【作者】 陈鹏；

【导师】 杨昆；

【作者基本信息】 天津医科大学 ， 卫生毒理学， 2011， 硕士

【摘要】 目的现今肺癌的发病率和死亡率增长迅速,已成为人类死亡的主要原因之一,各细胞类型中以肺腺癌的增加尤为明显。临床发现多为中晚期,手术及放化疗的总体疗效不甚乐观,易出现局部复发及远处转移。基因治疗是目前肺癌治疗的研究热点之一,其中自杀基因(suicidegene)治疗被认为是最有应用前景的基因治疗方法。目前,诸多治疗基因中单纯疱疹病毒胸苷激酶HSV1-tk基因研究最多,是最具前景的具有催化放射性核素的“自杀基因”。此外,HSV1-tk还兼具报告基因功能,故建立携带tk基因的肺腺癌裸鼠动物模型后,可在活体状态下能表达胸苷激酶ΓK。18F、124I、131I等标记的核苷类似物作为底物进行反应后通过PET显像实时监测HSV1-tk自杀基因在靶细胞内的表达情况,为今后进一步深入研究肺癌的自杀基因治疗研究和监测奠定基础。同时,也可以为下一步进行双报告基因报告显像跟踪监测骨髓间充质干细胞的活体修复等工作奠定可行性基础。内容将外源自杀基因特异地导入肿瘤细胞内并在靶细胞内高效且稳定的表达是能否成功基因治疗肿瘤的关键。对患者体内的自杀基因分布转导情况和治疗状态进行实时检测分析也是整个治疗过程中不可或缺的部分。故本研究拟在前期研究的基础上利用逆转录病毒载体介导HSV1-tk自杀基因兼报告基因转染肺腺癌A549细胞,经筛选阳性转导细胞,再利用稀释法单克隆建立一种能稳定表达HSV1-tk基因肺腺癌细胞株A549-tk,观察其与正常A549细胞的形态和生长状态的区别。建立能在活体内长期、稳定、高效表达HSV1-tk基因的肺腺癌裸鼠动物模型。方法利用分子克隆技术扩增带Bg1II、Sa1I酶切位点的HSV1-tk全基因片段,插入逆转录病毒载体pDON-AI-2-Neo中,与gag-pol表达质粒pGP和env表达质粒pE-ampho利用脂质体法转染293T包装细胞,形成pDON-AI-2-Neo-HSVI-tk重组报告基因假病毒颗粒,然后用其转染肺癌A549细胞,经G418筛选阳性表达,再经有限稀释法单克隆获得稳定表达株,RT-PCR检测证实A549-tk细胞的外源HSV1-tk基因在mRNA水平的稳定表达。通过长时间的传代培养进行细胞的生长、形态观察并绘制细胞生长曲线。将克隆细胞株种瘤于裸鼠后,比较正常A549瘤和A549-tk瘤两者之间的生长差异,取动物模型的成瘤做RT-PCR检测到HSV1-tk基因的表达.结果重组质粒pDON-AI-2-Neo-HSV1-tk经过酶切、测序鉴定证明与GenBank基因库公布的HSV1-tk基因序列(gi：59974)完全一致。并与gag-pol表达质粒pGP和env表达质粒pE-ampho通过包装细胞制备成感染性假病毒颗粒。经G418抗生素阳性筛选及有限稀释单克隆法培养出含HSV1-tk基因的单克隆细胞株,RT-PCR检测证实外源基因HSV1-tk成功导入A549细胞。比较A549-tk细胞株和正常A549两者的生长及形态特征并绘制细胞生长曲线,结果发现两种细胞的外形基本一致,生长特征也无明显差别(P>0.05)。A549-tk细胞株和A549细胞的成瘤率均为100%。比较了两种细胞肿瘤的生长特征,发现正常A549瘤和A549-tk两者之间的肿瘤的生长大小并无明显差异(P>0.05)。三次传代后经RT-PCR检测表明,成功建立了能稳定高效表达HSV1-tk基因的动物模型。结论1重组质粒pDON-AI-2-Neo-HSV1-tk能在肺腺癌细胞A549进行正确的翻译表达。2外源基因HSV1-tk在成功制备的A549-tk细胞株中可被长期稳定表达。3外源基因HSV1-tk的导入并没有影响到A549细胞的正常生理功能和形态。4外源HSV1-tk报告基因导入A549细胞后对肿瘤的生长状况无明显影响。5本实验成功建立了能稳定高效表达HSV1-tk基因的动物模型。更多还原

