【作者】 王志举；

【导师】 董子明；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 资料表明，肺癌是导致死亡的主要肿瘤类型之一，并且有上升趋势。肺癌常分为两种类型：小细胞肺癌（small cell lung cancer，SCLC）和非小细胞肺癌（non-small cell lung cancer，NSCLC）。其中NSCLC是肺癌中常见的类型，大约占肺癌发生率的80％，而且诊断时往往已经处于晚期。随着化疗药物的发展，紫杉醇（Paclitaxel）已经证明在治疗晚期NSCLC中具有显著的抗肿瘤活性。但是获得性耐药的出现，极大地限制了NSCLC的有效治疗。紫杉醇是从紫杉树皮中提取到的一种新型抗微管药物，其广泛的抗瘤谱和确切的疗效已经得到人们普遍的认同，目前主要用于治疗卵巢癌、乳腺癌、非小细胞肺癌、膀胱癌、头颈部肿瘤等。紫杉醇耐药机制比较复杂，目前尚不清楚，可能与多种机制有关。DNA聚合酶β（DNA polymerase beta，DNA polβ）广泛分布于哺乳类细胞中，是真核细胞主要的DNA损伤修复基因，其DNA序列高度保守，为一看家基因。DNA polβ的主要功能是参与DNA的碱基切除修复（base excision repair，BER）。其表达水平在整个细胞周期中相对稳定且不受细胞周期增殖调控。许多研究者在耐药肿瘤组织发现了DNA polβ的过表达，DN A polβ的过表达导致基因组不稳定性增加，使细胞获得突变表型，可能是介导肿瘤发生耐药的重要分子机制。有关DNA polβ在人肺腺癌耐药细胞中的表达状态以及其与多药耐药产生的关系的研究，国内外尚未见报道。肿瘤多药耐药（multidrug resistance，MDR）是影响肿瘤化疗临床疗效的主要障碍之一。探讨MDR产生机制、寻找克服肿瘤MDR逆转剂已经成为化疗药物发展的方向。体外建立多药耐药细胞系是研究肿瘤细胞产生多药耐药机制的重要手段，也是筛选新型耐药逆转剂的工具和方法。本研究首先采用逐步间歇增加剂量法，以紫杉醇为诱导药物，经过180d的诱导，建立了A549多药耐药细胞株-A549／TXL20，并对耐药细胞株进行了初步的生物学特性鉴定。接着，本文检测了A549／TXL20和A549细胞株中的DNApolβ及其相关耐药基因的mRNA表达水平；然后，采用RNA干扰（RNAi）技术，成功构建了DNA polβ靶向siRNA真核表达载体；最后，将DNA polβ靶向siRNA真核表达载体转染A549／TXL20细胞株，观察了其对A549／TXL20耐药逆转的作用，并探讨了其耐药逆转的机制。第一部分紫杉醇诱导人肺腺癌多药耐药细胞系的建立及其生物学特征方法1．以紫杉醇(Paclitaxel为诱导药物，人肺腺癌细胞系（A549）为诱导对象，采用逐步间歇增加紫杉醇药物浓度进行诱导，建立多药耐药细胞系A549／TXL20。2．采用MTT法检测A549／TXL20耐药指数，并同时对顺铂、长春新碱、5-氟脲嘧啶和丝裂霉素4种药物进行交叉耐药检测。3．采用细胞克隆形成法测定A549／TXL20对紫杉醇的敏感性。4．采用高效液相色谱测定细胞内紫杉醇的浓度。结果1．A549／TXL20形态不规则，较亲本细胞略小，核／浆比增加。2．A549／TXL20生长的倍增时间没有明显变化。3．A549／TXL20对紫杉醇的耐药指数为19.3，对顺铂的耐药指数为67.4，同时对长春新碱、5-氟脲嘧啶和丝裂霉素等抗癌药物有不同程度的耐药性。4．A549／TXL20对紫杉醇的敏感性降低。A549／TXL20细胞内紫杉醇的含量较亲本细胞下降。第二部分人肺腺癌多药耐药细胞系A549／TXL20和人肺腺癌细胞系A549中DNA polβ及相关耐药基因的表达方法1．提取人肺腺癌多药耐药细胞系A549／TXL20细胞及人肺腺癌细胞系A549细胞中的总RNA，通用引物逆转录出cDNA；2．以β-actin为内参照，对细胞的DNA polβ、mdr1、GST-π、mrp1、1rp及topoⅡ基因进行半定量PCR扩增；分析A549／TXL20及A549细胞中这些基因的相对表达水平。3．Western blot检测A549／TXL20及A549细胞系DNA polβ蛋白表达水平4．应用SPSS 12．0统计软件进行统计学处理，资料用均数±标准差表示，两组均数的比较采用独立样本的t检验，多组均数的比较采用方差分析，检验标准以p＜0.05为差异有显著意义。结果RT-PCR法对DNA polβ，mdr1，GST-π，mrp1，1rp和topoⅡ基因mRNA表达水平分析表明：1．人肺腺癌多药耐药细胞系A549／TXL20细胞中DNA polβ的基因mRNA表达量为0.9862±0.1768，人肺腺癌细胞系A549细胞中DNA polβ的基因mRNA表达量为0.2531±0.048，差异具有显著性，P＜0.05。表明人肺腺癌多药耐药细胞系A549／TXL20细胞中DNA polβ的基因mRNA表达量显著高于人肺腺癌细胞系A549细胞中DNA polβ的基因mRNA表达量(（P＜0.05）。2．人肺腺癌多药耐药细胞系A549／TXL20细胞中mdr1的基因mRNA表达量为1.3563±0.4953，人肺腺癌细胞系A549细胞中mdr1的基因mRNA表达量为0.4032±0.2506，差异具有显著性，P＜0.05。表明人肺腺癌多药耐药细胞系A549／TXL20细胞中mdr1的基因mRNA表达量显著高于人肺腺癌细胞系A549细胞中mdr1的基因mRNA表达量（P＜0.05）。3．人肺腺癌多药耐药细胞系A549／TXL20细胞中GST-π的基因mRNA表达量为1.6577±0.1892，人肺腺癌细胞系A549细胞中GST-π的基因mRNA表达量为0.5223±0.1769，差异具有显著性，P＜0.05。表明人肺腺癌多药耐药细胞系A549／TXL20细胞中GST-π的基因mRNA表达量显著高于人肺腺癌细胞系A549细胞中GST-π的基因mRNA表达量（P＜0.05）。4．人肺腺癌多药耐药细胞系A549／TXL20细胞中mrp1的基因mRNA表达量为1.2109±0.4361，人肺腺癌细胞系A549细胞中mrp1的基因mRNA表达量为0.5219±0.2646，差异具有显著性，P＜0.05。表明人肺腺癌多药耐药细胞系A549／TXL20细胞中mrp1的基因mRNA表达量显著高于人肺腺癌细胞系A549细胞中mrp1的基因mRNA表达量（P＜0.05）。5．人肺腺癌多药耐药细胞系A549／TXL20细胞中1rp的基因mRNA表达量为1.5783±0.1402，人肺腺癌细胞系A549细胞中1rp的基因mRNA表达量为0.6479±0.2642，差异具有显著性，P＜0.05。表明人肺腺癌多药耐药细胞系A549／TXL20细胞中1rp的基因mRNA表达量显著高于人肺腺癌细胞系A549细胞中1rp的基因mRNA表达量（P＜0.05）。6．