【作者】 李晋芸；

【导师】 赵素萍；

【作者基本信息】 中南大学 ， 耳鼻咽喉科， 2008， 博士

【摘要】 研究目的:鼻咽癌（NPC,nasopharyngeal carcinoma）是我国南方地区常见的恶性肿瘤,占头颈部恶性肿瘤首位,已严重地危害了人们的生命健康。病理学上95%以上的NPC为非角化癌,对放射治疗中度敏感,因此临床上首选的治疗手段是根治性放疗。虽然近年来随着放射物理学及放射生物学技术水平不断提高,放射技术不断改善。但放射疗效仍差强人意,总的5年生存率仅50-60%左右,且放射损害较大。NPC经第一次放射治疗后,引起局部组织的血管萎缩,导致鼻咽部供血不良,再次放疗的敏感性低、疗效差,且副作用大。鼻咽部部位隐蔽,周围有重要组织器官,手术暴露困难,手术效果不佳。因此NPC放疗后残灶及复发灶的治疗是临床上非常棘手的难题。本课题拟利用光动力治疗方法（PDT）,结合应激敏感的启动子Grp78特异性调控肿瘤凋亡基因VP₃的基因治疗NPC,观察该治疗方法对NPC的杀伤效率,探索鼻咽癌的治疗新策略,以提高鼻咽癌的治疗效果。研究方法1)利用PCR,酶切,连接等技术构建并鉴定四种真核质粒表达载体PcDNA3.1（-）Grp78p.VP₃-Myc-CMV,PcDNA3.1（-）Hre₂.Grp78p.VP₃-Myc-CMV,Pc DNA 3.1（-）CMV.VP₃-Myc以及PcDNA 3.1（-）VP₃-Myc-CMV2)将构建成功的四种真核质粒表达载体瞬时转染至鼻咽癌细胞株CNE-2中,通过RT-PCR,Western-Blotting鉴定凋亡素基因VP₃在NPC细胞株中的表达并利用CCK-8法观察VP₃对NPC的杀伤作用。3)摸索PDT杀伤鼻咽癌CNE-2细胞的最佳光敏剂浓度,最佳光敏剂孵育时间,以及激光辐射剂量,应用PDT治疗NPC,以不加5-氨基酮戊酸（5-ALA）和不用激光照射做为空白对照,单用5-ALA,单用激光做为阴性对照进行实验。利用CCK-8法观察各组细胞存活率.4)进行PDT联合基因治疗鼻咽癌的体外实验研究,以单用基因治疗,单用PDT治疗作为对照组,利用CCK-8法观察各组CNE-2细胞的存活率;利用Western-Blotting检测各组CNE-2细胞VP₃基因表达强度的变化;利用流式细胞仪检测各组CNE-2细胞的凋亡率;利用光镜和电镜观察各组CNE-2细胞形态学的变化。研究结果1)成功地构建了四种重组质粒载体即:PcDNA3.1（-）Grp78p.VP₃-Myc-CMV,PcDNA3.1（-）Hre₂.Grp78p.VP₃-Myc-CMV,Pc DNA 3.1（-）CMV.VP₃-Myc以及Pc DNA 3.1（-）VP₃-Myc-CMV2)将四种载体成功地转染至CNE-2细胞,在mRNA及蛋白水平检测到VP₃基因在CNE-2细胞中均有表达;在转染72小时后,转染Pc DNA 3.1（-）CMV.VP₃-Myc的细胞存活率（50.8±1.6）%,明显低于转染PcDNA3.1（-）Grp78p.VP₃-Myc-CMV（64.1±1.4）%,PcDNA3.1（-）Hre₂Grp78p.VP₃-Myc-CMV（62.3±0.8）%和PcDNA3.1（-）VP₃-Myc-CMV（95.5±1.0）%细胞的存活率（P＜0.05）。结果提示启动子调控下的VP₃基因对CNE-2细胞具有一定的杀伤作用。3)5-ALA与CNE-2细胞培养产生了内源性光敏剂原卟啉（PpⅨ）,PpⅨ均匀分布在CNE-2细胞的胞浆中,胞核区未见分布。5-ALA产生的PpⅨ在孵育6小时后在胞浆的表达强度最高,尤以浓度为1.0mmol/1时为显著。5-ALA介导的PDT对人鼻咽癌CNE-2细胞的杀伤作用随着5-ALA与CNE-2细胞孵育时间延长及5-ALA浓度的增加而增加,在孵育6小时,5-ALA浓度为1.0 mmol/L,激光功率为6.25J/cm²时,细胞存活率为（23.31±1.59）%,而单用激光时细胞存活率为（96.42±1.89）%,单用光敏剂5-ALA时细胞存活率为（97.23±2.01）%。结果提示PDT对鼻咽癌CNE-2细胞有较强的杀伤作用。4)应用PDT联合基因治疗CNE-2细胞后,CNE-2细胞的存活率（18.3±2.7）%明显低于单用基因治疗（74.4±0.9）%和单用PDT治疗（55.0±2.2）%的细胞存活率（P＜0.05）;PDT联合基因治疗后,细胞中VP₃基因蛋白表达的强度明显高于单用基因治疗组和PDT组（P＜0.05）;PDT联合基因治疗组内,Grp78p调控的VP₃基因蛋白表达的强度明显高于CMV调控的VP₃基因蛋白表达强度（P＜0.05）;PDT联合基因治疗后CNE-2细胞的凋亡率（68.3±2.3）%明显高于单用基因治疗（27.8±1.6）%和单用PDT（54.8±3.6）%的细胞凋亡率（P＜0.05）;形态学观察显示PDT联合基因治疗组出现明显的细胞坏死和凋亡。结果提示PDT联合基因治疗对鼻咽癌CNE-2有更强的杀伤作用。研究结论:1)首次成功地构建了含Hre₂反应元件,Grp78启动子调控的含VP₃基因完整编码序列的去除巨细胞启动子的质粒表达载体2)上游含巨细胞启动子或者Grp78p启动子的真核质粒表达载体转入鼻咽癌CNE-2细胞株后,VP₃基因对鼻咽癌细胞有一定的杀伤作用;而不含启动子的载体,VP₃基因对细胞无杀伤作用。3)5-ALA介导的PDT对鼻咽癌CNE-2细胞有很好的杀伤作用。4)PDT联合基因治疗较单独的基因治疗或者光动力治疗有更显著的杀伤鼻咽癌CNE-2细胞效应。更多还原

