CAJ | 학술논문

目的研究一种结合肿瘤基因治疗与病毒治疗优势的新型肿瘤治疗载体系统,即基因-病毒载体系统.方法利用病毒重组技术将抗癌基因插入肿瘤细胞特异性的增殖病毒基因组中,通过细胞病理作用、荧光显微镜、免疫酶链技术及电镜等技术分别观察病毒的杀伤效应、报告基因绿色荧光蛋白、抗癌基因小鼠白细胞介素12表达量及病毒复制情况.结果构建了一种新型基因-病毒载体系统,该载体系统腺病毒E1b-55 kDa蛋白缺失, 保留了腺病毒E1a蛋白.该载体系统具有肿瘤增殖病毒ONYX-015相似功能, 即它可在肿瘤细胞内复制及增殖,而在正常细胞内不能复制及增殖,从而特异性杀灭肿瘤细胞.它还具有更大的优势, 即该载体系统可携带各种抗癌基因以进一步提高抗肿瘤的疗效.应用该载体系统携带该报告基因绿色荧光蛋白可使绿色荧光蛋白在肿瘤细胞内高效表达, 其表达量明显高于传统基因治疗的腺病毒载体系,而在正常细胞内低表达, 与传统腺病毒载体系统相似或更低.应用该载体系统携带抗癌基因小鼠白细胞介素12,也产生类似结果,并在电镜中证实该载体系统携带抗癌基因小鼠白细胞介素12可在肿瘤细胞株中复制及增殖.结论基因-病毒载体是将抗癌基因插入肿瘤增殖病毒基因组,通过肿瘤增殖病毒在肿瘤细胞内特异性复制及增殖,从而数百倍乃至上万倍提高抗癌基因的表达量.它充分利用了肿瘤基因治疗与病毒治疗优势,进一步提高抗肿瘤的疗效,克服了传统基因治疗转染率低、表达量少及靶向性弱等缺点及病毒治疗的杀伤力低的缺点.它将成为肿瘤治疗最有希望的治疗方案之一.
목적연구일충결합종류기인치료여병독치료우세적신형종류치료재체계통,즉기인-병독재체계통.방법이용병독중조기술장항암기인삽입종류세포특이성적증식병독기인조중,통과세포병리작용、형광현미경、면역매련기술급전경등기술분별관찰병독적살상효응、보고기인록색형광단백、항암기인소서백세포개소12표체량급병독복제정황.결과구건료일충신형기인-병독재체계통,해재체계통선병독E1b-55 kDa단백결실, 보류료선병독E1a단백.해재체계통구유종류증식병독ONYX-015상사공능, 즉타가재종류세포내복제급증식,이재정상세포내불능복제급증식,종이특이성살멸종류세포.타환구유경대적우세, 즉해재체계통가휴대각충항암기인이진일보제고항종류적료효.응용해재체계통휴대해보고기인록색형광단백가사록색형광단백재종류세포내고효표체, 기표체량명현고우전통기인치료적선병독재체계,이재정상세포내저표체, 여전통선병독재체계통상사혹경저.응용해재체계통휴대항암기인소서백세포개소12,야산생유사결과,병재전경중증실해재체계통휴대항암기인소서백세포개소12가재종류세포주중복제급증식.결론기인-병독재체시장항암기인삽입종류증식병독기인조,통과종류증식병독재종류세포내특이성복제급증식,종이수백배내지상만배제고항암기인적표체량.타충분이용료종류기인치료여병독치료우세,진일보제고항종류적료효,극복료전통기인치료전염솔저、표체량소급파향성약등결점급병독치료적살상력저적결점.타장성위종류치료최유희망적치료방안지일.
Objective To develop a new kind of vector system called gene-viral vector, which combines the advantages of gene and virus therapies.Methods Using recombinant technology, an anti-tumor gene was inserted into the genome of replicative virus specific for tumor cells. The cell killing effect, reporter gene expression of the green fluorescence protein, anti-tumor gene expression of mouse interleukin-12 (mIL-12) and replication of virus were observed by the methods of cell pathology, fluorescence microscopy, ELISA and electron microscopy, respectively.Results A new kind of gene-viral vector system of adenovirus, in which the E1b-55 kD gene was deleted but the E1a gene was preserved, was constructed. The vector system, like the replicative virus ONYX-015, replicated and proliferated in tumor cells but not in normal ones. Our vector had an advantage over ONYX-015 in that it carried different kinds of anti-tumor genes to enhance its therapeutic effect. The reporter gene expression of the green fluorescence protein in tumor cells was much better than the adenovirus vector employed in conventional gene therapy, and the expression in our vector system was as low as or even less than that in the conventional adenovirus gene therapy system. Similar results were observed in experiments with this vector system carrying the anti-tumor gene mIL-12. Replication and proliferation of the virus carrying the mIL-12 gene in tumor cells were confirmed by electron microscopy.Conclusions Gene-viral vectors are new vectors with an anti-tumor gene inserted into the genome of replicative virus specific for tumor cells. Because of the specific replication and proliferation of the virus in tumor cells, expression of the anti-tumor gene is increased hundreds to thousands of times. This approach takes full advantages of gene therapy and virus therapy to enhance the effect on the tumor. It overcomes the disadvantages of conventional gene therapy, such as low transfer rate, low gene expression, lack of target tropism, and low anti-tumor activity. We believe that this is a promising means for future tumor treatment.