人肺腺癌多药耐药细胞系A549／TXL20细胞中topoⅡ的基因mRNA表达量为0.5247±0.0174，人肺腺癌细胞系A549细胞中topoⅡ的基因mRNA表达量为0.4925±0.3345。表明人肺腺癌多药耐药细胞系A549／TXL20细胞中topoⅡ的基因mRNA表达量与人肺腺癌细胞系A549细胞中topoⅡ的基因mRNA表达量没有显著差异（P＞0.05）。7．Western blot结果可以看出，A549／TXL20细胞内DNA polβ蛋白表达灰度值为94.3±0.36，明显高于其在A549细胞内的表达（56.82±0.53），两者相比差异有统计学意义（P＜0 05）。第三部分DNA polβ靶向SiRNA表达载体的构建和鉴定方法1．DNA polβ基因靶向的编码短发卡样siRNA的单链DNA模板的设计及合成。2．单链DNA模板的退火处理及纯化。3．加A处理，构建TA克隆及重组体的筛选和鉴定。4．亚克隆入pslience2．0-GFP载体及重组体的筛选和鉴定。结果1．基于DNA polβmRNA序列设计合成了2对编码小干扰RNA（siRNA）的单链DNA模板，并成功构建了DNA polβ靶向性siRNA真核表达载体pslience2．0-GFP-sipolb1。2．基于DNA polβmRNA序列设计合成了2对编码小干扰RNA（siRNA）的单链DNA模板，并成功构建了DNA polβ靶向性siRNA真核表达载体pslience2．0-GFP-sipolb2。3．基于DNA polβmRNA序列设计合成了2对编码小干扰RNA（siRNA）的单链DNA模板，并成功构建了DNA polβ对照siRNA真核表达载体pslience2．0-GFP-SC。第四部分重组DNA polβ靶向siRNA表达载体对人肺腺癌多药耐药细胞系A549／TXL20的耐药逆转作用及其机制的研究方法1．利用脂质体包裹将构建的DNA polβ靶向siRNA真核表达载体转染人肺腺癌多药耐药细胞系A549／TXL20，G418筛选，得到稳定沉默DNA polβ表达的转入DNA polβ靶向siRNA的人肺腺癌多药耐药细胞系A549／TXL20细胞株。2．采用MTT法检测转染DNA polβ靶向siRNA对A549／TXL20对紫杉醇和顺铂的耐药逆转作用。3．裸鼠腋窝皮下注射转染后的A549／TXL20细胞和未转染的A549／TXL20，建立裸鼠移植瘤模型，观察DNA polβ靶向siRNA对人肺腺癌耐药细胞系A549／TXL20移植瘤耐药性的作用。4．半定量RT-PCR检测转染细胞中DNA polβ、mdr1、GST-π及mrp1基因的mRNA表达。5．Western blot法检测转染细胞中DNA polβ的蛋白表达水平。6．体外哺乳类细胞HGPRT基因突变试验测定转化细胞的基因自发突变率。7．应用SPSS 12．0统计软件进行统计学处理，资料用均数±标准差表示，两组均数的比较采用独立样本的t检验，多组均数的比较采用方差分析，检验标准以P＜0.05为差异有显著意义。结果1．筛选得到稳定沉默DNA polβ表达的转入DNA polβ靶向siRNA的人肺腺癌多药耐药细胞系A549／TXL20细胞株。2．MTT法检测结果显示：转染pslience2．0-GFP-Sipolb1和pslience2．0-GFP-Sipolb2后，肿瘤细胞对紫杉醇和顺铂的IC₅₀较A549／TXL20细胞都有明显减少，出现了明显的耐药逆转，差异具有统计学意义。而转染对照质粒pslience2．0-GFP-SC和空质粒pslience2．0-GFP后，肿瘤细胞对紫杉醇和顺铂的IC₅₀较与A549／TXL20细胞没有显著差异。3．裸鼠耐药肺癌移植瘤模型中，sibolb1组和sibolb2组肿瘤生长率分别为20.26±0.25和20.87±0.39，较耐药组均明显降低，差异均有统计学意义；sibolb1组和sibolb2组瘤重分别为2310±131mg和2305±163mg，较耐药组（瘤重为3106+582mg）均明显降低，差异均有统计学意义（P＜0.05），抑瘤率分别为25.6％和25.8％。4．RT-PCR结果：转染对照siRNA（pslience2．0-GFP-SC）、空siRNA载体（pslience2．0-GFP）的A549／TXL20细胞和未转染的A549／TXL20细胞中DNA polβ和mdr1 mRNA呈现高水平的表达，但各组间差异没有统计学意义，P＞0.05。而转染polβ靶向siRNA载体pslience2．0-GFP-sipolb1和pslience2．0-GFP-sipolb2的A549／TXL20细胞中DNA polβ和mdr1的mRNA呈现低水平的表达，与未转染的A549／TXL20细胞中DNA polβ和mdr1的mRNA水平相比，显著性降低，差异具有统计学意义。表明polβ靶向siRNA能降低A549／TXL20细胞中的DNApolβ和mdr1的mRNA的表达；而转染polβ靶向siRNA载体pslience2．0-GFP-sipolb1、pslience2．0-GFP-sipolb2、对照siRNA载体（pslience2．0-GFP-SC）、空siRNA载体（pslience2．0-GFP）的A549／TXL20细胞与未转染的A549／TXL20细胞中GST-πmRNA、mrp1 mRNA及1rp mRNA均呈现高水平的表达。但各组间差异没有统计学意义，P＞0.05。表明polβ靶向siRNA对A549／TXL20细胞中的GST-π、mrp1及1rp的mRNA表达没有影响。5．Western blot结果可以看出，转染DNA polβ靶向siRNA载体（pslience2．0-GFP-sipolb1、pslience2．0-GFP-sipolb2）的人肺腺癌多药耐药细胞系A549／TXL20细胞，与转染对照siRNA载体（pslience2．0-GFP-SC）的A549／TXL20细胞、转染空载体（pslience2．0-GFP）的A549／TXL20细胞和未转染的A549／TXL20细胞相比，其细胞内DNA polβ蛋白表达灰度值均明显低于A549细胞内的表达水平，两者相比差异有统计学意义（P＜0 05）6．转染DNA polβ靶向siRNA载体（pslience2．0-GFP-sipolb1、pslience2．0-GFP-sipolb2）的人肺腺癌多药耐药细胞系A549／TXL20细胞，与转染对照siRNA载体（pslience2．0-GFP-SC）的A549／TXL20细胞、转染空载体（pslience2．0-GFP）的A549／TXL20细胞和未转染的A549／TXL20细胞相比，其自发突变率显著降低。结论1．经过180d紫杉醇的诱导，成功地建立了相对稳定的人肺腺癌多药耐药细胞系A549／TXL20，该细胞系对顺铂、丝裂霉素、氟尿嘧啶和长春新碱有交叉耐药性，可用于多药耐药性机制及耐药逆转的研究。