【作者】 杜雪莲；

【导师】 温泽清；

【作者基本信息】 山东大学 ， 妇产科学， 2009， 博士

【摘要】 第一部分利用寡核苷酸基因芯片技术筛选子宫内膜癌放射敏感性基因第一章人子宫内膜癌体外放射敏感性的检测【目的】子宫内膜癌（endometrial cancer）是妇科最常见的恶性肿瘤之一,其发病率在世界范围内呈逐年上升趋势。对存在高危因素的患者放疗联合手术治疗能显著提高子宫内膜癌疗效、降低盆腔及阴道残端的复发率,对合并有内科疾病的患者放疗是主要的治疗手段。因此,目前约2/3的子宫内膜癌患者需接受放疗,但在这些患者中仍有相当数量的病例在5年内复发。多数学者认为,患者对放疗的反应主要取决于子宫内膜癌个体固有的放射敏感性。本研究通过观察体外不同剂量X线照射后,4株人子宫内膜癌细胞的生存情况及影响因素,进行放射敏感性和敏感程度的初步研究,筛选出放射敏感性差异的细胞株,为下一步研究提供良好的实验基础和理想的实验材料。【方法】培养4株人子宫内膜癌细胞株Ishikawa（ISK,高分化腺癌）、RL95-2（中分化腺鳞癌）、HHUA（高分化腺癌）和KLE（低分化腺癌）,应用6MV X线照射细胞（剂量分别为0、1、2、3、4、6、8、10Gy）,观察细胞照射前后形态学变化,计算各组的克隆形成率,通过GraphPad prism软件,采用“多靶单击模型”来拟合剂量存活曲线,并计算放射敏感性参数外推值（N）、平均致死剂量（Do）、准阈剂量（Dq）和2Gy照射后细胞的存活分数（SF₂）。并用流式细胞术检测X线照射后ISK和KLE细胞周期的变化及凋亡细胞的比例。【结果】子宫内膜癌细胞照射后可见部分细胞形态变圆、脱落,呈凋亡改变。照射后的细胞种植于培养瓶后,培养约14～21d可形成肉眼可见的细胞克隆。四株子宫内膜癌细胞的SF₂值分别为0.572,0.510,0.446和0.328,其中ISK细胞的SF₂值最高,KLE细胞SF₂最低,二者之间存在明显差异（P=0.017）。细胞周期检测结果显示,照射前ISK细胞G₂/M期细胞比例明显低于KLE细胞（13.6%vs.37.0%,P＜0.05）。6Gy X线照射后,两组细胞均出现明显的G₂/M期阻滞,同时伴随S期比例下降。比较照射后12小时两组细胞中G₂/M期细胞增加的比例,在ISK细胞中高于KLE细胞（50.0%vs.14.1%,P＜0.05）。6Gy X线照射24小时后,与照射前比较2株细胞凋亡率均有增加,KLE细胞中凋亡细胞的比例（Annexin V+/PI-）从照射前的1.58%升高到9.25%,ISK细胞中由2.88%升高到4.60%,而KLE细胞的凋亡率明显高于ISK细胞（P＜0.05）。【结论】4株人子宫内膜癌细胞株中KLE细胞的放射敏感性最高,ISK细胞放射敏感性最低,这2株细胞被筛选出进入下一部分实验。第二章利用基因芯片技术筛选子宫内膜癌放射敏感性基因【目的】肿瘤患者对放疗的不同反应主要是由于其放射敏感性不同决定的,肿瘤本身固有的放射敏感性与辐射诱导的基因表达和基因调控有关,因此,通过基因表达谱差异研究可以发现与子宫内膜癌放射敏感性密切相关的基因,有利于制定子宫内膜癌患者个体化治疗方案。本部分研究的目的是通过应用含人类全基因组序列的寡聚核苷酸芯片对已筛选出的两个放射敏感性不同的子宫内膜癌细胞株进行检测,比较其接受照射前后基因表达谱的变化,并对筛选出的差异表达基因进行初步验证,以寻找与内膜癌放射敏感性密切相关的基因。【方法】1.应用北京博奥生物有限公司提供人类全基因组表达谱芯片22KHuman Genome Array检测4Gy X线照射后ISK细胞株及KLE细胞株的基因表达谱,分别筛选照射前后的差异表达基因。2.对两株细胞的差异表达基因进行主成分分析、GO分析及Pathway聚类分析,通过NCBI生物医学文献数据库检索基因信息及功能,寻找与子宫内膜癌放射敏感性相关的主效基因,探索放射耐受发生的分子机制。利用Molecule AnnotationSystem（MAS）系统进一步分析ISK和KLE细胞株接受X线照射后的基因表达谱变化,比较二者辐射诱导基因表达的差异。3.采用相对定量荧光实时PCR技术及蛋白印迹的方法对部分差异表达基因或编码的相应蛋白进行mRNA及蛋白水平的验证,以确定芯片检测结果的可靠性。【结果】1.ISK细胞株照射后与照射前比较差异表达的基因数目为227条,其中上调的基因共有70条,下调的有157条;KLE细胞株照射前后的差异基因数目为354条,其中上调的基因有121条,下调的233条。功能涉及不同的分子功能分类,参与多个生物学过程,主要涉及到DNA损伤修复、凋亡、生长因子、信号转导、细胞周期及细胞黏附等方面。2.用Gene Ontology聚类分析,ISK和KLE比较共筛选出53条差异在两倍以上的基因,其中已知基因48条,EST序列5条。上调表达基因24条,功能主要涉及DNA双链断裂修复、生长因子、凋亡抑制基因等,下调表达基因29条,主要与细胞周期、细胞黏附功能等有关。3.相对定量荧光实时PCR技术及蛋白印迹方法对部分差异表达在2倍以上的基因或编码的相应蛋白进行mRNA及蛋白水平的验证,显示实时PCR和蛋白印迹实验结果与芯片结果具有良好的一致性,进一步证实了芯片结果的可靠性。【结论】1.子宫内膜癌放射敏感性的分子机制极为复杂,是多基因协同作用的结果,涉及到DNA损伤修复、细胞周期调控、肿瘤细胞凋亡、信号转导、细胞粘附、应激反应等多种遗传学改变。2.聚类分析得出的差异表达基因可能与内膜癌放射敏感性密切相关。尤其是对在ISK细胞株中表达上调基因的进一步研究将有助于解释子宫内膜癌放射耐受发生的分子机制,为提高放射治疗的敏感性提供新的靶点及策略。第二部分子宫内膜癌肿瘤相关血管内皮细胞的分离提纯、鉴定以及基因表达谱的检测【目的】子宫内膜癌是妇科最常见的恶性肿瘤之一,虽然大部分早期患者预后较好,但晚期患者的复发率和死亡率仍较高,因此迫切需要探讨新的治疗途径。肿瘤的生长和转移与血管生成和内皮细胞的活动密切相关,抑制肿瘤血管生成,可减少肿瘤赖以生存的营养供应,从而抑制肿瘤生长。抗血管生成对于治疗和预防肿瘤的转移复发一种非常有前途的方法,但由于缺乏子宫内膜癌血管特异的靶点,因而疗效有限。本部分研究拟通过分离子宫内膜癌及正常子宫内膜中的人子宫内膜血管内皮细胞（human endometrial endothelial cells,HEECs）,检测其生物学特性及血管生成相关基因表达的改变,以期发现子宫内膜癌转移过程中,与血管生成密切相关的基因表达的改变,为内膜癌血管生成研究提供新的分子标记和治疗靶点。【方法】1.收集病理学证实的新鲜的子宫内膜癌组织和正常内膜组织标本,采用优化的CD31单克隆抗体交联免疫磁珠分选法,对人子宫内膜癌组织及正常内膜组织进行血管内皮细胞分选。对获得的HEECs应用免疫荧光法检测内皮细胞标记分子的表达情况,并用流式细胞术检测内皮细胞纯度。2.