E1A基因对鼻咽癌放射增敏作用及机制研究

发布时间：2018-03-13 15:36

本文选题：血管内皮生长因子　切入点：鼻咽癌　出处：《中南大学》2010年博士论文　论文类型：学位论文

【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。(P0.01)。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。
[Abstract]:Objective to investigate the sensitizing effect of adenovirus E1A gene on the radiotherapy of human nasopharyngeal carcinoma CNE-2Z cells and its related mechanism.
Methods human nasopharyngeal carcinoma CNE-2Z cells were treated with PBS, Ad- beta -gal, 48 hours after Ad-E1A, 6MV and 8Gy 0,2,4,6 were given X-ray irradiation, and flow cytometry was used to detect three groups of cell survival rate and the change of cell cycle by MTT method and clone analysis according to the cell survival curve recently, the calculation of radiosensitization effect of E1A gene on radiosensitivity of human nasopharyngeal carcinoma CNE-2Z cells, and the expression of RT-PCR was used to detect the VEGF levels of the three groups of cells and wild type p53. The detection of immunocytochemistry. The expression of the three groups of cells of VEGF protein.
Results RT-PCR results showed that Ad-E1A group can detect the expression of E1A gene by Ad-E1A. The cell survival rate after irradiation group was significantly lower than that of PBS control group and Ad- control group.Ad-E1A group -gal beta cell growth was slower than the PBS control group and -gal control group. Ad- beta cell survival curves showed that CNE-2Z cells in radiation Ad-E1A group the sensitization enhancement ratio were 1.37 (D0 ratio), 1.95 (Dq ratio), 1.46 (SF2).PBS control group and -gal control group Ad- beta cells after irradiation, the cell survival curve analysis showed no difference, D0, Dq, SF2 values were 1.57Gy and 1.53Gy, 1.82Gy and 1.78Gy, 0.89 and the results of 0.82.RT-PCR and the immunocytochemistry showed that the expression of Ad-E1A in PBS group than those in control group and Ad- control group -gal beta in human nasopharyngeal carcinoma CNE-2Z cells VEGF decreased significantly, RT-PCR results showed that Ad-E1A group than in control group and PBS control group Ad- beta -gal wild type p53 level in Ming Dynasty The flow cytology test showed that the cell apoptosis in group Ad-E1A + radiation therapy group was significantly higher than that in other groups.
Conclusion E1A gene can increase the sensitivity of human nasopharyngeal carcinoma cells to radiotherapy by down regulation of the expression of VEGF in human nasopharyngeal carcinoma CNE-2Z cells and up regulation of the expression of wild type p53.
Objective: To study the experimental study of radioactivity sensitization of adenovirus E1A gene to human nasopharyngeal carcinoma (NPC) animal model.
Methods: CNE-2Z cells in log phase 5 * 105/0.2mL, about 4 week old nude mice were inoculated in the right armpit subcutaneous tumor, seventh days subcutaneous tumors grew to 0.6-0.8cm, were randomly divided into 6 groups (n = 10) group:PBS, Ad- beta -gal group, radiation group, -gal+ radiation Ad- beta group, Ad-E1A group, Ad-E1A+ radiation group.Ad- beta -gal group /Ad-E1A group was given subcutaneous tumor in nude mice was 5 * 109PFU/50 L Ad-E1A/Ad- beta -gal injection at second weeks, 2 times a week for 2 consecutive weeks, radiotherapy group received 6MV-X irradiation at third weeks 2Gy per day for 5 consecutive days of.Ad- beta -gal /Ad-E1A+ + radiation group radiation group at the beginning of the second week, tumor bearing nude mice subcutaneous tumor was 5 * 109 PFU/50 L Ad-E1A/Ad- beta -gal injection, 2 times a week for 2 consecutive weeks, given 6MV-X irradiation at week third 2Gy per day for 5 consecutive days. After the first treatment, every 4 days with caliper 1 measurements of tumor volume, record The diameter and survival time of nude mice. When the diameter of tumor was over 2cm, the nude mice were sacrificed and the tumor tissues were isolated. The expression of VEGF and CD34 was analyzed by immunohistochemistry. RT-PCR was used to analyze the expression of wtp53, Western blot was used to analyze VEGF expression, TUNEL was used to analyze apoptosis.
Results: Ad-E1A+ radiotherapy group can significantly delay tumor growth time than the other group, the average tumor volume of nude mice Ad-E1A+ radiation group than single radiation group is 4.7 times smaller than Ad-E1A group, the small 5.3 times.Ad-E1A+ radiation group the survival rate of nude mice was significantly higher than that of the other treatment groups.Ad-E1A+ radiation group VEGF protein expression and microvessel density significantly compared with the other groups decreased. (P0.01) wtp53 gene in.Ad-E1A group and radiation group Ad-E1A+ expression was significantly increased the.TUNEL analysis results showed that Ad-E1A and Ad-E1A+ can group radiation group and radiation group in tumor tissue was observed. Apoptosis and apoptosis of tumor cells, Ad-E1A+ radiation group number was significantly higher than Ad-E1A group or single radiation group.
Conclusion: E1A gene can enhance the radiosensitivity of human nasopharyngeal carcinoma cells by inhibiting tumor angiogenesis and upregulating the expression of wtp53 and inducing apoptosis of tumor cells.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.63

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