【作者】 雷磊；

【导师】 姜泗长； 杨伟炎； 韩东一；

【作者基本信息】 中国人民解放军军医进修学院 ， 耳鼻咽喉—头颈外科， 2002， 博士

【摘要】 喉癌是一种较常见的恶性肿瘤，严重危害人类的健康。在我国，喉癌约占全身肿瘤的2％，占头颈部肿瘤的11—12％。目前临床上常用的治疗方法（手术、放疗和化疗）都存在一定的局限性，因此需要探索一种有效治疗喉癌的的新方法。军事医学科学院二所三室构建的携带人野生型p53基因和肿瘤免疫相关基因B7—1、GM—CSF的重组腺病毒（命名为BB—102）感染喉癌原代细胞后，能明显抑制癌细胞的增殖并诱导其凋亡，而且体外能诱导自体外周血淋巴细胞生成肿瘤杀伤性CTL细胞。本研究在此基础上，研究BB—102对种植喉癌细胞系的裸鼠的治疗效果，为进一步走向临床提供新的治疗方案。同时，我们还探讨了p14^ARF对喉癌细胞的抑制作用及对内源性p53的影响，以便加强自体p53水平，从而为进一步防治喉癌提供实验依据。据此，本研究完成以下工作： 1．腺病毒的扩增与质量检定 在293细胞中分别扩增BB—102和携带绿色荧光蛋白基因的对照重组病毒Ad-GFP；以氯化铯密度梯度超速离心法纯化病毒；以蚀斑分析法测定病毒滴度；按照《中国生物制品规则》之通则，检测重组腺病毒BB—102的质量。结果表明：Ad-GFP和BB—102的滴度分别为1.1×10¹¹、2.4×10¹⁰；重组腺病毒BB—102的质量符合《中国生物制品规则》的要求；重组腺病毒介导的目的基因可以在喉癌细胞中有效表达。 2．重组腺病毒介导人野生型p53、B7—1和GM—CSF基因对喉癌的临床前研究 建立喉癌裸鼠模型，瘤内注射BB—102、Ad-GFP和PBS，在裸鼠体内进行人喉癌的基因治疗的临床前研究，对肿瘤组织进行测量、病理学检查、摘 要免疫组化等研究。结果表明：在实验组与对照组之间经多因素析因方差分析，肿瘤重量和瘤体积具有显著性差异。经治疗后，实验组肿瘤中未发现大变个P53的表达，Ki67阳性表达率极低，悦明BB—102在喉癌细胞中有效表达并能显著抑制喉癌细胞在裸鼠体内增殖。同时光镜和透射电镜下可见肿瘤细胞的坏死、i)M亡及炎性细胞浸润。 3．pl4叩’基因抑制喉癌细胞生长及其对内源性p53表达的影响 采用基因转染技术，将全长野生型 PI4“”CDNA转染到人喉鳞状细胞癌HepZ细胞中，利用流式细胞仪、RT—PCR和 WesternEloting等万法研究其对细胞周期、内源性野生型p53表达的影响。结果显示：转染N叩’基因的Hep习细胞的生长受到明显抑制，其克隆形成能力为转染空载体的 5 7 ％。细胞分析表明，转染刊“”CDNA48 ’J＼时后，处在＊G；和 G。／M期的细胞皆增加了一倍。同时，Western Bloting分析结果表明，内源性野生型p53表达明显卜升。 根据以上比较实验，我们得到如下结论： BB－102在喉癌基回治疗中具有较好的应用前景，可望发展成临床卜的一种抗癌剂。同时，pl4“”基因的失活可能是喉癌发生过程中的一个重要因素，野生型p14的恢复，上调喉癌HepZ细胞中内源性野生型p53的表达并抑制喉癌细胞生长，进一步发挥p53肿瘤抑制作用，有可能成为临床基因治疗的又一新靶点。更多还原

【Abstract】 Laryngeal carcinoma is a very common malignant tumor, which is seriously detrimental to human health. It has been estimated that the incidence of laryngocarcinoma is 2% of all human tumors and 11-12% of the head and neck tumors. Conventional therapeutic methods such as surgical treatment ,radiotherapy and chemotherapy are being used currently, but all have certain limitations. It therefore ,is needed to develop a new kind of therapy for effective treatment of laryngeal carcinoma.In Department of Experimental Hematology of Beijing Institute of Radiation Medicine, they constructed a recombinant adenovirus carrying human wild -type p53 gene and tumor immunity-related genes -B7-1 and GM-CSF, named.as BB-102, and observed that proliferation of the human laryngocarcinoma cells was inhibited markedly and apoptosis was induced after infection with BB-102.Besides,such infected cells could also induce in vitro autologous peripheral blood lymphocytes to transform into tumor-killing cytotoxic T lymphocyte (CTL).Hence, in the present study the effect on cotransduction of human wide-type p53,B7-l and GM-CSF genes mediated by recombinant adenovirus on nude mice model bearing laryngocarcinoma were studied to explore the biological value in the therapy of human laryngeal carcinoma. Along with the study, we also explore the inhibitory effect of p!4ARF on the cell growth of laryngeal carcinoma and the expression on endogenous p53 to intensify the autologous level of p53 , thus providing experimental evidence for new therapeutic measures of laryngeal carcinoma.Construction and production of recombinant adenovirusesRecombinant plasmids pBB-102 (containing human wild-type p53, GM-CSF and B7-1 genes) were constructed by molecular cloning. Recombinant adenoviruses BB-102 were constructed by homologous recombination in 293 cells. BB-102 and Ad-GFP (control virus) were amplified in 293 cells on a large scale and purified by ultra-centrifugation in CsCl step gradient solutions. The viral particles were determined by OD260nm and the purity was evaluated by the ratio of OD260 nm/OD280 nm. The viral liters were determined by plaque assays.Preclinical study of recombinant adenovirus in the treatment of human laryngeal squamous carcinomaNude mice model bearing laryngocarcinoma were established using human laryngeal squamous carcinoma cell line (Hep-2). Large amounts of recombinant adenovirus were injected into the tumor. Changes in carcinoma administrated with recombinant adenovirus were observed under light and electron microscopes. The difference between experimental and control groups was statistically significant. The necrosis of the tumor cells and inflammatory cellular infiltration were found under light and electron microscopes. The morphology of cells infected with BB-102 was analyzed for evidence of apoptosis by transmission electron microscopy. It has been shown that wild type P53 protein can inhibit cell growth and induce apoptosis, while B7-1 and GM-CSF have no such effects. So the high level expression of p53 gene mediated by BB-102 is reasonably the major cause of cell growth inhibition and apoptosis.Effect of p!4ARF gene on cell growth of human laryngeal tumor cells and expression of endogenous p53 proteinp!4ARFcDNA was transferred to the cell line Hep-2 of squamous cell carcinoma of the larynx by gene transfer to study the cell cycles and the expression of endogenous wild type p53 by using flow cytometry, RT-PCR and Western-blotting. Expression of p!4ARF significantly affected the Hep-2 cell growth. The clone-forming efficiency of the Hep-2 cells transferred p!4ARF was 57%,compared with empty vector pcDNA3. The numbers of cells transferred p!4ARF at the Go/G,,G2/M phase as twice as the control after 48 hours transferred with p!4ARF cDNA . The expression of endogenous wild type p53 significantly enhanced.As mentioned above, the results showed that BB-102 has significant efficacy on suppressing tumor cell growth and inducing their apoptosis, which suggested that BB-102 might be further devel更多还原