【作者】 卿菁；

【导师】 赵素萍；

【作者基本信息】 中南大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 鼻咽癌是一类以放射治疗为主的恶性肿瘤,如何提高肿瘤的局控率及生存率、减轻放射治疗对正常组织的损伤、提高患者的生存质量一直是该病的研究重点,本文将分章讨论E6.BARF1启动子(简称BARF1p)调控CD/UPRT.UL49的表达对鼻咽癌的靶向放射-基因研究。目的设计BARF1基因的启动子序列,验证该序列是否具有启动子活性,通过比较该启动子在人鼻咽正常上皮细胞NP69和人鼻咽癌细胞CNE-2中的启动子活性,验证所设计的BARF1基因的启动子是否具有肿瘤相对特异性。方法结合启动子预测软件结果(TRANSFAC和MATINSPECTOR)及引物设计软件(Primer premier5.0),设计BARF1基因的启动子序列,从B95-8细胞株中提取基因组DNA,PCR扩增所设计的BARF1基因的启动子,将扩增的启动子连接在荧光素酶报告基因载体上,通过脂质体介导,瞬时转染入NP69和CNE-2细胞细胞中,验证所设计的启动子是否具有启动子活性,同时比较该启动子在NP69细胞和CNE-2细胞中的的启动子活性的差异,验证该启动子是否具有肿瘤相对特异性,差异的比较采用SPSS10.0的t检验。结果所设计的BARF1基因的启动子在CNE-2细胞中具有启动子活性,但是活性较低,而在NP69细胞中不具备启动子活性,且该启动子在CNE-2细胞中的启动子活性远高于NP69细胞,差异有统计学意义(p<0.05)。结论本研究设计的BARF1基因的启动子具有启动子活性,且该启动子具有肿瘤相对特异性。目的建立稳定表达自杀基因CD/UPRT.UL49的CNE-2细胞自杀基因修饰细胞株。方法构建真核基因表达质粒pcDNA3.1 (-)E6.BARF1p.CD/UPRT.UL49,经脂质体法转染CNE-2细胞,G418筛选出阳性克隆细胞并扩大培养,抽提阳性克隆细胞的总蛋白质,通过Western-blotting检测目的基因表达。结果自杀基因CD/UPRT.UL49在CNE-2转染细胞内稳定表达。结论本组通过脂质体转染技术,成功地建立了稳定表达CD/UPRT.UL49基因的CNE-2细胞株。目的体外实验观察CD/UPRT.UL49/5-FC系统对鼻咽癌CNE-2细胞的杀伤效应。方法给予野生型CNE-2细胞和稳定转染CD/UPRT.UL49的CNE-2细胞株不同剂量的丫射线照射,同时联合不同剂量前体药物5-Fc作用48h后,用MTT法检测细胞的杀伤效应；再给予野生型CNE-2细胞和稳定转染CD/UPRT.UL49的CNE-2细胞株2Gy的γ射线照射,联合200μg／ml前体药物5-FC作用48h后,流式细胞仪检测前体药物对表达CD/UPRT.UL49基因的CNE-2细胞的杀伤作用,并检测CD/UPRT UL49基因对CNE-2细胞的旁杀效应。统计分析采用SPSS10.0软件进行t检验及方差分析。P<0.05表示有统计学意义。结果表达自杀基因CD/UPRT.UL49的CNE-2细胞的生长受到不同程度的抑制,当仅有10%的CNE-2细胞表达CD/UPRT基因时,可以杀死37.46%的细胞。结论CD/UPRT.UL49/5-FC自杀基因／前体药物系统对鼻咽癌CNE-2细胞具有直接杀伤和旁杀效应。目的通过建立裸鼠CNE-2动物模型,观察自杀基因表达载体pcDNA3.1(-)E6.BARF1p.CD/UPRT.UL49在前体药物干预下对鼻咽癌放射治疗的增敏效应。方法CNE-2细胞接种裸鼠皮下建立鼻咽癌动物模型,进行脂质体包裹质粒DNA瘤内注射的原位基因(in situ)治疗实验,待肿瘤长至0.8-1.0cm时,将裸鼠随机分组,记录肿瘤体积,比较不同处理组的抑瘤效应,病理切片证实肿快性质,抽提肿瘤组织RNA, RT-PCR反应,确定CD/UPRT.UL49基因在肿瘤组织中的表达。结果成功建立鼻咽癌动物模型,病理切片证实肿块为低分化鳞癌,通过原位基因治疗实验发现,各处理组均能抑制肿瘤生长,较对照组的抑瘤效应更明显(P<0.01)。结论E6.BARF1p.CD/UPRT.UL49自杀基因载体具有放射诱导和相对肿瘤特异性双重特性,对鼻咽癌CNE-2细胞的杀伤效应,为鼻咽癌的基因-放射治疗开辟了新思路。更多还原