2．首次揭示人肺腺癌多药耐药细胞系A549／TXL20中DNA polβ基因的mRNA表达水平和蛋白表达均显著高于亲本A549细胞，表明DNA polβ基因的过表达可能参与了A549／TXL20细胞多药耐药的发生。3．A549／TXL20细胞存在mdr1、GST-π、mrp1及1rp基因的高表达，topoⅡ基因没有明显变化。提示mdr1、GST-π、mrp1及1rp的过表达亦可能参与了A549／TXL20细胞多药耐药的发生。4．成功构建2个DNA polβ靶向siRNA表达载体pslience2．0-GFP-sipolb1和pslience2．0-GFP-sipolb2，并成功转染人肺腺癌多药耐药细胞系A549／TXL20，可显著沉默细胞中DNA polβ基因的表达。5．DNA polβ靶向siRNA体外实验能显著逆转肿瘤细胞对紫杉醇和顺铂的耐药性，体内实验可显著降低裸鼠耐药肺癌移植瘤的肿瘤生长率。6．DNA polβ靶向siRNA能显著降低A549／TXL20细胞中的DNA polβ和mdr1的mRNA的表达和降低DNA polβ的蛋白表达，并能显著降低其自发突变率。7．综合研究结果显示：采用逐步间歇增加药物浓度法，用紫杉醇成功诱导出相对稳定的人肺腺癌多药耐药细胞A549／TXL20，其耐药机制可能与DNA polβ及mdr1、GST-π、mrp1及1rp过表达有关联；DNA polβ靶向siRNA可以显著逆转A549／TXL20的耐药性。DNA polβ过表达可能通过诱导mdr1基因过表达及提高细胞自发突变率而导致A549／TXL20产生多药耐药性。更多还原

【Abstract】 Data have showed that pulmonary carcinoma has been one of the major tumor types resulting in death, and at present there is an upward tendency. Pulmonary carcinoma is often divided into two types: small cell lung cancer （SCLC） and non-small cell lung cancer （non-small cell lung cancer, NSCLC）. NSCLC is the common type of lung cancer, which is about 80% of lung cancer. Furthermore, pulmonary carcinoma can not be perceived until in its advanced stage. Along with the development of chemotherapy drugs, paclitaxel has been proved to be the significant anti-tumor activity against the advanced non-small cell lung cancer. However, the emergence of obtained drug resistance has impeded the effective treatment of NSCLC greatly. Paclitaxel is a new type of anti-tubule drugs which is extracted from yew barks, and its broad spectrum of anti-tumor effect has been generally recognized. At present, paclitaxel has been used for the treatment of ovarian cancer, breast cancer, non-small cell lung cancer, bladder cancer, head and neck tumors. The mechanism of resistance of paclitaxel is more complex and it is not clear, it may be have a variety of mechanisms.DNA polymerase beta （DNA polβ） is an important repair gene for DNA damage in eukaryote cells. As a housekeeping gene, the DNA sequence of DNA polβgene is highly conservative. The main function of DNA polβis to excise and repair DNA base mutation, and the expression level of DNA polβis relatively stable in the whole cell cycle without being regulated by proliferation of cell cycle. It has been reported that DNA polβgene overexpressions have been found in many tumor tissues. The overexpressions of DNA polβgene which lead to the increase of genomic instability and the acquisition of mutation phenotype may be an important molecular mechanism for mediating the formation of multidrug resistance.However, at present the studies on the expression of DNA polβgene in the human lung adenocarcinoma resistance cells, and its relation to the occurrence of MDR have not been reported yet both at home and at abroad.The multidrug resistance （MDR） is one of the major obstacles in successful chemotherapy of cancer. The investigation of resistant mechanism developed by tumor and the search