【作者】 郭万峰；

【导师】 郭国祯； 王升启；

【作者基本信息】 第四军医大学 ， 放射医学， 2005， 博士

【摘要】 肺癌是最常见的恶性肿瘤之一,严重威胁着人类的健康。根据组织学分类,肺癌分为小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC)两种,NSCLC又包含多种类型。NSCLC约占支气管肺癌的80%,其5年生存率不足10%。大多数患者在诊断时都已达到中晚期,不能进行手术,只能进行放疗和/或化疗。目前放射治疗多采用直线加速器进行分割照射,每次2Gy,总剂量约为60Gy。虽然可以给予较大的剂量,但是辐射引起的副作用影响患者的预后和生存质量。因此,迫切需要发展新的治疗模式。许多科学家认为将来肺癌的治疗方向将偏重于靶向治疗和靶向治疗与常规治疗相结合的综合治疗模式。目前,传统的细胞毒性药物和分子化合物的研究进展并不理想。如果想提高肺癌的疗效,必须寻找新的靶标和分子化合物。 同一组织来源的细胞系基因表达具有明显的异质性,以往的研究认为基因表达和放射治疗的疗效以及辐射敏感性密切相关。SCLC和NSCLC是肺癌的两大类,它们的辐射敏感性明显不同。所以,我们选择了两种具有代表性的肺癌细胞系作为研究对象,一种是肺腺癌细胞A549,另一种是小细胞肺癌细胞NCI-H446。我们对两种细胞的辐射敏感性和辐射反应进行了检测,通过细胞克隆形成实验检测了两种细胞的辐射存活曲线;使用流式细胞仪检测了两种细胞受到5Gy照射后不同时间点的细胞周期变化和细胞凋亡的情况。 人类基因组完成后,DNA芯片已经成了研究转录组的主要方法。鉴于辐更多还原

