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

Combination Therapy of Experimental Murine Hepatoma with mIL-12 Gene and MHCⅠ Gene Mediated by Liposome

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-2KbcDNA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-2Kb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-2Kb转染的MM45T.Li细胞,RT-PCR检测有mIL-12和H-2KbcDNA的特异扩增片段,WesternBlot检测显示有57kDaH-2Kb蛋白表达,ELISA检测mIL-12分泌量达48ng/ml/106细胞。经LF2000介导pcDNA3/mIL-12与pcDNA3/H-2Kb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,?

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(μg∶μg). 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/106 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.