NP及HN和F基因抗人喉癌的实验研究

The study of recombinant plasmid of NP,HN and F gene therapy on the transplantable model of human laryngeal squamous carcinoma in nude mice

目的:探讨基因治疗喉癌的新方法。方法:采用RT-PCR方法及基因重组技术,将新城疫病毒(NDV)的NP、HN及F基因克隆于真核表达质粒pcDNA3中。将体外培养的人喉上皮癌细胞系Hep-2细胞注射给15只裸鼠,制作人喉癌裸鼠模型。肿瘤细胞接种3周后,随机将裸鼠分成2组:实验组(8只),将pcDNA3-NP、pcDNA3-HN及pcDNA3-F混合注射于肿瘤局部进行治疗;对照组(7只):将同量空质粒pcDNA3注射于肿瘤局部进行治疗。将2组治疗后的肿瘤组织在光镜和电镜下进行组织病理学及超微结构观察;提取裸鼠各种组织染色体DNA为模板,以成功克隆NP基因的引物为引物,进行PCR检测是否存在NP基因;取裸鼠血清,以NDV V4株作为抗原,进行血清酶联免疫吸附(ELISA)测定。结果:实验组与对照组间的鼠重、瘤重、瘤体积和抑瘤率的差异均有统计学意义(均P<0.01)。对照组裸鼠死亡1只,其余裸鼠呈恶液质状态,肿瘤呈结节状,表面有破溃。实验组肿瘤较对照组小,肿瘤呈圆形,质地较硬,与皮肤及深部组织无粘连;光、电镜下可见肿瘤细胞坏死与凋亡。实验组未检测到NP基因;实验组血清中抗NDV的免疫球蛋白为阳性,对照组为阴性。结论:NP、HN及F基因具有抗肿瘤作用。

Objective： To explore a new method in the gene therapy of human laryngocarcinoma. Method.. The NP, HN and F gene of the Newcastle disease virus were cloned into the pcDNA3 by RT-PCR. The human laryngeal squamous carcinoma model in nude mice had been established by transplantation of the Hep-2 cell line . The nude mice had been grouped two in the third week of the Hep-2 cell line had been transplanted. The admixture of the pcDNA3-NP and the pcDNA3-HN and pcDNA3-F were injected into the tumor in the experimental group, the equal weights pcDNA3 was injected into the tumor in the control group. The microstructural and ultra micro structural changes of carcinoma were observed under light and eletro microscopes. The chromatosome DNA were extracted from all tissue of the nude mice, the DNA was tested to do template by PCR, whether or not exist the NP gene, The nude mice serums were examined by the method of enzyme-linked immunosorbent assay（ELISA）for the antigen of the DNA V4. Result： There were statistically significant difference in the mice weight, the tumor weight, the tumor volume and the restrained tumor rate between the experimental group and the control group （each P 〈0. 01）. A nude mouse died in the control group, the others were very asthenic. The tumor looked like nodosity and the tumor surface were defeated. The tumor in the experimental group were smaller than that in the control group, the tumors were round shape and hard and not adhered on the skin and deep part constitution. The necrosis and apoptosis of the tumor cell had been found under light and eletron microscopes. The NP gene had not been explored in all tumor tissue by PCR in the experimental group. The experimental group was positive in ELISA,The control group was negative. Conclusion; The NP, HN and F gene could play a role in treating tumor.