【作者】 卿毅；

【导师】 王东；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2009， 博士

【摘要】 宫颈癌是我国女性发病率较高的恶性肿瘤之一,每年新增宫颈癌病例130000余例、死亡50000余例,其发病率与死亡率分别占我国妇科肿瘤的第一位和第二位,对中晚期或术后复发的宫颈癌患者,放射治疗是主要的治疗方法。1998年,我院全国首家应用体外照射联合中子后装治疗宫颈癌,肿瘤局部控制率和5年存活率分别为85.2%和88.7%,取得了良好的疗效,但在治疗过程中,我们观察到部分宫颈癌患者存在放射抵抗的问题,而且在临床实践中也发现治疗过程中有耐放射性改变,因此,研究影响宫颈癌细胞放射敏感性的因素具有十分重要的意义。DNA损伤修复是影响放射敏感性的重要因素,因此深入研究DNA修复基因的表达谱,有助于阐明宫颈癌抵抗放疗的分子机制,为寻找提高宫颈癌放疗敏感性的分子靶点提供实验基础。但DNA损伤修复机制十分复杂多样,小数量的基因不能阐述清楚,需要参与DNA损伤修复过程基因组的大量信息,这样才能真正了解其分子机制。而基因芯片技术是一个强有力的获得癌细胞中成千上万的基因表达的综合性信息的工具,通过该技术可为研究DNA损伤修复基因致宫颈癌放疗抵抗的分子机制提供有效方法。因此本研究首先检测了DNA损伤修复基因APE1在宫颈癌中的表达情况,分析APE1与宫颈癌的临床病理因素及锎-252中子放疗的预后的关系,初步探讨了DNA损伤修复基因APE1与宫颈癌放射抵抗的相关性。然后通过诱导建立宫颈癌耐放射细胞株,采用基因芯片技术对宫颈癌放DNA损伤修复信号通路基因表达谱进行研究,筛选出GADD45α等差异表达基因,构建GADD45α表达载体并初步证明其增加宫颈癌放疗敏感性的作用。研究目的1.探讨DNA损伤修复基因APE1与宫颈癌放射抵抗的相关性;2.探讨DNA损伤修复相关基因致宫颈癌放射抵抗的机制;3.初步探讨GADD45α表达载体增强宫颈癌放疗敏感性的作用。研究内容和方法1. DNA损伤修复基因APE1在宫颈癌中的表达及其与锎-252中子放疗预后的关系:采用采用免疫组化方法APE1在宫颈癌中的表达情况,分析APE1与宫颈癌的临床病理因素及锎-252中子放疗的预后的关系;2.诱导并建立放射线照射耐受株:应用直线加速器和锎-252中子后装治疗机对宫颈癌Hela细胞株进行剂量个体化的反复放射线照射,剂量呈梯度增加,最后使其耐放射性具有一定的稳定遗传能力,建立耐中子射线细胞株HelaNR和耐X射线细胞株HelaXR,并通过克隆形成分析、超微结构观察、细胞倍增时间、细胞周期分布和凋亡这些指标检测其耐放射特性。3.耐放射宫颈癌细胞株DNA损伤修复相关差异表达基因的筛选:采用DNA损伤信号通路基因芯片(OHS-029)检测Hela、HelaNR和HelaXR细胞的基因表达谱,分析其差异表达基因,并采用western blot和Real-time PCR技术验证基因芯片结果的可靠性。4. GADD45α表达载体增强宫颈癌放疗敏感性的初步实验研究:构建特异性pcDNA3.1-GADD45α真核表达载体,Real-time PCR检测转染后宫颈癌细胞GADD45αmRNA表达情况,流式细胞仪检测转染后细胞凋亡情况。研究结果1. DNA损伤修复基因APE1在宫颈癌中的表达及其与锎-252中子放疗预后的关系:宫颈癌组织APE1表达水平明显高于正常宫颈组织和CIN病例(P<0.01)。APE1在正常宫颈组织和CIN病例均呈胞核表达,宫颈癌组织中APE1呈胞核表达(59)、单纯胞浆表达(8)或核浆共同表达(22)。APE1表达强度与FIGO分期、病理分级和淋巴结转移情况有关(P<0.05),与年龄和病理分型无关。APE1亚细胞定位情况与FIGO分期、病理分级有关(P<0.01),与淋巴结转移情况无关。生存分析显示在APE1核表达组(中位生存时间70.9月)和APE1低表达组(中位生存时间75.8月)的生存时间明显长于APE1浆表达组(中位生存时间57.8月)和APE1高表达组(中位生存时间56.5月)(P=0.025,0.001)。2.诱导并建立放射线照射耐受株:(1)克隆形成分析结果发现耐放射株与亲本株相比,SF2值、D0(平均致死剂量)、Dq(准域剂量)值均明显增高,结果说明诱导的耐放射细胞株的放射敏感性降低了,其耐放射性具有一定的稳定遗传能力。(2)通过电子显微镜观察细胞,可发现耐放射细胞的形态与结构均发生了变化,细胞表面可见伪足样突起;细胞浆内可见大量空泡,核蛋白体、线粒体、染色体、粗面内质网、核仁均出现了形态变化,细胞骨架排列紊乱。(3)Hela, HelaNR和HelaXR的细胞倍增时间分别是(28.62±2.77) h、(33.12±3.67) h和(36.94±3.16) h,放射耐受株的细胞倍增时间明显长于亲本株(P <0.05)。(4)我们用流式细胞仪检测细胞周期分布的结果显示,相对于亲本株Hela,中子耐受细胞株HelaNR和X射线耐受细胞株HelaXR的各周期百分比变化并不明显;但在接受4Gy射线照射后,亲本株G2期分布明显升高,G1期分布减少,而放射耐受细胞株G2升高不明显;在接受16Gy射线照射后,放射耐受细胞株G2期也明显升高,但亲本株G2期升高更加明显。这也说明放射耐受细胞株较亲本株的放射敏感性更低。(5)凋亡检测结果显示,随着照射剂量的增加,细胞的凋亡率也升高,但在相同剂量射线照射后,放射耐受株的细胞凋亡率明显低于亲本株细胞,两者间有显著性差异(P <0.05),表明在相同放射剂量的照射下,放射耐受株对放射线更加抗拒,进一步证明了诱导的耐放射株具有放射抵抗性。3.耐放射宫颈癌细胞株DNA损伤修复相关差异表达基因的筛选:与亲本株Hela细胞相比,耐放射株HelaNR和HelaXR细胞基因表达改变的总趋势是一致的,表明中子射线和X射线照射诱导Hela细胞发生放射抵抗,引起相似的DNA损伤修复基因表达的改变。筛选亲本株与耐放射株差异在两倍以上的基因:HelaNR细胞有24个差异表达基因,其中19个上调,有5个下调;HelaXR细胞有41个差异表达基因,其中38个上调,有3个下调。通过生物信息和参考文献挖掘得到GADD45α和BTG2等可能与宫颈癌放射耐受密切相关的基因,并采用western blot和Real-time PCR检测了Hela、HelaNR和HelaXR细胞中GADD45α和BTG2两个基因的蛋白和mRNA表达情况,结果显示:在HelaNR和HelaXR细胞中,GADD45α表达下调,BTG2表达上调,与基因芯片的结果是一致的。4. GADD45α表达载体增强宫颈癌放疗敏感性的初步实验研究:通过测序和在PUBMED上做BLASTn比对,结果表明,重组质粒中插入的序列与已知GADD45α基因序列同源性达到99%,我们成功构建pcDNA3.1-GADD45α真核表达载体,Real-time PCR检测结果显示转染后细胞GADD45αmRNA含量明显增加(P<0.05),说明pcDNA3.1-GADD45α真核表达载体能增加GADD45α基因在这3个细胞株中的表达。