【作者】 魏瑞；

【导师】 何剪太；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 目的：(1)观察人肺腺癌细胞株A549与人肺腺癌耐DDP细胞株A549/DDP的放射生物学特性,探讨肺腺癌耐DDP细胞株的耐药性对其放射敏感性的影响。(2)建立A549与A549/DDP两组细胞株的双向凝胶电泳图谱,识别并鉴定其差异表达的蛋白质,筛选肺腺癌耐药、耐辐射相关蛋白质。方法：(1)使用6MV-X射线对A549和549／DDP进行照射,采用克隆形成实验拟合细胞存活曲线,根据D0、Dq, N及SF2值等放射生物学参数,比较2株肿瘤细胞的放射敏感性。流式细胞仪(FCM)检测细胞周期,检测肿瘤细胞的凋亡率,分析2株肿瘤细胞的细胞周期分布、凋亡率与放射敏感性之间的关系。(2)收集A549与A549/DDP总蛋白质,应用二维凝胶电泳(2-DE)分离2组总蛋白质,采用PDquest7.1软件进行匹配和差异分析,识别两组之间表达差异蛋白点。从胶中切取差异蛋白点进行胶内原位酶解、酶解产物进行MALDI-TOF-MS分析,获取肽质量指纹图,数据库搜索鉴定蛋白质。使用Western免疫印迹对其中3个蛋白质进行验证,免疫组化技术对上述3个差异蛋白质在肺腺癌的化疗耐药组及化疗敏感组的差异表达水平进行验证。结果：(1)2株肿瘤细胞D0、Dq、N及SF2值大小为A549<A549/DDP,放射敏感性为A549>A549/DDP。流式细胞仪检测到6Gy照射后2组细胞出现G2/M期阻滞,且A549较A549/DDP阻滞明显,照射后24小时、48小时、72小时A549及A549/DDP2组细胞有显著性差异(p<0.05)。流式细胞仪检测到明显的凋亡峰、肿瘤细胞凋亡率,并且2种肿瘤细胞株凋亡峰值依次为A549>A549/DDP,与其放射敏感性高低一致。(2)成功地进行了肺腺癌细胞株A549与A549/DDP蛋白质的分离,建立了分辨率较高、重复性较好的A549与A549/DDP细胞蛋白质的2-DE图谱,识别了45个差异表达的蛋白质点,鉴定了27个差异表达蛋白质,这些蛋白质按功能主要可以分为六类：①基本代谢相关的酶类：如ATP synthase subunit beta, mitochondrial、Egl nine homolog 1、Delta(3,5)-Delta(2,4)-dienoyl-COA isomerase, mitochondrial、Triosephosphate isomerase、Proteasome subunit alpha type-6、Cytochrome b5 type B、Fatty acid-binding protein, epidermal、Hemoglobin subunit beta等；②信号传导相关蛋白：如Cofilin-1、Keratin,typeⅠcytoskeletal 18、Keratin,typeⅡcytoskeletal 1等；③与解毒和转录翻译相关蛋白：如Peroxiredoxin-4、Elongation factor 1-delta、Membrane-associated progesterone receptor component 2、Putative RNA-binding protein 3、Probable ATP-dependent RNA helicase DDX6等；④分子伴侣：如Heat shock protein beta-1、10 KDa heat shock protein, mitochondrial等；⑤细胞结构相关蛋白质：如Vimentin、Tubulin beta chain、Tubulin beta-2A chain、PDZ and LIM domain protein 1、Myosin regulatory light chain MRLC2、Histidine triad nucleotide-binding protein 1、Histone H2B type 1-C/E/F/G/I等；⑥钙结合蛋白：Annexin A2、Annexin A4。Western免疫印迹分析证实了差异蛋白质：Heat shock protein beta-1、Annexin A4、Vimentin在A549与A549/DDP细胞中的差异表达水平。免疫组化技术亦证实了对上述3个差异蛋白质在肺腺癌的化疗耐药组及化疗敏感组的差异表达水平。结论：(1)肺腺癌耐药细胞株的耐药性对其放射敏感性具有一定影响,肺腺癌耐药细胞株放射敏感周期G2／M期降低,凋亡率降低,从而导致其放射敏感性降低,辐射抵抗性增加。(2)27个差异表达蛋白质为研究肺腺癌耐药、耐辐射机制提供了实验依据。更多还原

【Abstract】 Objective (1) To observe radiobiological characteristics of A549 and A549/DDP cell lines, and investigate the impact of the drug resistance on the radioresistance in human lung adenocarcinoma cell line. (2) To establish 2-dimensional electrophoresis(2-DE) maps of A549 and A549/DDP cell lines, to screen Multidrug Resistance (MDR) and radioresistance related proteins in human lung adenocarcinoma.Methods (1) Two tumor cell lines A549 and A549/DDP were irradiated by 6MV-X ray, then the radiobiological characteristics of the two lines were analyzed by the method of colony-forming assay According to radiosensitivity parameters Do, Dq, N and SF2. Compare the radiosensitivity of the two tumors cell lines. Flow cytometry (FCM)was used to detect the cell cycle distribution and the apoptosis index of tumor cells. The relationship between radiosensitivity and the cell cycle、radiation-induced apoptosis in two tumor cell lines were analyzed. (2)The total proteins of A549 and A549/DDP cells were obtained, and were extracted and separated by