for new and potent reversal agents of MDR are the focus of the development of new chemotherapeutic agents. To establish MDR cell line in vitro is an important method to study the MDR mechanism and search the new reversal agents of MDR.In this study, at first, human lung adenocarcinoma MDR cell lines （A549/TXL20） were established in vitro by exposure to stepwise increased concentrations of the paclitaxel in a cell culture medium for nearly 180d, and biological characteristics of the drug-resistance cell line were valued preliminarily. Then, the mRNA expressions of DNA polβand some drug-resistance related gene were measured. Meanwhile, on account of the overexpression of DNA polβin human lung adenocarcinoma MDR cell line A549/TXL20, we utilized RNA interference （RNAi） technique to construct DNA polβtargeted siRNA eukaryotic expression vectors, and then transfected the vectors into A549/TXL20 cell lines to observe the silencing effects of DNA polβtargeted siRNA on the expressions of DNA polβ, and to research the effect of DNA polβtargeted siRNA eukaryotic expression vectors on its MDR reversal and reversal mechanism after transfected into A549/TXL20.PartⅠEstablishment and biological characteristics of human lung adenocarcinoma multidrug resistance cell lines induced by paclitaxel Methods1. A human lung adenocarcinoma multidrug-resistant cell lines （A549/TXL20） was established in vitro by exposure to stepwise increased concentrations of the paclitaxel in a cell culture medium.2. Methyl tetrazolium assay was performed to value the chemoresistance indexes of cell lines and to measure the cross-resistance of the cell lines to cisplatin, mitomycin, 5-fluouracil （5-FU） and vincristin respectively.3. Drug sensitivity of cell lines to paclitaxel was evaluated by colonyforming assay4. Drug concentration in cultured cells of paclitaxel was analyzed by solid-phase of extraction high performance liquid chromatographyResults1. Compared with A549 cell lines, the decrease of cellular size, and the increase of N/C ratio were found in the A549/TXL20 cell lines.2. The growth property of A549/TXL20 didn’t change significantly compared with A549.3. The resistant cells, A549/TXL20, were 19.3 folds more resistant to paclitaxel and 67.4 folds more resistant to cisplatin than the parent cells. The resistant cells also demonstrated cross-resistance to mitomycin, vinblastine, and 5-fluouracil （5-FU）.4. Compared with the A549 cell lines, an unreasonably higher level of drug-resistance and lower drug concentration was detected in A549/TXL20 cells after exposure to the drug in a culture medium.PartⅡThe expression of DNA polymeraseβand multidrug resistance-related genes （mdrl, mrpl,GST-π, lrp, and topoⅡ）in a human lung adenocarcinoma MDR cell lines A549/TXL20 and a human lung adenocarcinoma cell lines A549 Methods1. Total RNA was extracted from human lung adenocarcinoma multidrug-resistant cell lines A549/TXL20 and human