Ad-OSM对鼻咽癌抑癌效应的实验研究

发布时间：2018-05-26 13:34

本文选题：OSM + 腺病毒　；参考：《苏州大学》2012年硕士论文

【摘要】：研究背景及目的：鼻咽癌(nasopharyngeal carcinoma，NPC)是我国南方、越南、菲律宾等其它南亚国家高发肿瘤之一，广东、广西、福建、湖南等省为国内高发区，，部分地区高达30/10万～50/10万，调整死亡率分别为6.47/10万、4.92/10万、3.28/10万、3.22/10万，亦居世界首位（调整死亡率2.88/10万）。男性发病率约为女性的2～3倍，40～50岁为高发年龄组。鼻咽癌的发病率和死亡率均随年龄增长而上升。鼻咽癌的发生发展是一个多因素作用、多基因参与、多阶段病变发生的极其复杂的癌变过程，基因治疗作为一种高效性、特异性、靶向性的治疗手段已是医学界广泛关注的热点。近年来研究发现，抑瘤M（Oncostatin M,OSM）基因对肿瘤细胞具有生长抑制作用和靶向诱导凋亡的功能。本项实验拟采用以腺病毒为载体的OSM基因，观察其对该鼻咽癌细胞及裸鼠移植瘤的抑癌效应，并对其可能机理进行了探讨，为将来运用Ad OSM应用于临床提供实验依据。方法：体外：将Ad-OSM体外感染CNE-2Z鼻咽癌细胞，运用RT-PCR及Westernblot检测OSM基因在CNE-2Z细胞中的转录及表达；MTT法和Annexin-V-PE/7-AAD双染色的FCM检测PBS、Ad-GFP、Ad-OSM体外抑制鼻咽癌细胞生长和诱导凋亡的抑癌增效效应；PI染色的FCM检测Ad-OSM对鼻咽癌细胞周期的影响；运用RT-PCR检测P21、P27、Bcl-2、Survivin等以分析Ad-OSM诱导肿瘤细胞凋亡可能的分子机制；Western blot检测Ad-OSM对CNE-2Z细胞中cleaved Caspase-3的表达的影响。体内：用CNE-2Z细胞构建裸鼠移植瘤，在鼻咽癌的裸鼠移植瘤动物模型上，运用免疫组化方法检测CNE-2Z鼻咽癌移植瘤生长相关因子的表达：（1）P21、P53、Bax、Caspase-3、等促凋亡因子；（2）Bcl-2、Cox-2等凋亡抑制因子。结果：RT-PCR和Western blot检测证实腺病毒介导的OSM基因可以在CNE-2Z细胞中稳定转录及表达；Ad-OSM体外能明显抑制CNE-2Z鼻咽癌癌细胞的生长，引起G1期阻滞和诱导细胞凋亡（P0.05）。体内也能明显抑制CNE-2Z裸鼠鼻咽癌移植瘤的生长。分子机制检测结果表明：Ad-OSM能明显上调CNE-2Z鼻咽癌细胞P21、P27、P53、Bax、Caspase-3和下调Cox-2、Bcl-2、Survivin等细胞周期和凋亡相关蛋白的表达。结论： 1、Ad-OSM能抑制CNE-2Z人鼻咽癌细胞及其移植瘤的生长和诱导其凋亡（P＜0.05）。 2、Ad-OSM能显著上调P21、P27、P53、Bax、Caspase-3和下调Survivin、Cox-2、Bcl-2等细胞周期和凋亡相关蛋白表达的效应，最终引起cleaved Caspase-3表达，可能是OSM表达对鼻咽癌细胞体内外生长具有抑癌效应的重要机制之一。
[Abstract]:Background and objective: nasopharyngeal carcinoma (NPC) is one of the most prevalent tumors in South China, Vietnam, the Philippines and other South Asian countries. Guangdong, Guangxi, Fujian and Hunan provinces are high incidence areas in China, with some areas as high as 30 to 100, 000 to 50, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000. The adjusted mortality rate was 647 / 100, 000 / 100, 000 / 100, 000 / 100, 000 or 32, 200 / 100, 000 respectively, ranking first in the world (adjusted mortality rate is 28. 88 / 100, 000). The incidence of male was about 2 times as high as that of female, and 4050 years old was the age group with high incidence. The morbidity and mortality of nasopharyngeal carcinoma increased with age. The occurrence and development of nasopharyngeal carcinoma (NPC) is an extremely complicated process of carcinogenesis involving many genes and multistage lesions. Gene therapy is a kind of high efficiency and specificity. Targeted therapy has become a hot topic in the medical field. In recent years, it has been found that the tumor suppressor M(Oncostatin Mastoma gene can inhibit the growth of tumor cells and induce apoptosis. The aim of this study was to observe the inhibitory effect of adenovirus OSM gene on nasopharyngeal carcinoma cells and xenografts in nude mice, and to explore its possible mechanism, and to provide experimental evidence for the application of Ad OSM in clinical practice in the future. Methods: CNE-2Z nasopharyngeal carcinoma cells were infected with Ad-OSM in vitro. RT-PCR and Westernblot were used to detect the transcription and expression of OSM gene in CNE-2Z cells. Annexin-V-PE/7-AAD double staining FCM was used to detect the inhibitory effect of Ad-GFP-Ad-OSM on the growth and apoptosis of nasopharyngeal carcinoma cells in vitro. FCM staining and Pi staining were used to detect the effect of Ad-OSM on the cell cycle of nasopharyngeal carcinoma. In order to analyze the possible molecular mechanism of apoptosis induced by Ad-OSM, RT-PCR was used to detect the expression of cleaved Caspase-3 in CNE-2Z cells by using P21 P27, Bcl-2 and survivin. Western blot was used to detect the effect of Ad-OSM on the expression of cleaved Caspase-3 in CNE-2Z cells. In vivo: CNE-2Z cells were used to construct xenografts in nude mice. The expression of growth related factors (GRF) in transplanted tumor of CNE-2Z nasopharyngeal carcinoma (NPC) was detected by immunohistochemical method. The apoptotic inhibitory factors, such as P53-P53-BaxCaspase-3, and the apoptosis-promoting factor, Bcl-2Cox-2, were detected by immunohistochemical method in nude mice model of transplanted tumor of nasopharyngeal carcinoma (NPC). Results RT-PCR and Western blot analysis showed that adenovirus mediated OSM gene could stably transcribe and express in CNE-2Z cells. Ad-OSM could significantly inhibit the growth of CNE-2Z nasopharyngeal carcinoma cells in vitro and induce G1 phase arrest and apoptosis. The growth of nasopharyngeal carcinoma xenografts in nude mice with CNE-2Z was also inhibited in vivo. The results of molecular mechanism analysis showed that the cell cycle and apoptosis-related protein expression of CNE-2Z NPC cell line P21P27P27P53P53P5axCaspase-3 and Cox-2Bcl-2survivin could be down-regulated. Conclusion: 1Ad-OSM could inhibit the growth and induce apoptosis of CNE-2Z human nasopharyngeal carcinoma cells and its transplanted tumor (P < 0.05). (2) Ad-OSM could significantly up-regulate the expression of Caspase-3 and down-regulate the expression of cell cycle and apoptosis-related proteins such as survivvin-Cox-2OSM, which may be one of the important mechanisms of inhibiting the growth of nasopharyngeal carcinoma cells in vivo and in vitro.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

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