应用人类全基因组表达谱芯片22K Human Genome Array检测子宫内膜癌肿瘤相关HEECs的基因表达谱的改变,筛选不同于正常HEECs的差异表达基因。并对差异表达基因进行主成分分析、GO分析及Pathway聚类分析,通过NCBI生物医学文献数据库检索基因信息及功能,寻找与子宫内膜癌血管生成相关的基因。应用相对定量荧光实时PCR技术及蛋白印迹方法对部分差异表达在2倍以上的基因或编码的相应蛋白进行mRNA及蛋白水平的验证。3.对分离的肿瘤相关HEECs和正常HEECs进行体外培养,分别应用MTT法、划痕实验、Transwell侵袭小室模型、Matrigel胶体外管腔形成实验来比较二者增殖、迁移、侵袭和管腔形成能力。【结果】1.分选获得HEECs的纯度均在95%以上。体外培养呈现典型的内皮细胞形态特征,免疫荧光证实此内皮细胞表达CD31、CD34和vWF,而肿瘤上皮标记分子角蛋白8、造血细胞标志物CD14、CD45及平滑肌细胞肌动蛋白抗体检测,均为阴性表达,从而排除了肿瘤细胞、淋巴细胞及组织平滑肌细胞污染的情况。2.与正常内膜中的HEECs相比,肿瘤相关HEECs中共有317条基因差异超过2倍以上,其中191条为上调,126条为下调。上调基因主要包括:细胞外基质功能相关的基因,如MMP10、MMP9、LAMB1、SPP1、LOX及LGALS3BP;与肌动蛋白细胞骨架功能有关的基因如CAPG、CAPZA1和TMSB10;细胞周期调节基因MAPRE1、CDC25B、CCNB2等。下调超过5倍的25条基因,其中包括具有潜在抗血管生成和增殖作用的基因如周期素依赖性蛋白激酶抑制剂2A（CDKN2A）、溶质传递家族29-2（SLC29A2）;神经生长因子受体相关蛋白1（NGFRAP1）;凋亡诱导基因如凋亡伴生斑点像蛋白质包含看板卡（PYCARD）以及细胞黏附分子连环蛋白1（CTNNB1）等。Pathway分析有显著性差异的通路中占主导地位有细胞周期、细胞黏附分子（CAMs）、黏着斑、细胞外基质及受体相互作用通路等。相对定量荧光实时PCR技术及蛋白印迹方法对部分差异表达在2倍以上的基因或编码的相应蛋白进行mRNA及蛋白水平的验证,结果显示实时PCR和蛋白印迹实验结果与芯片结果具有良好的一致性。3.在体外培养条件下,肿瘤相关HEECs和正常对照HEECs增殖率无显著性差异（P=0.173）。在同样条件下,肿瘤相关HEECs在划痕实验修复面积与对照相比明显提高（P=0.006）,在侵袭实验中穿过膜的细胞数也显著增加（P=0.033）。HEECs在Matrigel胶上排列成多中心连接的血管腔样结构,肿瘤相关HEECs形成的血管腔样结构明显多于正常HEECs（P=0.026）。【结论】采用优化的免疫磁珠分选法高纯度地分离纯化出子宫内膜癌和正常内膜组织中的HEECs,分离的内皮细胞可用于内膜癌血管生成的相关研究。HEECs体外培养生物学特性表明:肿瘤相关HEECs迁移、侵袭能力明显增强,与正常内皮细胞相比具有更强的血管生成能力。基因芯片检测结果显示肿瘤相关HEECs与正常HEECs比较基因表达水平存在明显差异,这些差异基因为内膜癌血管生成研究提供了新的分子标记和治疗靶点。更多还原

【Abstract】 PartⅠIdentification of Differentially Expressed Genes Response to Ionizing Radiation in Human Endometrial Cancer Cell Lines with Distinct Radiosensitivities by Oligonucleotide MicroarraysChapterⅠExperimental Study of Radiosensitivity for Human Endometrial Cancer Cell LinesOBJECTIVE:Endometrial cancer is one of the most common gynecological malignancies worldwide,which morbidity is increasing year by year.High-risk patients are at risk for local-regional relapse and therefore adjuvant radiotherapy is essential for local control.Although there are two-thirds of patients need adjuvant radiotherapy,quite a few patients recurrent within 5 years.It is generally believed that the efficacy of this therapeutic modality depends on the individual inherent radiation sensitivity.The aim of the present study is to investigate the radiosensitivities of 4 human endometrial cancer cell lines ISK,RL95-2,HHUA and KLE and select two cell lines with different radiosensitivity for later analysis.METHODS:Human endometrial cancer cells from the ISK（well differentiated）, RL95-2（moderately differentatied）,HHUA（well differentiated） and KLE（poorly differentiated） cell lines were irradiated by 6MV X-rays with various doses:0,1,2,3, 4,6,8,10Gy,and plating efficiency was calculated from the postirradiated cells by colony formation assay.Survival curves were determined by ’single hit multi target theory’ and radiosensitivity parameters were calculated by GraphPad prism software. Cell cycle distributions and apoptosis after irradiation were analyzed using a FACScan flow cytometer.RESULTS:After irradiation,the cells became round