进一步用流式细胞仪检测0、4和16Gy X射线照射后Hela细胞的凋亡情况,结果显示随着照射剂量的增加,细胞的凋亡率也升高,但在相同剂量X射线照射后,对照组的细胞凋亡率与脂质体组无显著差异(P>0.05),转染组的细胞凋亡率明显高于对照组和脂质体组,它们之间有显著性差异(P <0.05),表明转染pcDNA3.1-GADD45α质粒,增加Hela细胞GADD45αmRNA表达,可增加X射线照射后细胞的调亡率。结论1. APE1表达强度和亚细胞定位情况与宫颈癌的发生、发展和锎-252中子放疗的预后有关,APE1表达强度与宫颈癌的侵袭和转移有关,提示APE1的DNA损伤修复功能可能是导致宫颈癌放疗抵抗的重要因素;2.我们采用梯度增加剂量照射宫颈癌Hela细胞进行诱导的方法,建立了耐中子射线细胞株HelaNR和耐X射线宫颈癌细胞株HelaXR,并通过检测其放射生物学特性,证明了诱导的耐放射株具有放射抵抗性;3.通过基因芯片技术检测宫颈癌Hela细胞株和诱导的耐放射亚株HelaNR及HelaXR的基因表达谱,筛选出与宫颈癌放射抵抗相关的DNA损伤修复信号通路差异表达基因,为宫颈癌放疗耐受的干预及基因放射治疗提供可能的靶点。4.构建pcDNA3.1-GADD45α真核表达载体,转染入宫颈癌细胞,可显著增加其GADD45α基因的表达,并且能显著增加X射线诱导的细胞凋亡,初步说明了提高宫颈癌细胞GADD45α基因表达可增加其对放射线的敏感性。更多还原

【Abstract】 Cervical cancer is one of the most common malignancies in China, with a high incidence and mortality and more than 130,000 new cases reported and 50,000 women dying of the disease per year. Radiotherapy is particularly effective for patients with cervical cancers at an advanced stage or that cannot be cured surgically. In 1998, we first treated cervical cancer with 252Cf rays combined with X-rays in China. It obtained satisfactory clinical effect, tumor local control rate was 85.2% and 5-year survival rate was 88.7% respectively. But we observed the existence of radioresistance phenomenon in some primarily cervical cancer patients and secondary radioresistance in course of treatment. Therefore, it was very important that we investigated influencing factors of radiosensitivity in cervical cancer.The activity of the DNA damage repair pathway is one of the most important factors leading to radioresistance in tumors, including cervical cancer. In many human tumor lines, radiosensitivity correlated with DNA damage induction and repair, so the research of DNA damage repair gene expression profiles is helpful to elucidate the mechanism of radioresistance in cervical cancer and supports to look for the targets of improving radiosensitivity. But the mechanism of DNA damage repair so complex that it couldn’t be explained by a little of genes and need much information of DNA repair genes. DNA microarray technology is a powerful technique to detect the biological response of thousands of genes and can be used to study the mechanism that DNA damage repair gene leads to radioresistance in cervical cancer. Hence, we first investigate the expression feature of APE1, a DNA damage repair gene, and its correlation with clinicopathology and prognostic significance after 252Cf radiotherapy in cervical cancer. Then, long-term 252Cf neutron ray and X-ray irradiation of Hela cells was used to generate two radioresistant cell sub-lines, HelaNR and HelaXR, which provided a model system for studying the radioresistance mechanisms of cervical cancer cells. Whereafter, microarray analysis was used to identify the gene expression patterns of two radioresistant sub-lines derived from Hela cells and the differentially expressed genes were selected, for example GADD45αand BTG2. Finally, we constructed the eukaryotic expression vector of GADD45αgene and investigated its role in radiosensitivity