【Abstract】 Objective:Nasopharyngeal carcinoma（NPC）is one of the most malignant tumors in the South China and it ranks the first in head and neck cancer, which damaged the health of the people seriously.Since more than 95% of the NPC are non-cancroids which is moderately radiosensitive,radical radiation therapy is the first choice of treatment in clinic.With the development of radio-physics and radiobiology,the radiation effect is still far from satisfaction.The general 5-years’ survival rate is only up to 50%-60%and damage to the tissue companying with the radiation is serious.After radiation therapy,the blood vessels around the pharynx nasalis atrophied and blood-supply to pharynx nasalis is poor which decreased curative effect and sensitivity to the second radiation. Meanwhile the place where NPC originated is concealed and there are lots of important tissues and organs around it.It is hard to be exposured by surgical operation and the effect of surgical operation is not good as well.The treatment of NPC recidivists after radiation therapy is a tough problem in clinic.In this study we combined the photodynamic therapy （PDT）with apoptotic gene therapy together to observe killing effect on NPC in vitro and explore a new NPC treatment strategy to improve the effect of the NPC therapy.Methods:1)To construct four plasmid expression vectors PcDNA3.1（-） Grp78p.VP₃- Myc -CMV,PcDNA3.1（-）Hre₂.Grp78p.VP₃- Myc-CMV, Pc DNA 3.1（-）CMV.VP₃-Myc and Pc DNA 3.1（-）VP₃-Myc -CMV by PCR,enzyme restriction and ligation techniques.2)To transfect the Vectors PcDNA3.1（-）Grp78p.VP₃- Myc -CMV, PcDNA3.1（-）Hre₂.Grp78p.VP₃-Myc-CMV,Pc DNA 3.1（-）CMV.VP₃ -Myc and PcDNA3.1（-）VP₃-Myc-CMV into CNE-2 cell line by liposome. Total RNA and protein were extracted and VP3 gene expression was detected by RT-PCR and Western-blotting;to investigate the survival rate of CNE-2 after gene therapy by CCK-8 assay. 3)To explore the best concentration and the best incubation time of the photosensitizer 5-ALA and the best dose of laser radiation and observe the rate of cytocide after PDT.4)To combined PDT with gene therapy together,study the survival rate and apoptotic rate of CNE-2,the expression of VP3 and the morphosis change among PDT/gene therapy group,PDT group and gene therapy groupResults:1)PcDNA3.1（-）Grp78p.VP₃- Myc -CMV,PcDNA3.1（-） Hre₂.Grp78p.VP₃- Myc-CMV,Pc DNA 3.1（-）CMV.VP₃-Myc and Pc DNA 3.1（-）VP₃-Myc -CMV were constructed successfully and identified by