【Abstract】 Lung cancer causes many deaths worldwide and is the leading cause of cancer death in China and the most common malignant cancer that threatens human being’s health. The long-term survival of lung cancer patients treated with conventional therapies remains poor and has changed little in decades. Lung cancer is classified according to the histology of the cancer cells into SCLC (small cell lung cancer) and NSCLC (non-small cell lung cancer), the latter consisting of several types. NSCLC accounts for nearly 80% of all bronchogenic neoplasms and is characterized by a particularly poor prognosis with almost 90% of patients dying from it within 5 years of diagnosis. Most NSCLC patients are inoperable at diagnosis and submitted to radiotherapy and/or chemotherapy. Radiotherapy is usually delivered by an external beam from a linear accelerator. Standard therapy for unresectable disease consists of approximately 60 Gy, with the dose divided among 30 sessions over a period of six weeks. Although higher doses have been used, the more the toxic effects of radiation in normal organs have been found. Concurrent chemotherapy may increase the efficiency of radiotherapy by sensitizing the tumor to ionizing radiation (IR), but it can also increase the adverse effects (particularly esophagitis). Because of the poorprognosis, there is an urgent requirement for research in the development of new therapies, such as gene therapy for lung cancer. Many scientists think future directions for lung cancer treatment are heavily weighted toward targeted therapies - namely, those aimed at molecular abnormalities involved in the pathogenesis of lung cancer- rather than traditional cytotoxic agents and targeted therapies combined with standard therapy. Since little further progress is expected with the use of traditional cytotoxic agents and compounds, such as those that target protein kinase C, epithelial growth factor receptor, vascular endothelial growth factor, cyclooxygenase-2, and farnesyl transferase, new agentsand approaches must be evaluated if we are to advance therapy for lung cancer.Previous studies reported that cell lines of the same pathological origin could be highly heterogeneous in gene expression. Many studies have reported correlations between gene expression and radiotherapeutic response and in vitro radiosensitivity. SCLC and NSCLC represent the two major categories of lung cancer that differ in their sensitivity to IR. We selected two lung cancer cell lines, one representative of adenocarcinomas (A549) and one variant of small cell lung cancer (NCI-H446). Radiosensitivities of both cell lines were measured with a standard clonogenic assay, cell cycle distributions and apoptosis were detected with FCM and radiation survival curves derived from colony formation assays of two cell lines were found.DNA microarrays have become the main technological workhorse for the transcripotome after the completion of the human genome. Given the complexity of IR-induced responses, microarray as a powerful tool for investigation small intracellular changes offers new opportunities to identify a wider range of genes and signaling pathways involved in the IR response. Understanding the mechanism of IR response at the molecular level will help us to provide valuable information for designing effective radiotherapy of various tumor types and improving radiocurability of tumor cells. To better understand the IR-mediatedgene expression changes that might results in different response to IR, oligonucleotide microarray were designed by our laboratory. To validate the microarray results, RT-PCR was performed using the RNA obtained 6, 24 and 48 h after irradiation.Antisense oligonucleotides have been extensively studied as a research tool in determining gene function and as a novel therapeutic approach to treatment of various human diseases. Microarray results found that MDM2 is interesting, which has been shown to be a critical component of the responses to IR. The design of the ASO complementary to the MDM2 mRNA was completed by a computational neural network model, secondary structure of RNA prediction and molecular mechanisms of action of antisene drug. Five anti-MDM2 ASOs were synthesized in our laboratory and transfected the A549 cells in the presence of Lipofectin and demonstrated specifically knocked-down MDM2 expression, as demonstrated at both mRNA and protein levels. The cell survival and proliferative ability, apoptosis and radiosensitivities of treated cells were detected by MTT assays, FCM and clonogenic assays respectively.Currently, RNAi is a discovered knockdown phenomenon that is induced by double-stranded RNA (dsRNA) in organism and mammalian cells. The mechanism of RNAi is not fully understood, but recent genetic and biochemical studies have revealed some details at the molecular level. The activity of siRNA is high even at low concentrations, without apparent toxicity. In the present study, RNAi strategy was employed to reduce MDM2 in A549 cells. Plasmid pPUR/U6 was the gift of ZY. Shen, University of New Mexicoschool of Medicine. Six RNAi oligos were designed according to the sequence of effective ASOs and principles of siRNA design. Equal amount of sense and antisense template were annealed, and subcloned into the Apal and EcoRI sites of pPUR/U6 vector toconstruct MDM2-siRNA plasmids, Escherichia coli DH5a competent cells were transformed with the resultant plasmids. Plasmid DNA was purified using a commercial DNA purification kit and confirmed by DNA sequence analysis. Two out of three siRNA plasmids were constructed successfully. A549 cells grown in 6-we 11 plates were transfected 3u,g siRNAs by using Lipofectamine? 2000 according to the manufacturer’s protocol. pPUR/U6 without insert was used as a control. The cells were selected with puromycin. MDM2 expression was determined by RT-PCR and Western Blotting and the transfected cells response to IR was detected by FCM and MTT assay.The studies found that both lung cancer cell lines that differ in then-sensitivity to IR. The A549 cell line displayed the surviving fraction at 2Gy (SF2) was 0.64 in our colony-forming assays. The NCI-H446 cell line can be considered as relatively sensitive to radiation with a SF2 of 0.51 in our colony-forming assays. By FCM, a significant G2 phase arreast and DNA fragment were found in NCI-H446 cells after IR. Therefore, both cell lines were selected for the analyses. The radiation-related oligonucelotide microarray was fabricated successfully, which has 143 genes, including 127 genes involved in stress response, cell cycle progression, DNA damage and repair, apoptosis and proliferation, together with 11 housekeeping genes and 2 rice genes and 3 luciferase genes as control. Using selected lung cancer cell lines, we identified genes showing altered expression in lung cancer cell lines, as well as several potential ionizing radiation resistance candidate genes. We set a cutoff of > 1.5-fold for "induction" and <0i75 for "repression" in all experiments. According to these criteria, 34 differential expression genes were identified. To verify the expression of the genes identified in microarray experiments, RT-PCR was performed using the RNA at the 6h, 24h and 48h after IR. We tested seven genes ( MDM2, p53, , XRCC5, Bcl-2, PIM2 ,NFKBIA and Cyclin Bl) for RT-PCR, and found that the results were inaccordance with those from the microarray data except for NFKBIA gene, eventhough the value for each mRNA level might be different between the twomeasurements. Among the regulated genes, some might be predicted to result ineffects on DNA repair, some on cell cycle arrest, others on apoptosis. Ofnotable interest was MDM2, which has been shown to be a critical component ofthe responses to IR. In our studies, we found that MDM2 was induced in A549 cellsand reduced in NCI-H446 cells after IR. IR can activate p53 to arrest the cell cycle andto stimulate apoptosis. However, p53 is nr the only factor that determinesradiosensitivity. p53 and MDM2 form an autoregulatory feedback loop. The levelof MDM2 expression determines the extent to radiation induces an increase in theactivity of the p53 tumor suppressor. Therefore, the ability of MDM2 to conferradioresistance to tumors is beginning to be assessed. Now, it is important todetermine whether increased levels of MDM2 correlate with poor clinicaloutcome and whether MDM2 can be targeted to increase the efficacy ofradiotherapy. By antisense technology, inhibition of MDM2 using ASO5 hassignificant enhanced radiosensitivity in reducing survival and increasing apoptosisof A549 cells. By RNAi, inhibition of MDM2 using siRNAs has significantlyenhanced the efficacy of IR in reducing survival and increasing cell death ofA549 cells, especially for siRNAl. Although gene silencing by" transfection ofsiRNA is effective in mammalian cells, it reduces gene expression for only a shortperiod. Loss of long-term gene silencing was found despite the cells resistant topuromycin.As described and discussed, Highlights of our study were fabrication of radiation-related oligonucleotide microarray and the detection of a few genes with cell proliferation, such as MDM2, BCL-2, PKCz and PIM2 expression levels更多还原