AdFOXMlshRNA在鼻咽癌和乳腺癌基因治疗中的疗效分析

发布时间：2018-02-26 22:44

本文关键词： 转录因子FOXM1 腺病毒载体AdFOXM1shRNA RNA干扰肿瘤基因治疗鼻咽癌乳腺癌　出处：《湖南大学》2013年博士论文　论文类型：学位论文

【摘要】：癌症是21世纪困扰人类的难题之一，给人类健康和生命安全带来严重威胁。其中，鼻咽癌是中国南方和东南亚最常见的恶性肿瘤之一，每年大约十万人中就有20例鼻咽癌发生，其发病率和死亡率均居头颈恶性肿瘤首位。乳腺癌是女性排名第一的常见恶性肿瘤，是导致女性死亡的重要原因，且发病率呈逐年上升的趋势，成为全球公共卫生的一大难题。由于传统的治疗癌症的方法如化疗，放疗存在毒副作用等严重缺陷，寻找癌症治疗的新方法和新靶点已成为近年来的研究热点。 FOXM1属于Forkhead Box转录因子家族，参与调节细胞增殖、细胞衰老、DNA损伤修复、器官形成等很多重要的生理过程。大量文献表明FOXM1在多种人类恶性肿瘤中都有过表达的现象，其中包括乳腺癌，但是FOXM1在鼻咽癌中的作用还未见文章报道。本文中我们研究发现FOXM1在鼻咽癌组织和细胞株，以及乳腺导管癌组织和细胞株中均存在高水平的表达。另外，有研究发现FOXM1在维持肿瘤细胞的增殖、分化、凋亡和转移的平衡方面扮演了重要的角色。这些证据预示着FOXM1有可能成为抗肿瘤新方法的靶标。腺病毒是目前基因治疗的主要载体，，具有宿主范围广、感染效率高、滴度高、安全性好，能容纳大片段的外源DNA等优点。RNA干扰是基因治疗中常用的分子工具之一，然而小分子干扰RNA导入靶细胞的效率高低是其成功应用的关键。为了解决这一问题，本课题中我们构建了一个腺病毒介导的shRNA表达系统，命名为AdFOXM1shRNA，用以在肿瘤细胞中特异性的干扰FOXM1的表达，进而研究该载体在鼻咽癌和乳腺癌基因治疗中的初步的疗效分析。通过实验，我们得到了以下结果。首先，摸索本研究所选择的三株细胞中合适的病毒使用剂量，这三株细胞分别是鼻咽癌细胞CNE以及乳腺癌细胞MDA-MB-231和ZR-75-30。实验发现用10pfu/cell的对照病毒AdLacZ感染这三株细胞都几乎能达到100%的感染效率。并且，实验证明在该剂量下我们所构建的AdFOXM1shRNA载体能够非常有效的干扰鼻咽癌和乳腺癌细胞中FOXM1的表达。接着，我们考察了干扰FOXM1的表达对肿瘤细胞增殖能力的影响。与预期的一样，鼻咽癌细胞CNE和乳腺癌细胞MDA-MB-231， ZR-75-30感染AdFOXM1shRNA后，由于FOXM1的表达下降，细胞周期相关基因的表达也随之下降，细胞周期被阻断，细胞的增殖能力被严重抑制。然后，我们检验了FOXM1在肿瘤的发生过程中是否起作用。通过软琼脂克隆形成实验我们看到，感染AdFOXM1shRNA的鼻咽癌细胞CNE和乳腺癌细胞ZR-75-30的克隆形成率大大降低，细胞的非贴壁依赖性生长的能力被抑制；而动物实验的结果显示，FOXM1的缺失降低了乳腺癌细胞ZR-75-30生成肿瘤的能力。两组实验分别在体外和体内证实了肿瘤的发生有赖于FOXM1的存在。最后，我们通过建立裸鼠移植瘤模型考察了FOXM1对肿瘤生长的影响。鼻咽癌和乳腺癌的体内实验的结果表明，瘤内注射AdFOXM1shRNA能够降低肿瘤组织中FOXM1的表达水平，进而抑制肿瘤的生长，显示出FOXM1对于肿瘤的生长的重要性，预示着AdFOXM1shRNA应用于鼻咽癌和乳腺癌基因治疗的潜在可能。
[Abstract]:Cancer is one of the most common malignant tumors in the 21st century , which poses a serious threat to human health and life safety . Among them , nasopharyngeal carcinoma ( NPC ) is one of the most common malignant tumors in South China and Southeast Asia . There are many important physiological processes involved in the regulation of cell proliferation , cell senescence , DNA damage repair , organ formation , and so on . In this paper , we have found that FoxM1 plays an important role in the maintenance of tumor cell proliferation , differentiation , apoptosis and metastasis . Adenovirus is the main carrier of gene therapy . It has the advantages of wide host range , high infection efficiency , high titer , good safety , and can accommodate large segments of foreign DNA . In order to solve this problem , we constructed an adenovirus - mediated shRNA expression system , which is named as Adjoint M1 shRNA , which is used to specifically interfere with the expression of FoxM1 in tumor cells , and further studies the preliminary therapeutic effect of the vector in the gene therapy of nasopharyngeal carcinoma and breast cancer . First , we groped for the appropriate dose of virus in three cells selected by the Institute . These three cells were nasopharyngeal carcinoma cell CNE and breast cancer cell MDA - MB - 231 and ZR - 75 - 30 , respectively . As expected , the expression of cell cycle related genes decreased and the cell cycle was blocked , and the cell proliferation ability was severely inhibited . The results showed that the expression of ZR - 75 - 30 in nasopharyngeal carcinoma cell CNE and breast cancer cell line ZR - 75 - 30 was significantly decreased and the ability of cell adhesion - dependent growth was inhibited ; and the results of animal experiment showed that the deletion of FoxM1 reduced the ability of ZR - 75 - 30 to generate tumor . Both groups demonstrated that the occurrence of tumor depended on the presence of FoxM1 in vitro and in vivo . Finally , we investigated the effect of FoxM1 on tumor growth by establishing a nude mouse xenograft model . The results of in vivo experiments of nasopharyngeal carcinoma and breast cancer show that the tumor growth can be reduced by injecting the Adjoint M1 shRNA into the tumor , which indicates the importance of FoxM1 for the growth of the tumor , and indicates the potential for the application of the AdFoxM1 shRNA in the gene therapy of nasopharyngeal carcinoma and breast cancer .

【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R450;R737.9;R739.63

【共引文献】