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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 of gene therapy for laryngeal cancer. Methods: NP, HN and F genes of Newcastle disease virus (NDV) were cloned into eukaryotic expression plasmid pcDNA3 by RT-PCR and gene recombination. In vitro cultured human laryngeal carcinoma cell line Hep-2 cells were injected into 15 nude mice and a laryngeal cancer nude mouse model was made. The tumor cells were randomly divided into two groups: experimental group (n = 8), pcDNA3-NP, pcDNA3-HN and pcDNA3-F were injected into the local tumor for treatment. Control group (n = 7) The same amount of empty plasmid pcDNA3 injection in the tumor local treatment. The tumor tissues of two groups were observed under light microscope and electron microscope for histopathological and ultrastructural observation. The chromosomal DNA of various tissues of nude mice was extracted as template, and the primers of cloned NP gene were used as primers to detect the existence of PCR NP gene. The serum of nude mice was taken and the NDV V4 strain was taken as the antigen for determination of serum enzyme-linked immunosorbent assay (ELISA). Results: There was significant difference in weight, tumor weight, tumor volume and tumor inhibition rate between the experimental group and the control group (all P <0.01). The control group of 1 death of nude mice, the other nude mice showed catecholamines, the tumor was nodular, ulcerated on the surface. The tumor in the experimental group was smaller than the control group, the tumor was round, the texture was hard, and there was no adhesion with the skin and the deep tissue. The necrosis and apoptosis of tumor cells were observed under light and electron microscope. The NP gene was not detected in the experimental group; the immunoglobulin of anti-NDV in the serum of the experimental group was positive and the control group was negative. Conclusion: NP, HN and F genes have anti-tumor effect.