to radiotherapy in cervical cancer.Objective1. To explore the relationship between APE1 and radioresistance in cervical cancer.2. To investigate the mechanism that DNA damage repair gene leads to radioresistance in cervical cancer.3. To investigate primarily the function that eukaryotic expression vector of GADD45αgene enhance radiosensitivity in cervical cancer.Materials and Methods1. The expression feature of APE1, a DNA damage repair gene, and its correlation with clinicopathology and prognostic significance after 252Cf radiotherapy in cervical cancer: The expression of APE1 was detected by immunohistochemistry technique in 89 cases of cervical cancer (treated by 252Cf), 15 cases cervical intraepithelial neoplasia(CIN) and 10 cases of normal cervical tissue, and its association with clinicopathological data as well as prognosis was analyzed.2. The generation of two radioresistant cell sub-lines: Hela cells were treated with fractionated 252Cf neutron and X-rays, with a cumulative dose of 75 Gy each, over 8 months, yielding the sub-lines HelaNR and HelaXR. Radioresistant characteristics were detected by clone formation assay, ultrastructural observations, cell doubling time, cell cycle distribution, and apoptosis assay.3. The screening of differentially expressed genes relate to DNA damage repair genes in cervical cancer: Gene expression patterns of the radioresistant sub-lines were studied through microarray analysis and verified by western blotting and real-time PCR.4. The investigation of eukaryotic expression vector of GADD45αgene enhancing radiosensitivity in cervical cancer: we constructed the eukaryotic expression vector of GADD45αgene. Then, The expression of GADD45αmRNA was detected after eukaryotic expression vector of GADD45αgene was transfected into cervical cancer cell strains by real-time PCR and the apoptosis was detected after eukaryotic expression vector of GADD45αgene was transfected into Hela cells by FCM. Results1. The expression feature of APE1, a DNA damage repair gene, and its correlation with clinicopathology and prognostic significance after 252Cf radiotherapy in cervical cancer: The expression of APE1 in cervical cancer is higher significantly than that in normal cervical tissue and CIN(P<0.01). In normal cervical tissue and CIN, the APE1 express was located in the nucleus. In cervical cancer, the APE1 express was located in the nucleus (59), cytoplasm (8) or nucleus and cytoplasm (22), the location of APE1 was related with FIGO stage and pathological grade (P<0.01), and not related with lymph node metastasis. The level of APE1 express related with FIGO stage, pathological grade and lymph node metastasis (P<0.05), and not related with age and pathological type. The Kaplan-Meier survival analysis showed that the survival time of the group of APE1 nucleus expression (median survival time is 70.9 months) and the group of APE1 low expression (median survival time is 75.8 months) is longer significantly than that of the group of APE1 cytoplasm