【Abstract】 OBJECTIVEThe current incidence and mortality of lung cancer has grown rapidly, It has become one reason of the major human deaths, the most prominent of which is the Increase of lung adenocarcinoma. the overall effect of Chemotherapy and drug treatment is less optimistic. The cancer is prone to form local recurrence and distant metastasis,. Gene therapy for lung cancer is an important issue for treatment of lung cancer, in which suicide gene (suicidegene) therapy is considered the most promising method of gene therapy.At present, herpes simplex virus (HSV1-tk) genetic research is the most of many therapeutic genes and the most promising of radioactive nuclides with catalytic "suicide gene". In addition, HSVl-tk has reporter gene features. Thus establishing the nude mice animal model of lung cancer of carrying tk gene, then expressing thymidine kinase TK in vivo, intaked in a amount of exogenous of radionuclides such as 18F, 124I,131I, etc. substrate nucleoside analogues labeled. We can monitor with real-time monitoring the expression of tk suicide gene in target cells by PET imaging which is basis for further in-depth study for the lung cancer suicide gene therapy research. Also, We can for the next double reporter gene imaging of double tracking and monitoring of bone marrow mesenchymal stem cells in vivo feasibility of laying foundation repairCONTENTThe exogenous suicide gene is transduced into human tumor cells in different places and the stable、efficient expression successfully in target cells are the keys of gene therapy for cancer. In addition, the real-time detection of the distribution of suicide gene in vivo with patients and treatment status to the entire treatment process is an integral part. In this study, we transfect HSV1-tk suicide gene and reporter gene into lung adenocarcinoma A549 cells with retroviral vector-mediated,based on previous study.By screening positive transduced cells, re-use dilution to establish the monoclonal lung adenocarcinoma cell line A549-tk which can stable express HSV1-tk gene, Observion the difference of the morphology and growth status between the normal A549 cells and A549-tk cells,. Established lung cancer model of nude mice with carrying tk gene, testing whether the TK gene can express long-term stability in vivoMETHODSHSV1-tk gene of fragment with restriction site of Bgl II, Sall is amplified by Molecular cloning. The retroviral vector pDON-AI-2-Neo which HSV1-tk gene is inserted into, and the plasmid pGP expressing gag-pol and the plasmid pE-ampho expressing env are transfected 293T packaging cells by liposome to form artificial recombinant virus particles carrying pDON-AI-2-Neo-HSV1-tk reporter gene. Then it is transfected A549 lung cancer cells.The transfected cells by G418 selecting positive expression, then with limiting dilution Monoclonal is formed stable expression strain.RT-PCR testing confirmed the expression of exogenous HSV1-tk gene in A549-tk cells is stability in the mRNA level. The morphology observation and cell growth curve are finished with the long passage of cell culture growth.. Cloned tumor cell lines are implanted into nude mice, studying the difference between the normal A549 and A549-tk tumor tumor growth, RT-PCR detects HSV1-tk gene expression of A549-tk tumorRESULTSThe HSV1-tk gene of plasmid pDON-AI-2-Neo-HSVl-tk is consistent with HSV1-tk gene sequences (gi:59974) published by GenBank gene pool with digesting and sequencing.It is usd to merged the plasmid pGP expressing gag-pol and the plasmid pE-ampho expressing envl which were prepared false infectious particles by packaging celPositive selection by the antibiotic G418 and limiting dilution method cultivate a monoclonal cell lines expressing HSV1-tk gene, and RT-PCR confirmed HSV1-tk gene is transfected into A549 cells successfullyWe found that the same shape of two cells are basically, and no significant difference in growth characteristics (P> 0.05) by compareing morphology and the cell growth curve between A549-tk cell line and normal A549 growthA549-tk tumor growth was 100%, that is no different with A549 cells on tumorigenicity.We found that the tumor size and tumor growth between the normal A549 and A549-tk tumors was no significant difference (P> 0.05) by compareing the growth characteristics of two cell tumors. After three passages, RT-PCR analysis showed that the successful establishment of stable and efficient expression of HSV1-tk gene in an animal modelCONCLUTION1 The plasmid pDON-AI-2-Neo-HSVl-tk can be translated and expressed correctly in adenocarcinoma of lung A549 cell.2 HSV1-tk gene in A549-tk cells can be stability expressed after long-term successfully.3 the exogenous gene HSV1-tk imported did not affect the normal physiological function and morphology of A549 cells.4 After exogenous HSV1-tk reporter gene into A549 cells, there is no significant effect for the state on tumor growth.5 The experiment is successfully established an animal mode expressing HSV1-tk gene stablely and efficiently.更多还原