in morphology and appeared anoikis comparing with normal cell.Colonies formed after 10 days of incubation.The SF₂ values were 0.572,0.510,0.446,and 0.328,respectively.Significant difference was found between ISK and KLE cells（P=0.017）.The cell cycle distribution showed lower fractions of G₂/M phase cells in radioresistant cell line ISK before radiation compared with radiosensitive cell line KLE（13.6%vs.37.0%,P＜0.05）.Irradiation caused cell cycle arrest at G₂/M phases in both cell lines.12 hours after radiation, higher fractions of G₂/M phase cells were found in ISK compared with KLE（50.0% vs.14.1%,P＜0.05）.Apoptosis assessment also showed significant elevation in the percentage of early apoptosis cells in KLE cells（P＜0.05）.CONCLUSION:From detection of clonogenic survival curves for the four cell lines exposed by X-ray radiation,we select two cell lines with different radiosensitivity.Cell line KLE was classified as radiation-sensitive,and cell lines ISK were classified as relatively radiation resistant. ChapterⅡDifferential Expression Profiling of Genes Response to Ionizing Radiation in Endometrial Cancer Cell LinesOBJECTIVE:The efficacy of radiotherapy depends on the individual inherent radiosensitivity which related to gene expression induced by irradiation.Identification of the genes differentially expressed between radiosensitive and radioresistant cancers may provide new insights into the mechanisms underlying clinical radioresistance and improve the efficacy of radiotherapy.The aim of the present study is to examine global expression patterns induced by X-ray irradiation of two cell lines with different radiosensitivities and elucidate the further molecular events involved in radiosensitivity of endometrial cancer.METHODS:Microarray experiments were performed using 22K Human genome oligonucleotide microarrays containing 21,329 well-characterized Homo sapiens genes to screen gene expression changes after X-ray exposure in ISK and KLE cells.Differential expression was determined using the combined basis of t-test with P＜0.05 and fold changes（either up or down） of≥2-fold.All differentially expressed genes were analyzed using Molecular Annotation System 5.0 which integrates three pathway resources—KEGG,BioCarta and GenMAPP.To show the reproducibility of the microarray analysis,10 genes were selected at random,and these changes were validated by real-time RT-PCR and Western blot.RESULTS:227 and 354 genes that exhibited≥2-fold difference were identified in ISK and KLE,respectively.However,only 53 genes showing differences more than double the median expression value between the two groups were defined as radiosensitivity（or radioresistance） related genes.Among these,genes associated with DNA-repair,apoptosis,growth factor,signal transduction,cell cycle and cell adhesion were obviously predominant.The validity of the expression level of 10 randomly selected genes was confirmed by real-time PCR