lung adenocarcinoma cell lines A549, and was reversely transcribed to cDNA.2. DNA polβ, mdrl, GST-π, mrp1, lrp and topoⅡgenes were amplified by semi-quantitative PCR whileβ-actin was performed as inner control. The mRNA expression levels of these genes in human lung adenocarcinoma multidrug-resistant cell lines A549/TXL20 and human lung adenocarcinoma cell lines A549 were analyzed.3. Western blot assay was preformed to measure the expression of DNA polβprotein in human lung adenocarcinoma multidrug-resistant cell lines A549/TXL20 and human lung adenocarcinoma cell lines A549.4. Statistical analysis SPSS 12.0 was used to perform t test, analysis of variance P value less 0.05 was considered as statistically significant.ResultsThe expressions of mRNA of DNA polβ, mdrl, GST-π, mrpl, lrp and topoⅡgenes by RT-PCR assay indicated: 1. The mRNA expression levels of DNA polβgene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 0.9862±0.1768, whereas the expressions of DNA polβin human lung adenocarcinoma cell lines A549 were 0.2531±0.048. The results showed that DNA polβgene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 was significantly higher than those in human lung adenocarcinoma cell lines A549（P＜0.05）.2. The mRNA expression level of mdr1 gene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 1.3563±0.4953, whereas the expressions of mdrl in human lung adenocarcinoma cell lines A549 were 0.4032±0.2506. The results showed that mdrl gene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 were significantly higher than those in human lung adenocarcinoma cell lines A549（P＜0.05）.3. The mRNA expression level of GST-πgene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 1.6577±0.1892, whereas the expressions of GST-πin human lung adenocarcinoma cell lines A549 were 0.5223±0.1769. The results showed that GST-πgene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 were significantly higher than those in human lung adenocarcinoma cell lines A549（P＜0.05）.4. The mRNA expression level of mrp1 gene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 1.2109±0.4361, whereas the expressions of mrpl in human lung adenocarcinoma cell lines A549 were 0.5219±0.2646. The results showed that mrpl gene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 were significantly higher than those in human lung adenocarcinoma cell lines A549（P＜0.05）.5. The mRNA expressions level of 1rp gene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 1.5783±0.1402, whereas the expressions of 1rp in human lung adenocarcinoma cell lines A549 were 0.6479±0.2642. The results showed that 1rp gene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 were significantly higher than those in human lung adenocarcinoma cell lines A549（P＜0.05）.6. The mRNA expressions level of topoⅡgene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 0.5247±0.0174, and the expressions of topoⅡin human lung adenocarcinoma cell lines