠皮下,观察致瘤性的变化,在荷瘤Balb/C小鼠瘤内注射上述脂质体-DNA复合物,观察瘤体生长情况及小鼠生存期的改变。结果：LF2000介导pcDNA3/GFP转染MM45T.Li细胞的最佳条件为脂质体与DNA为3：1（μg：μg）,转染效率达30%,经LF2000介导pcDNA3/mIL-12与pcDNA3/H-2K^b转染的MM45T.Li细胞,RT-PCR检测有mIL-12和H-2K^bcDNA的特异扩增片段,WesternBlot检测显著有57kDaH-2K^b蛋白表达,ELISA检测mIL-12分泌量达48ng/ml/10^6细胞,经LF2000介导pcDNA3/mIL-12与pcDNA3/H-2K^b转染的MM45T.Li细胞,其致瘤性下降;荷瘤Balb/C小鼠瘤内注射该脂质体-DNA复合物,FACS检测显示小鼠脾脏淋巴细胞中CD3^ ,CD4^ 和CD8^ 的数量治疗组较对照组高,肿瘤生长相对缓慢,且pcDNA3/mILp-12与pcDNA3/H-2K^b联合治疗具有一定的正协同效应。结论：mIL-12基因与MHCⅠ基因联合治疗小鼠肝癌具有正协同效应,增强了抗肿瘤效果。

Combination therapy of experimental murine hepatoma with mIL-12 gene and MHC I gene mediated by liposome

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.