【作者】 周蓉蓉；

【导师】 田勇泉； 肖志强；

【作者基本信息】 中南大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 目的探讨腺病毒E1A基因对人鼻咽癌CNE-2Z细胞放射治疗的增敏作用及相关机制。方法选用人鼻咽癌CNE-2Z细胞为靶细胞,分别用PBS,Ad-β-gal, Ad-E1A作用48小时后,用6MVX线分别给与0、2、4、6和8Gy剂量照射后,用MTT法和流式细胞仪检测三组细胞成活率及细胞周期变化,并进行克隆分析绘制细胞存活曲线,计算放射增敏比来观察E1A基因对人鼻咽癌CNE-2Z细胞放射敏感性的影响,同时用RT-PCR法检测三组细胞的VEGF水平的表达及野生型p53的表达。细胞免疫化学方法检测三组细胞的VEGF蛋白的表达。结果RT-PCR结果显示Ad-E1A组可以检测到E1A基因的表达。经照射后Ad-E1A组的细胞存活率明显低于PBS对照组和Ad-β-gal对照组。Ad-E1A组的细胞的生长速度明显慢于PBS对照组和Ad-β-gal对照组。细胞存活曲线结果显示Ad-E1A组的CNE-2Z细胞放射增敏比分别为1.37(D0值比)、1.95(Dq值比)、1.46(SF2比)。PBS对照组和Ad-β-gal对照组的细胞照射后细胞存活曲线分析结果显示无差异,D0、Dq、SF2值分别为1.57Gy及1.53Gy、1.82Gy及1.78Gy、0.89及0.82。RT-PCR结果和细胞免疫化学结果显示Ad-E1A组比PBS对照组和Ad-β-gal对照组的人鼻咽癌CNE-2Z细胞的VEGF的表达明显下降,RT-PCR结果显示Ad-E1A组比PBS对照组和Ad-β-gal对照组的野生型p53水平明显上升。流式细胞学实验显示Ad-E1A组+放射治疗组细胞的凋亡明显高于其他组。结论E1A基因通过下调人鼻咽癌CNE-2Z细胞VEGF的表达和上调野生型p53的表达来增加人鼻咽癌细胞对放射治疗的敏感性。目的：探讨腺病毒E1A基因对人鼻咽癌动物模型放射增敏的实验研究。方法：取对数生长期的CNE-2Z细胞5×105/0.2mL,接种于4周龄左右裸鼠右前肢腋部皮下致瘤,第7天裸鼠皮下肿瘤长至直径0.6-0.8cm时,随机分为6组(每组10个)：PBS组,Ad-β-gal组,放射组,Ad-β-gal+放射组,Ad-E1A组,Ad-E1A+放射组。Ad-β-gal组/Ad-E1A组在第2周开始给予荷瘤裸鼠皮下肿瘤内滴度为5×109PFU/50μL的Ad-E1A/Ad-β-gal注射,每周2次,连续2周,放射组在第3周给予6MV-X照射,每天2Gy,连续5天。Ad-β-gal+放射组/Ad-E1A+放射组在第2周开始,给予荷瘤裸鼠皮下肿瘤内滴度为5×109 PFU/50μL的Ad-E1A/Ad-β-gal注射,每周2次,连续2周,在第3周给予6MV-X照射,每天2Gy,连续5天。第1次治疗后,每隔4天用卡尺测量1次肿瘤的体积,记录其直径,裸鼠的生存时间。当肿瘤的直径超过2cm时,处死裸鼠,分离肿瘤组织,采用免疫组织化学方法分析VEGF和CD34表达,RT-PCR分析wtp53的表达,Western blot分析VEGF的表达,TUNEL分析细胞凋亡。结果：Ad-E1A+放射组较其他组能明显延缓移植瘤生长时间,Ad-E1A+放射组裸鼠的肿瘤平均体积比单独放射组小4.7倍,比单独Ad-E1A组的小5.3倍。Ad-E1A+放射组的裸鼠生存率显著高于其他治疗组。Ad-E1A+放射组的VEGF蛋白表达和微血管密度较其他各组明显下降。(P<0.01)。Ad-E1A组和Ad-E1A+放射组的wtp53基因表达明显上调。TUNEL分析结果显示Ad-E1A组和Ad-E1A+放射组及放射组的肿瘤组织内均可明显观察到凋亡细胞。并且,Ad-E1A+放射组肿瘤细胞凋亡数目明显高于Ad-E1A组或单独放射组。结论：E1A基因通过抑制肿瘤血管的形成和上调肿瘤组织中wtp53的表达及诱导肿瘤细胞的凋亡来提高人鼻咽癌细胞对放射的敏感性。更多还原