A549 were 0.4925±0.3345. The results showed that topoⅡgene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 were similar to those in human lung adenocarcinoma cell lines A549, and there was no significant difference of topoⅡgene mRNA between the A549/TXL20 cell lines and A549 cell lines （P＞0.05）.7. Western blot showed that the expressions of DNA polβprotein in the A549/TXL20 celllines were 94.3±0.36, and significantly higher than those in A549 cell lines （56.82±0.53）, （P＜0.05）.PartⅢThe construction and identification of DNA polβtargeted siRNA expression vectorMethods1. Single strand DNA templates encoding short hairpin siRNA against DNA polβwere designed and synthesized.2. Both single strand DNA templates encoding short hairpin siRNA against DNA polβwere annealed and purified;3. Target DNA templates encoding short hairpin siRNA against DNA polβwere added to adenine （A）, and T-A clone were constructed, and then the recombinant was screened and identified.4. Target DNA templates encoding short hairpin siRNA against DNA polβwere subcloned into expression vector pslience2.0-GFP and then the recombinant was screened and identified.Results1. According to DNA polβsequence, two pairs of single strand DNA template encoding small interference RNA （siRNA） were designed and synthesized, and DNA polβtargeted siRNA eukaryotic expression vectors pslience2.0-GFP-sipolbl were successfully constructed.2. According to DNA polβsequence, two pairs of single strand DNA template encoding small interference RNA （siRNA） were designed and synthesized, and DNA polβtargeted siRNA eukaryotic expression vectors pslience2.0-GFP-sipolb2 were successfully constructed.3. According to DNA polβsequence, two pairs of single strand DNA template encoding small interference RNA （siRNA） were designed and synthesized, and DNA polβcontrol siRNA expression vector pslience2.0-GFP-SC were successfully constructed.PartⅣThe effect of DNA polβtargeted siRNA eukaryotic expression vectors on its MDR reversal and reversal mechanism after transfected into A549/TXL20Methods1. DNA polβtargeted siRNA eukaryotic expression vectors were transfected into human lung adenocarcinoma MDR cell lines A549/TXL20 by lipofectin reagent. After selection of G418, A549/TXL20 cell lines with stable overexpressions of polβtargeted siRNA were obtained.2. Methyl tetrazolium assay was performed to value the MDR reversal by polβtargeted siRNA to A549/TXL20 cell lines on paclitaxel and cisplatin respectively.3. The naked mouse models of grafting tumor were established by injecting subcutaneously transfected A549/TXL20 cells and untreated A549/TXL20 cells in axillas, and observed the effects of polβtargeted siRNA on grafting tumor.4. Semi-quantitative RT PCR was performed to detect the expressions level of mRNA of DNA polβ, mdr1, GST-πand mrp1 genes in transfected cells.5. Western blot assay was preformed to measure the expression of DNA polβprotein in transfected