iNOS转基因诱导裸鼠移植瘤细胞凋亡及其与p53表达相关性的实验研究

APOPTOSIS OF TRANSPLANTED TUMOR CELLS INDUCED BY GENE TRANSFECTION OF iNOS IN NUDE MICE AND THE CORRELATION WITH p53 EXPRESSION

目的观察诱生型一氧化氮合酶(inducible nitric oxide synthase,iNOS)基因转染对人喉鳞癌细胞裸鼠移植瘤生长的影响,研究在体人喉鳞癌细胞中iNOS与突变型p53基因的表达,探讨二者的相关性及促进肿瘤细胞凋亡的机制。方法将iNOS基因转染人喉鳞癌Hep-2细胞并筛选出阳性克隆细胞进行扩增,制备荷瘤细胞小鼠模型,致瘤小鼠随机分为3组,转染组,转染空载体组,未转染组。应用逆转录-聚合酶链反应检测各组喉鳞癌组织中iNOS与p53 mRNA的表达,免疫组织化学检测各组喉鳞癌组织中iNOS与p53蛋白的表达,流式细胞术检测各组喉鳞癌组织中iNOS与p53蛋白、细胞凋亡及细胞周期的变化。结果转染组iNOS与p53蛋白及其mRNA的表达明显高于转染空载体组和未转染组,且iNOS与p53的表达呈明显的正相关(P<0.01)。转染空载体组和未转染组iNOS与p53蛋白及其mRNA的表达无明显差别。同转染空载体组和未转染组相比,转染组喉癌细胞发生了明显的G0/G1期阻滞,同时出现了明显的凋亡峰,瘤体积出现了明显的减小,转移发生率明显降低。结论iNOS基因转染能明显增加iNOS mRNA的表达从而增加了iNOS蛋白的表达,iNOS蛋白的表达使瘤细胞内合成大量的一氧化氮,从而诱导p53基因的大量表达,促进了肿瘤细胞凋亡,推测p53基因诱导肿瘤细胞凋亡的机制可能同细胞发生G0/G1期周期阻滞有关。

Objective To observe the influence of gene transfection of inducible nitric oxide synthase （iNOS） to the transplanted human laryngeal squamous carcinoma in nude mice, to investigate the expression and the correlayion of iNOS and p53, and to proke into the possible apoptosis mechanism. Methods Transfect the gene of iNOS to Hep-2 human laryngeal cancer cell,sieve out positive clone cell and amplify. Prepare bearing cancer mice, the 15 mice were divided into three groups, group I （transfec iNOS）, group Ⅱ （transfect no-load）, group Ⅲ（transfect nothing）, iNOS and p53 mRNA level was determined by reverse transcription- polymerase chain reaction （RT- PCR）. Flow cytometry （FCM） was used to measure the distribution of cell cycles and the protein level of iNOS and p53. It was also used to detect cell apoptosis. The protein level of iNOS and p53 was also determined by immunohistochemistry. Results The mRNA and protein level of iNOS and p53 in group I were significantly higher than that in group Ⅱ and Ⅲ. The high expression of p53 was positive correlation with high expression of iNOS（P〈0.01）. The mRNA and protein expression of iNOS and p53 in group Ⅱ and Ⅲ had no significant difference. Compared to group Ⅱ and Ⅲ,the gene transfection of iNOS of group I caused significantly G0/G1 cell cycle arrest, meanwhile group I showd a visible peak of apoptosis, the volume become smaller, the rate for transferring become lower too. Conclusion Gene transfection of iNOS evidently increased the mRNA level of iNOS in Hep-2 human laryngeal cancer cell,then increased the protein level of iNOS,the high expression of iNOS induced more NO synthesis, further, massive NO induced high expression of gene of p53, meanwhile induced apoptosis in the cell. It was speculated that the high apoptosis rate was related to G0/G1 cell cycle arrest.