【作者】 蔡鑫章；

【导师】 赵素萍；

【作者基本信息】 中南大学 ， 耳鼻咽喉科学， 2007， 博士

【摘要】 鼻咽癌（Nasopharyngeal carcinoma，NPC）是我国南方常见的恶性肿瘤之一，在头颈部恶性肿瘤中占首位。鼻咽癌原发部位隐蔽，不易观察，且与鼻腔、鼻窦和颅内相毗邻，所以临床症状出现较晚而且各异；鼻咽癌也是头颈肿瘤中转移率最高的。这些都导致了鼻咽癌常常容易被误诊或漏诊，影响治疗效果。鼻咽癌的发病人群以40～50岁的青壮年多见，一旦发病对社会、经济和家庭造成较大影响。因此，鼻咽癌的早期诊断和治疗的研究一直是我国耳鼻咽喉—头颈外科学研究的重点之一。目前鼻咽癌的治疗还是以放射治疗为主。对放疗不敏感、放疗后复发以及中晚期的患者，可采取化学药物治疗，但这只是辅助性治疗或姑息性治疗。鼻咽癌的手术治疗也只在一些特殊情况下作为辅助方法采用。除了以上三种常规治疗，近年来也有作者尝试应用光动力疗法和微波热疗治疗鼻咽癌，但也只能作为辅助手段。重组DNA技术的发展使肿瘤的基因治疗成为可能。根据肿瘤发生机理制定的基因治疗方案已成为肿瘤治疗的热点。早期研究者主要运用反义核酸技术干预肿瘤相关基因的表达，取得了一定效果。近年来，RNA干扰技术在哺乳动物体内的应用，更为肿瘤的基因治疗开辟了一个新的视野。目前应用于肿瘤基因治疗的RNA干扰技术大多是以非编码的小干扰RNA（siRNA）为效应分子。对另一种非编码RNA——microRNA（miRNA）的应用研究才刚刚起步。miRNA是机体内源性的RNA干扰触发器，它们可以通过在经典的RNA干扰途径降解靶基因mRNA、也可以直接抑制靶基因的蛋白翻译、还可以通过快速脱腺苷化降解mRNA。miRNA已经被看作生物体内固有的基因调控子中最大的一个家族。本研究的目的是模拟天然miRNA构建表达miRNA前体的质粒，实现调控肿瘤相关基因——人端粒酶逆转录酶（hTERT）基因和血管内皮生长因子（VEGF）基因的表达，开展对鼻咽癌的基因治疗研究。整个研究内容分三部分进行：第一部分调控目标基因的miRNA表达质粒的构建和有效质粒筛选。目的是确定针对hTERT和VEGF基因的miRNA的有效作用位点，观察构建的候选质粒对鼻咽癌中两个靶基因的调控效应，为后续的体外和体内实验奠定基础。结果针对hTERT和VEGF基因分别构建了3个质粒，每个基因各筛选出了1个有效质粒，分别是：pcDNA6.2-GW／EmGFP-miR-hT2035、pcDNA6.2-GW／EmGFP-miR-V1025；并确定了2035～2055为hTERT的有效作用位点，1025～1045为VEGF基因的有效作用位点；将两个有效质粒瞬时转染CNE-2细胞后，应用实时荧光定量RT-PCR观察到miRNA使hTERT和VEGF基因的mRNA水平分别下调为77.3％和79.3％；应用Westernblotting观察到两个基因的蛋白表达分别下调73.2％和80.2％；应用CCK-8细胞增殖抑制实验和流式细胞术发现：miRNA对hTERT基因的调控还可以使肿瘤细胞增殖受到抑制，最高抑制率可达到51.1％，细胞周期受阻于G₀～G₁期，诱导肿瘤细胞凋亡。第二部分观察调控hTERT和VEGF的miRNA表达质粒对CNE-2细胞的抑制作用。首先利用第一部分筛选出的两个有效的miRNA重组质粒，构建了能够同时调控hTERT和VEGF两个基因的质粒，即pcDNA6.2-GW／EmGFP-miR-hTERT-VEGF。将该质粒和第一部分获得的两个质粒以及阴性质粒分别转染CNE-2细胞株，经抗生素筛选后成功建立4种稳定表达miRNA质粒的CNE-2细胞株。应用实时荧光定量RT-PCR和Westernblotting观测到调控hTERT表达的质粒和同时调控两个基因的质粒分别下调hTERT的mRNA达87.6％和89.1％，下调蛋白水平达89.4％和80.5％；观测到调控VEGF的质粒和同时调控两个基因的质粒分别下调VEGF的mRNA水平达83.7％和87.5％，下调蛋白水平达87.7％和81.6％。软琼脂集落实验证实靶向hTERT的质粒和同时靶向两个基因的质粒都能够抑制CNE-2细胞的集落形成能力。第三部分裸鼠体内实验。将第二部分获得的4种稳定表达具有调控作用的miRNA质粒的CNE-2细胞株和野生型CNE-2细胞株，分别注入裸鼠皮下，成功建立了人鼻咽癌裸鼠移植瘤模型。通过绘制肿瘤生长曲线、计算肿瘤抑制率以及免疫组化等方法，观察体内实验的效果。结果发现调控hTERT、VEGF和同时调控两个基因的3种质粒对裸鼠移植瘤生长抑制率分别为64.8％、68.3％、69.2％。本研究最后得到以下结论：（1）成功构建了调控hTERT和VEGF基因表达的microRNA质粒载体，并筛选出有效下调目标基因表达水平的miRNA质粒；hTERT基因位点2035～2055、VEGF基因位点1025～1045是microRNA调控的有效作用位点。（2）体外实验发现，miRNA表达质粒能够有效调控hTERT和VEGF基因的表达，使其mRNA和蛋白表达均下调；对hTERT的调控还可抑制CNE-2细胞的集落形成能力。（3）成功构建了人鼻咽癌裸鼠移植瘤模型；并发现用于调控hTERT表达、调控VEGF表达和同时调控hTERT和VEGF表达的三种miRNA重组质粒都能有效抑制肿瘤生长，为进一步的临床研究打下了基础。更多还原