A549/TXL20 cells and untreated A549/TXL20 cells.6. Gene spontaneous mutation rate in transfected cells was assayed by HGPRT gene test.7. The statistical analysis SPSS 12.0 software was used to perform t test, the analysis of variance. P value less than 0.05 was considered as statistically significant.Results1. The A549/TXL20 cell lines with stable overexpressions of polβtargeted siRNA were obtained.2. Compared with A549/TXL20 cell lines, the IC₅₀ value of A549/TXL20 cells transfected with sipolbl and in A549/TXL20 cells transfected with sipolb2 both to paclitaxel and to cisplatin were significantly decreased. Meanwhile, there was no significant difference of IC₅₀ value to paclitaxel and cisplatin in A549/TXL20 cells transfected with SC and A549/TXL20 cells transfected with empty vectors compared with A549/TXL20 cell lines.3. In the naked mouse models of human lung adenocarcinoma MDR cell line A549/TXL20, the tumor growth rate of sibolbl group and sibolb2 group were 20.26±0.25 and 20.87±0.39 respectively, which were significantly lower than that in A549/TXL20 group. Compared with A549/TXL20 group, the tumor weight of sibolb1 group and sibolb2 group were 2310±131mg and 2305±163mg respectively, which decreased significantly, （P＜0.05）; The tumor inhibitory rate of sibolbl group and sibolb2 group were 25.6% and 25.8% respectively.4. RT-PCR indicated that there existed mRNA overexpression of DNA polβand mdr1 genes in untreated A549/TXL20 cells and in A549/TXL20 cells transfected with control siRNA vectors （pslience2.0-GFP-SC） and empty vectors （pslience2.0-GFP） respectively, but there was no significant difference among them, P＞0.05. However, compared with A549/TXL20 cells, the mRNA expression levels of in DNA polβand mdrl genes in A549/TXL20 cells transfected with pslience2.0-GFP-sipolb1 and pslience2.0-GFP-sipolb2 respectively, were significantly decreased, and these results showed that DNA target siRNA may decrease mRNA expression levels of DNA polβand mdrl mRNA in A549/TXL20 cells. There existed mRNA overexpressions of GST-π, mrp1, and 1rp genes in untreated A549/TXL20 cells and in A549/TXL20 cells transfected with pslience2.0-GFP-sipolb1, pslience2.0-GFP-sipolb2, control siRNA vectors （pslience2.0-GFP-SC） and empty vectors （pslience2.0-GFP） respectively. The results showed that DNA target siRNA had little effect on mRNA expressions of GST-π, mrp1 and lrp genes.5. Western blot showed that there existed mRNA overexpression of DNA polβprotein in untreated A549/TXL20 cells and in A549/TXL20 cells transfected with control siRNA vectors （pslience2.0-GFP-SC） and empty vectors （pslience2.0-GFP） respectively, but there was no significant difference among them, P＞0.05. However, compared with A549/TXL20 cells, the expression levels of in DNA polβprotein in A549/TXL20 cells transfected with pslience2.0-GFP-sipolb1 and pslience2.0-GFP-sipolb2 respectively, were significantly decreased, and these results showed that DNA target siRNA may decrease expression levels of DNA polβprotein in A549/TXL20 cells.