expression (median survival time is 57.8 months) and the group of APE1 high expression (median survival time is 56.5 months) (P=0.025, 0.001).2. The generation of two radioresistant cell sub-lines: (1) Under equal doses of 252Cf neutron ray and X-ray irradiation, the D0, Dq, and SF2 values of the HelaNR and HelaXR cells were higher than those of the Hela cells, indicating that the sub-lines were more radioresistant than the parent cells. (2) In Hela cells, microvilli were present on the surface and the cytoplasm contained abundant mitochondria and ribosome. In the radioresistant sub-lines, swelling of mitochondria, vacuolization, dilatation of the endoplasmic reticulum, and myelin figures were observed. Thus, in the HelaNR and HelaXR sub-lines, the ultrastructural changes induced by long-term irradiation persisted even > 2 months after the last radiation treatment. (3) The cell doubling times of the HelaNR and HelaXR cells (33.12±3.67 h, and 36.94±3.16 h, respectively) were longer than those of the parent Hela cells (28.62±2.77 h). (4) In Hela cells, exposure to a radiation dose of 4 Gy significantly increased the proportion of cells in G2 and decreased the proportion of cells in G1. The proportion of G2-arrested Hela cells was even greater following 16-Gy irradiation. In the HelaNR and HelaXR sub-lines, however, the proportion of cells in G2 did not increase after irradiation with 4 Gy. At 16 Gy, cells of both sub-lines became arrested in G2 but the proportion was much lower than in Hela cells. Thus, cells of the radioresistant sub-lines probably arrested in the G1 phase of the cell cycle. (5) At 0 Gy, the apoptosis rate was 0.93–2.71% for all three cell lines. At 4 and 16 Gy, the apoptosis rate of Hela cells was much higher than that of the radioresisitant sub-lines (4Gy: 9.32 vs 3.84, 7.94 vs 5.43; 16Gy: 22.47 vs 7.28, 20.03 vs 11.1, p < 0.05). Thus, the sub-lines HelaXR and HelaNR were more radioresistant than the parental Hela cell line.3. The screening of differentially expressed genes relate to DNA damage repair genes in cervical cancer: Using stringent criteria for array analysis (≥2-fold change in expression), we identified 113 genes related to DNA damage signaling pathways that were differentially expressed in the parental Hela cells compared to radioresistant HelaXR and HelaNR cells. Of the 24 genes significantly altered by at least 2-fold in HelaNR cells, 19 were up-regulated and 5 down-regulated. Likewise, of the 41 genes significantly altered by at least 2-fold in HelaXR cells, 38 were up-regulated and 3 down-regulated. For the two radioresistant sub-lines, the similar overall trend in gene-expression changes indicated that long-term exposure to 252Cf neutron and X-rays had resulted in a similar induction of genes involved in DNA damage signaling pathways. The number of genes that underwent homologous recombination and nonhomologous end-joining, processes that result in altered gene expression, was higher in HelaXR cells (8 and 3, respectively) than in HelaNR