and/or Western blotting.CONCLUSION:The molecular mechanisms of radiosensitivity in endometrial cancer are very complex and involve expression changes of multiple genes associated with DNA-repair,cell cycle,apoptosis,signal transduction,cell adhesion and stress reaction.The differential gene expression changes that occur after radiation in the two cell lines will not only provide insight into molecular mechanisms of radioresistance in endometrial carcinoma,but also as a means to find potential targets to achieve further gains in therapeutic benefit. PartⅡIsolation,Purificantion and Gene Alterations in Tumor-associated Human Endometrial Endothelial CellsOBJECTIVE:Endometrial cancer is one of the most common malignancies of the female genital tract.Although early-stage endometrial cancer can be treated surgically,mortality in women with advanced disease has remained largely unchanged despite improvements in surgical technique,chemotherapy regimens,and radiation protocols.Therefore,novel therapeutic strategies are highly needed.It is now well known that tumor growth and proliferation are associated with angiogenesis. The growth of malignant tumor can be reduced through inhibiting angiogenesis.The therapeutic effect of antiangiogenesis is limited by deficiency of specific target for endometrial cancer.In the present study,we aim to immunopurify endothelial cells from freshly resected specimens of endometrial cancers and normal endometrial tissues and investigate the gene expression profile using microarrays.The growth characteristics and the capacity of immigration,invasiveness,and tube formation of isolated human endometrial endothelial cells（HEECs） are examined additionally.The alterations in gene expression profile in tumor-associated human endometrial endothelial cells（HEECs） may allow opportunities for developing new therapeutic approaches to inhibit angiogenesis in endometrial cancer.METHODS:Endothelial cells were isolated from 3 freshly collected endometrial cancer tissues and 3 normal endometria tissues with anti-CD31 conjugated magnetic microbeads.Global expression patterns of purified HEECs were analyzed using 22K Human genome oligonucleotide microarrays.All differentially expressed genes were analyzed using Molecular Annotation System 5.0 which integrates three pathway resources—KEGG,BioCarta and GenMAPP.Real-time RT-PCR and Western blot were used to validate the reproducibility of microarray analysis.We also performed in vitro culture and identified the endothelial origin,as well as observed the functional characteristics in angiogenesis by MTT,Wound Healing Assays,Transwell Cell Invasion Assay and Tube Formation Assay of HEECs from the two different sources.RESULTS:Flow cytometry revealed that the immunopurification technique yielded endothelial cell purity of＞95%in all samples.HEECs separated from normal and malignant endometrial tissue presented similar appearance in culture medium under