（P＜0.05）.6. Compared with A549/TXL20 cells transfected with control siRNA vectors （pslience2.0-GFP-SC） and empty vector （pslience2.0-GFP） and untreated A549/TXL20 cells, the spontaneous mutation rate of HGPRT gene in A549/TXL20 cells transfected with pslience2.0-GFP-sipolb1, pslience2.0-GFP-sipolb2 significantly decreased. Conclusions1. The paclitaxel-induced human lung adenocarcinoma MDR cell lines A549/TXL20 are successfully established which are cross-resistance to cisplatin, mitomycin, 5-FU and vincristin, and may be used as an experimental system for the search of a means to overcome drug resistance and elucidate possible mechanisms of acquired MDR involved in human lung adenocarcinoma.2. It is the first time to reveal that the mRNA and protein expression levels of DNA polβgene in human lung adenocarcinoma MDR cell lines A549/TXL20 are significantly higher than those in human lung adenocarcinoma cell lines A549, which indicates that overexpressions of DNA polβmight confer the development of drug resistance in A549/TXL20 cell lines.3. The overexpressions of mdr 1, GST-π, mrp1 and 1rp genes existed in human lung adenocarcinoma MDR cell lines A549/TXL20, and there is no significant difference of expressions of topoⅡgene between A549/TXL20 cells and A549 cells. The results indicate that the overexpressions of mdr 1, GST-π, mrp1 and lrp genes might be related to the formation of A549/TXL20 MDR.4. DNA polβtargeted siRNA eukaryotic expression vectors （pslience2.0-GFP-sipolb1, pslience2.0-GFP-sipolb2） are successfully constructed and successfully transfected into human lung adenocarcinoma MDR cell lines A549/TXL20, which can significantly inhibit the mRNA expressions of DNA polβgene.5. DNA polβtargeted siRNA may result in reversal resistance to paclitaxel and cisplatin of A549/TXL20 in vitro study, and may decrease tumor growth rate of human lung adenocarcinoma grafting tumor of naked mouse significantly in vivo study.6. DNA pol targeted siRNA may decrease the mRNA expression levels of DNA polβand mdr1 genes in A549/TXL20 cell lines, decrease the expressions of DNA polβprotein, and decrease spontaneous mutation rate of HGPRT gene significantly.7. The results of comprehensive studies show that human lung adenocarcinoma multidrug-resistant cell lines （A549/TXL20） are established in vitro by exposure to stepwise increased concentrations of the paclitaxel in a cell culture medium, whose multidrug resistant mechanism may have relation to mRNA overexpressions of DNA polβ, mdr 1, GST-π, mrpl and lrp genes; DNA polβtargeted siRNA may reverse the drug resistant characteristics of A549/TXL20 cell lines to some extent. The overexpressions of DNA polβgene may result in formation of A549/TXL20 MDR by inducing the mRNA overexpressions of mdr 1 gene and increasing the spontaneous mutation rate of HGPRT gene.更多还原