analysis of DNA sequence2)The killing effect of VP₃ gene on NPC:The above four vectors were transfected to CNE-2 successfully.Expression of VP₃ gene in transfected CNE-2 cell line was confirmed from mRNA and protein level. VP3 gene has a certain cytocide effect on CNE-2.The survival rate of CNE-2 induced PcDNA3.1（-）CMV.VP₃ -Myc（50.8±1.6）%is significantly higher than that of CNE-2 induced PcDNA3.1（-）Grp78p.VP₃-Myc -CMV（64.1±1.4）%,PcDNA3.1（-）Hre₂.Grp78p.VP₃-Myc-CMV（62.3±0.8） %and Pc DNA3.1（-）CMV.VP₃-Myc-CMV（95.5±1.0）%（P＜0.05）3)The killing effect of PDT on NPC:5-ALA produced endogenous PpIX in CNE-2 and PpIX was observed evenly in the cytoplasma,but not in the nuclear region.The expression intensity of 5-ALA was highest after 6 hours’ incubation in concentration 1.0mmol/L.The survival rates of the CNE-2 which is mediated by 5-ALA PDT decreased with the time passing or the 5-ALA concentration increasing.The survival rate of the cells only treated by 5-ALA or light irradiation had no significant difference compared with that of the blank control group.4)The killing effect of 5-ALA-PDT combined with gene therapy on NPC:Expression of VP3 in CNE-2 which were transfected with PcDNA3.1（-）Grp78p.VP3-Myc -CMV and PcDNA3.1（-）Hre2.Grp78p. VP3-Myc-CMV is higher than that in CNE-2 transfected with PcDNA3.1（-）CMV.VP3-Myc with PDT（P＜0.05）.The cell survival rate of CNE-2 treated with 5-ALA-PDT and gene therapy（18.3±2.7）%is lower than that of CNE-2 treated with gene therapy（74.4±0.9）%or 5-ALA-PDT（55.0±2.2）%only（P＜0.05）,while the apoptosis rate of CNE-2 treated with 5-ALA-PDT and gene therapy（68.3±2.3）%is higher than that of CNE-2 treated with gene therapy（27.8±1.6）%or 5-ALA-PDT（54.8±+3.6）%only（P＜0.05）.Apoptotic body can be obviously observed in gene therapy or gene therapy/PDT groups.Conclusion:1)PcDNA3.1（-）Grp78p.VP3-Myc-CMV and PcDNA3.1（-）Hre2 Grp78p.VP3-Myc-CMV were successfully constructed.2)PcDNA3.1（-）Grp78p.VP3-Myc-CMV,PcDNA3.1（-）Hre2Grp78p VP3-Myc-CMV and PcDNA3.1（-）CMV.VP3-Myc were induced to CNE-2 cells successfully and can kill the CNE-2 cells3)5-ALA-PDT has a good killing effect on nasopharyngeal carcinoma and it is a great treatment strategy to nasopharyngeal carcinoma4)5-ALA-PDT/gene therapy is a much better treatment strategy to NPC comparing with PDT or gene therapy only.And this strategy can promote the effect of cytocide of PDT and gene therapy with each other.更多还原