phase contrast microscope.Cell cultures presented flattened monolayers,with typical cobblestone morphology.All purified cells were characterized as endothelial cells on the basis of expression of the classical endothelial markers vWF,CD31,and CD34 as shown by immunofluorescence examination.In addition,the cells were negative when stained with antibodies recognizing the epithelial cell markers（keratin 8 and 6A）,hematopoietic cells（CD14 and CD45）,and smooth muscle cell actin.Microarray analyses revealed distinct gene expression pattems and consistent up-regulation of certain endometrial endothelial marker genes across patient samples. 317 genes that exhibited≥2-fold differences,including 191 up-regulated genes and 126 down-regulated one,were identified in tumor-associated HEECs.Several proteins involved in extracellular matrix function,such as MMP10,MMP9,LAMB1,SPP1, LOX,and LGALS3BP,had increased expression in tumor vasculature.Several proteins responsible for actin cytoskeleton organization and regulation,such as CAPG, CAPZA1 and TMSB10,were also up-regulated.MAPRE1,CDC25B and CCNB2, genes correlated with regulation of cell cycle,were also elevated in tumor endothelium.There were 25 genes with≥5-fold decrease in tumor endothelium, including several genes with potential antiangiogenic or antiproliferative roles,such as Cyclin-dependent kinase inhibitor 2A（CDKN2A） and Solute cartier family 29 member 2（SLC29A2）,nerve growth factor receptor associated protein 1（NGFRAP1）, apoptosis-associated speck-like protein containing a CARD（PYCARD）,and cellular adhesion,molecule Catenin beta 1（CTNNB1）.Pathway analysis showed that pathways of Cell cycle,Cell adhesion molecules （CAMs）,focal adhesion,and extracellular matrix（ECM）-receptor interaction were obviously predominant.The results of the microarray analysis were confirmed by quantitative real-time PCR,immunohistochemistry,and/or western blotting. After cells were cultured for 24,48,72 and 96 h,there was no significant difference in proliferation rate of tumor-associated HEECs and normal HEECs （P=0.173）.Moreover,although the tumor-associated HEECs didn’t show faster proliferation than normal HEECs,they exhibited enhanced migration ability （P=0.006）,potent invasiveness（P=0.033）,and elevated tube formation in vitro （P=0.026）.CONCLUSION:HEECs can be efficiently isolated from endometrial cancer and normal endometrial tissues by immunomagnetic methods and these cells were verified for angiogenesis research in endometrial cancer.The present study shows that the tumor-associated HEECs exhibited enhanced migration ability,potent invasiveness,and elevated tube formation in vitro.Microarray analysis show that tumor and normal endothelium differ at molecular level,and additional characterization of this gene expression database will provide insights into the angiogenesis of endometrial cancers and might be of great benefit for finding potential therapeutic targets.更多还原