cells (2 and 1, respectively). Furthermore, the number of cell cycle arrest and mismatch repair genes expressed was higher in HelaXR cells (7 and 3, respectively) than in HelaNR cells (3 and 1, respectively) whereas the number of base excision repair genes expressed in the two sub-lines was almost the same (4 in HelaXR cells and 5 in HelaNR cells). Thus, genes encoding double-strand break (DSB) repair, mismatch repair, and cell cycle arrest functions were more highly expressed in cells resistant to X-rays than in cells resistant to 252Cf neutron rays. Two genes, BTG2 and GADD45α, were chosen for further analysis and validation of the microarray data by Western blotting and real-time PCR. As shown by Western blotting, BTG2 protein expression was up-regulated in the resistant sub-lines, especially in HelaNR cells, while the expression of GADD45αprotein was down-regulated in both resistant sub-lines. PCR analysis showed that BTG2 and GADD45αmRNA expression paralleled that of the respective proteins.4. The investigation of eukaryotic expression vector of GADD45αgene enhancing radiosensitivity in cervical cancer: Through gene sequencing and BLASTn in PUBMED, we determined that eukaryotic expression vector of GADD45αgene was constructed successfully. The expression of GADD45αmRNA increased significantly after eukaryotic expression vector of GADD45αgene was transfected into cervical cancer cell strains (P<0.05). Apoptosis rate for almost 0.81–1.91% was found at 0Gy dose point in three groups,which was increased with higher radiation doses. A significant difference was observed between the transfected group and control group, lipofectin group (p < 0.05). There was no significant difference between control group and lipofectin group (p> 0.05).Conclusion1. The dystopic express of APE1 might play a pivotal role in carcinogenesis and progression of cervical cancer, and the express of APE1 might estimate the prognosis after 252Cf radiotherapy. It showed that the DNA damage repair function of APE1 might correlate with radioresistance in cervical cancer.2. Hela cells were irradiated with fractionated 252Cf neutron and X-rays, yielding two radioresistant cell sub-lines HelaNR and HelaXR. The radioresistant sub-lines were more radioresisitant to parental Hela cells by detecting their radioresistant characteristics.3. Microarray analysis was used to identify the gene expression patterns of two radioresistant sub-lines derived from Hela cells and the differentially expressed genes relate to DNA damage repair genes in cervical cancer were selected. It supported to look for the targets of improving radiosensitivity in cervical cancer.4. The eukaryotic expression vector of GADD45αgene was constructed. The expression of GADD45αmRNA and radiation-induced apoptosis increased significantly after eukaryotic expression vector of GADD45αgene was transfected into cervical cancer cell strains. It showed that eukaryotic expression vector of GADD45αgene might enhance radiosensitivity in cervical cancer.更多还原