two-dimensional gel electrophor-esis(2-DE). The images of the gels were acquired by the scanner and then analyzed by using PDquest7.1 software to find the differentially expression protein-spots in each group. Then the differentially expressed protein-spots was incised from the gels and digested by trypsin.The peptide mass fingerprintings(PMF) was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and the proteins were identified by data searching in the Mascot-database. Western blot and immunohistochemical method were used to determine the differential expression levels of the 3 proteins.Results (1) Do and Dq and N and SF2 were ranked in the following descending order:A549<A549/DDP, while the radiosensitivity:A549> A549/DDP. G2 phase arrest was detected by FCM after irradiation of 6Gy in both groups, the mean percentage of G2 phase cells was higher in A549 group than in A549/DDP group (P<0.05) at 24h,48 h and 72h. The result of FCM assay detected obvious apoptosis peak. The apoptosis peak of the two tumor cell lines:A549> A549/DDP, in accord with the height of radiosensitivity. (2)The proteins were separated successfully for proteome research,2-DE maps of total proteins with high resolution and reproducibility from A549 and A549/DDP were established. A total of 45 differential protein spots in the 2 cell lines were found, and 27 differential expression proteins were identified by MALDI-TOF-MS. The differential expressional proteins could be divided into six main groups based on their functions:①metabolic enzymes:ATP synthase subunit beta, mitochondrial、Egl nine homolog 1、Delta(3, 5)-Delta(2,4)-dienoyl-COA isomerase, mitochondrial、Triosephosphate isomerase、Proteasome subunit alpha type-6、Cytochrome b5 type B、Fatty acid-binding protein,epidermal、Hemoglobin subunit beta.②proteins relative to signal transduction:Cofilin-1、Keratin,typeⅠcytoskeletal 18、Keratin,typeⅡcytoskeletal 1.③proteins involved in drug detoxification or Translation:Peroxiredoxin-4、Elongation factor 1-delta、Membrane-associated progesterone receptor component 2、Putative RNA-binding protein 3、Probable ATP-dependent RNA helicase DDX6.④chaperones:Heat shock protein beta-1、10 KDa heat shock protein, mitochondrial.⑤Proteins related to cellular structure:Vimentin、Tubulin beta chain、Tubulin beta-2A chain、PDZ and LIM domain protein 1、Myosin regulatory light chain MRLC2、Histidine triad nucleotide-binding protein 1、Histone H2B type 1-C/E/F/G/I.⑥calcium- binding Proteins:Annexin A2、Annexin A4. Western blot and immunohistochemical method showed that Heat shock protein beta-1、Annexin A4、Vimentin were differential expression proteins in A549 and A549/DDP, which was consistent with the results from the comparative proteomic analysis.Conclusion (1) Drug resistant human lung adenocarcinoma cell line have reduced G2/M period percentage and the rate of apoptosis, and then increased radioresistance. (2) 27 differential expression proteins in human lung adenocarcinoma are useful for studying the multidrug resistance and radioresistance mechanism of lung adenocarcinoma.更多还原