【Abstract】 Objective:The purpose of this study determined the effect and mechanism of Ad-E1A gene therapy in vitro radiosensitivity to nasopharyngeal carcinoma cell.Methods:The human nasopharyngeal carcinoma CNE-2Z cell lines were investigated. The recombinant adenovirus vector containing E1A gene was used for this study. After CNE-2Z cells was treated with PBS, Ad-β-gal and Ad-E1A for 48h, the three groups were irradiated in different doses at 0、2、4、6 and 8Gy, The cytotoxicity was determined by MTT assay and cell cycle was analysis by flow cytometry. Clone forming assays were carried out, cell survival curves were drawn and the sensitivity enhancing ratio (SER) was calculated. The VEGF expression was evaluated by RT-PCR assay and immunocytochemical analysis. The wtp53 express was evaluated by RT-PCR.Results:RT-PCR confirmed that E1A gene had been integrated into positively transfected cells and stably expressed. Significant cell deaths by IR were observed in a dose dependent manner in the three group CNE-2Z cells. After transduction of the E1A gene into CNE-2Z cells, the sensitivity of these cells to radiation was enhanced than the PBS treated group and Ad-β-gal treated group. Cell growth inhibition in Ad-E1A treated group by IR was strongly enhanced than Ad-β-gal treated group and PBS treated group. Cell survival curves showed that the SER of Do, Dq and SF2 value was 1.37,1.95 and 1.46 with Ad-E1A treated group The Do, Dq and SF2 value was 1.57Gy,1.82Gy,0.89 in PBS treated group and 1.53Gy,1.78Gy,0.82 in Ad-β-gal treated group,respectively. RT-PCR assay and immunocytochemical analysis showed VEGF expression was downregulation in Ad-E1A treated group. Moreover, the expression of wtp53 gene was markedly enhanced in Ad-E1A treated group. The Flow cytometry showed more apotosis can be detected in the cells treated with Ad-E1A plus radiotherapy than those of the other treated groups.Conclusions:E1A gene therapy can effectively enhance the nasopharyngeal carcinoma cell sensitivity to the radiotherapy by down regulated VEGF expression and enhanced the expression of wtp53. Objective:To determine the effect of Ad-E1A gene therapy in vivo radiosensitivity to nasopharyngeal carcinoma.Methods:CNE-2Z cells (2 x 105) were injected subcutaneously into nude mice resulted in tumor development (1-3 mm) 6 days later. The tumor-bearing mice were then randomly divided into six groups (10 mice per group) for PBS treatment or treatment with radiotherapy, Ad-E1 A, or Ad-β-gal alone or radiotherapy in combination with Ad-E1A or Ad-β-gal. Animals were treated with Ad-E1A or Ad-β-gal (5 x 109 plaque forming units) by intratumoral injection twice weekly for 2 weeks at beginning of week 2. Animals treated with radiotherapy in combination with Ad-E1A or Ad-β-gal were received 2 Gy radiotherapy daily for 5 days following the first week of treatment with Ad-E1A or Ad-β-gal. Control animals received PBS therapy or radiotherapy only after tumor cells were injected. When the sizes of tumors exceeded 2 cm, the mice were killed and the tumors underwent immunohistochemical analysis for VEGF and CD34 expression and RT-PCR analysis for wtp53 expression and TUNEL assay for apoptosis and Western blot analysis for VEGF expressionResults:The growth delay time (TGD) was longest in the Ad-E1A plus radiotherapy group. Tumors treated with Ad-E1A plus radiotherapy were 4.7-fold smaller than those treated with radiotherapy alone and 5.3-fold smaller than those treated with Ad-E1A alone. The survival rate of tumor-bearing mice treated with Ad-E1A plus radiotherapy was significantly higher than that of other treated groups. The vessel density and the VEGF expression were significantly lower in the tumors treated with Ad-E1A plus radiotherapy than those treated with radiotherapy alone, Ad-E1A alone, Ad-β-gal alone or Ad-β-gal plus radiotherapy (P<0.01). wtp53 expression were significantly increased in the tumors treated with Ad-E1A plus radiotherapy than those treated with radiotherapy alone, Ad-β-gal alone or Ad-β-gal plus radiotherapy. TUNEL staining revealing apoptosis can be detected in Ad-E1A group, radiotherapy group, Ad-E1A plus radiotherapy group, and more apoptosis can be detected in the tumors treated with Ad-E1A plus radiotherapy than those of the other treated groups.Conclusion:E1A gene therapy can effectively enhance the nasopharyngeal carcinoma sensitivity to the radiotherapy by down-regulating VEGF expression increasing wtp53 expression and inducing apoptosis. These findings may pave the way for efficient radiation-gene therapy to NPC in future.更多还原