【Abstract】 Nasopharyngeal carcinoma is a kind of malignant tumor treated mainly by radiotherapy, how to improve regional control rate and survival rate, reduce the radiation damage to normal tissue and improve the quality of life of patients has always been the focus of this study, therefore, this article is going to discuss the research on targeting radio-gene therapy through the expression of CD/UPRT.UL49 mediated by E6.BARFlp(referred to as BARFlp) in nasopharyngeal carcinoma.Objective To design the of BARF1 gene promoter sequence, verify whether this sequence has promotive activity by comparing the promoter activity in human nasopharyngeal epithelial cells NP69 and that in normal human nasopharyngeal carcinoma cells CNE-2, and confirm whether the promoter of BARF1 gene has relative specificity of tumor.Methods Combining results of promoter prediction software (TRANSFAC and MATINSPECTOR) and primer design software (Primer premier 5.0), we designed the sequence of BARF1 gene promoter, extracted the genomic DNA from B95-8 cell lines, amplified the designed BARF1 gene promoter by PCR, connected the amplified promoter to the luciferase reporter gene vector, then transfected this vector into NP69 cells and CNE-2 cells transiently through liposome way, and verified its promoter activity, also compared the promoter activity in NP69 cells with that in CNE-2 cells, to verify whether this promoter has tumor relative specificity, t-test of SPSS 10.0 was used to detect the difference between them.Results BARF1 gene promoter showed promoter activity in CNE-2 cells, but the activity is low, yet there is no promoter activity detected in NP69 cells, and the promoter activity showed in CNE-2 cells is much higher than that in human nasopharyngeal epithelial cells NP69, difference is significant(p<0.05).Conclusion The promoter sequence of BARF1 gene has promotive activity, and also has tumor relative specificity.Objective To construct nasopharyngeal carcinoma CNE-2 cell lines expressing stable suicide gene.Methods The plasmids of pcDNA3.1 (-) E6.BARF1p. CD/UPRT.UL49 was transfected into CNE-2 cells through lipofectamine, and the transfected CNE-2 cells were selected by G418 to get the cells expressing CD/UPRT.UL49 gene. The protein produced by the suicide gene was tested by Western-blotting in CNE-2 cells.Results Suicide genes was expressed stably in CNE-2 cells. Conclusions We constructed CNE-2 cell lines expressing stable suicide gene through lipofectamin transfection.Objective To observe killing effect towards CNE-2 cells through the expression of suicide gene and prodrug system of CD/UPRT.UL49/5-FC in vitro gene therapy.Methods Given different quantity of Y radiation combined with different quantity of prodrug 5-Fc to wild CNE-2 cells and CNE-2 cells transfected with CD/UPRT.UL49 respectively, after 48h, the killing effects was tested by MTT method; again given 2Gy radiation and 200μg/ml prodrug 5-Fc to wild CNE-2 cells and CNE-2 cells transfected with CD/UPRT.UL49 respectively, after 48h, the killing and bystander effects on CNE-2 cells transfected with CD/UPRT.UL49 was tested by flow cytometry (FCM). T test and ANOVA were used to detect the differences. P＜0.05 represents significant.Results The growth of CNE-2 cells expressing suicide genes was suppressed more or less, and such a suppression effect of CD/UPRT.UL49 was of dose dependence to the 5-FC. Though there were only 10 percent of the cells expressing CD/UPRT.UL49 gene, it was demonstrated that 37.46 percent of the cells were killed. Conclusion The suicide gene and prodrug system of CD/UPRT.UL49/5-FC has direct cytotoxic and bystander effects to CNE-2 cells.Objective To observe the enhancement of the sensitization of the NPC to radiation under the intervention of prodrugs by plasmid vector expressing CD/UPRT gene in situ and in vivo gene-radiotherapy.Method CNE-2 cells in log phase were inoculated subcutaneously in nude mice to construct a nude mouse model of NPC,and an in situ gene therapy was performed with plasmid packed by plasmid. When the size of tumors reaching 0.8-1.0cm, the nude mice were randomized to different grouped, the size of the tumor was recorded and the suppression of tumor was compared among different treating groups. Biopsy of the tumor were performed to find the difference between treatment groups.Then targeted RNA was isolated from tumor tissues and RT-PCR was performed to define the expression of suicide gene.Results In situ gene therapy experiment showed that compared with cotroll group,growth of transplant tumour of each treatment group was distinctly suppressed(P<0.01).Conclusions The suicide gene E6.BARF1p.CD/UPRT. UL49/prodrug system, is both radiosensitive and tumor specifically effective in anticancer therapy, and exert killing effect to CNE-2 cells under radiation. From this study, we established a new strategy for radio-gene therapy of NPC.更多还原