【Abstract】 Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in the Southern of China, and it ranks the first in head-neck cancer. The place where NPC originates from is covert and it is difficult to be discovered. The nasopharynx is neighbor to nose cavity, nasal sinuses and encephalic structure, so clinic symptoms appear late, and early signs are multiple. There is the highest incidence of distant metastasis for NPC among head and neck cancer. For the reasons stated above, NPC is easy to be misdiagnosed or fail to be diagnosed, and it’s curative effect is not satisfactory. The maximum age incidence is the fouth to fifth decade. It brings great suffering to patient’s family and society. It is very important to research early diagnosis and therapy of NPC.Nowaday, the major therapeutic modality of NPC is radiotherapy. The chemotherapy only is used in the patients who aren’t sensitive to radiotherapy or the patients who have recurrence tumors after previous radiotherapy, or patients who are found lately. As an adjunctive therapeutic modality, operation only is limited to the special conditions. In recent years, some authors attempt to use photodynamic therapy and microwave hyperthermia therapy to treat NPC, but those methods also are regarded as adjunctive therapy.With the development of DNA recombination, the gene therapy is developed fastly. The gene therapy based on the pathogenesis of carcinoma has become popular. Former researchers mostly used antisense oligodeoxynucleotides (ASODN) to treat NPC, and acquired some achievements. Recently as application of RNA interference (RNAi) in mammal, it provides a new way of gene therapy for carcinoma.Nowaday, siRNA is used as effective molecule to apply to gene therapy for carcinoma. But an other small RNA——microRNA (miRNA) has attracted researcher’s great attention. MicroRNA belongs to a class of non-coding RNAs that act as endogenous triggers of the RNA interference pathway. It can induce RISC to cleave the target gene mRNA though the classic RNAi pathway. It also can suppress translation of target gene, and can direct rapid deadenylation of targe mRNAs to lead to a rapid mRNA decay. MiRNA has been regarded as the biggest family of genome regulators.The purpose of this study is to construct recombination plasmids which express precursor of miRNA——pre-miRNA by simulating natural miRNA, and to attempt to regulate the expression of cancer-related genes——hTERT and VEGF, and to determine gene therapy in NPC. The experiment is divided into three sections:Section one: To construct the plasmid which regulates target genes, and t(?) s(?)reen effective plasmid, and to confirm efficient positions in which miRNA direct against hTERT gene and VEGF gene, and to observe regulative effect of candidate plasmid. These studies will provide the foundation for follow-up experiments in vitro and in vivo. For each gene, three candidate plasmids are constructed and one effective vector is picked up, namely: pcDNA6.2-GW/EmGFP-miR-hT2035, pcDNA6.2-GW/EmGFP-miR-V1025. We find that the loci from 2035 to 2055 are effective to regulate hTERT gene, and the loci from 1025 to 1045 are effective to regulate VEGF gene. After the plasmids are transfected CNE-2 cell, the levels of mRNA of hTERT and VEGF gene are down-regulated to 77.3% and 79.3% respectively, the expression of protein of two genes are also down-regulated to 73.2% and 80.2% respectively, miRNA regulation of hTERT gene results in inhibition of tumor cell proliferation and cell detention at G0～G1 stage and cell apoptosis.Section two: To observe inhibitory effect of CNE-2 cell by regulating expression of hTERT and VEGF gene, by chaining miR-hTERT and miR-VEGF, we construct another plasmid: pcDNA6.2-GW/EmGFP-miR- hTERT-VEGF. Four plasmids, including the miRNA plasmid above and pcDNA6.2-GW/EmGFP-miR-hTERT and pcDNA6.2-GW/EmGFP-miR-VEGF and negative control plasmid, are used to transferred to CNE-2 cell line separately, and to generate stable cell lines that constitutively express miRNA by antibiotics selection. The effects of stable transfection of the miRNA plasmids are observed by qRT-PCR, Western blotting, soft agar colonies formation assay. Result find that four cell lines express stably miRNA, and miRNA regulate expression of targe gene in stable cells.Section three: In vivo, the test of nude mice is carried. Four stable cell lines and wild type CNE-2 cell line are inoculated to subcutis of nude mice, animal models are established. The tumor volume and tumor morphology are observed, and the protein expression is detected by immunohistochemistry. Result find that in addition to negative plasmid, all three plasmids can inhibite growth of the transplanted tumor of nude mice.Conclusions:(1)We construct successfully the recombination plasmid vectors which target hTERT gene and VEGF gene; the plasmid vectors which express miRNA can down-regulate the expression of hTERT gene and VEGF gene efficiently; the miRNA of hTERT gene loci from 2035 to 2055 is effective to down-regulate hTERT gene, the miRNA of VEGF gene loci from 1025 to 1045 is effective to down-regulate VEGF gene;(2)In vitro miRNA plasmids of hTERT and of VEGF can regulate effectively expression of hTERT and VEGF genes, and down-regulate the mRNA and protein level of hTERT and VEGF, and detent the CNE-2 cell at G0-G1 stage, and induce cell apoptosis.(3)In vivo, we establish animal model of NPC in nude mice; the result proved that all of three recombination plasmids which regulate hTERT、VEGF、hTERT and VEGF respectively can efficiently inhibit the growth of the transplanted tumor in mude mice. All results prompt that it might be effective for gene therapy of nasopharyngeal carcinoma.更多还原