【作者】 郑靖芳；

【导师】 刘培淑； 刘劲松； Robert C. Bast Jr.；

【作者基本信息】 山东大学 ， 妇产科学， 2010， 博士

【摘要】 [研究背景与目的]卵巢癌是妇科肿瘤中死亡率最高的恶性肿瘤。由于缺乏有效的早期筛查手段,早期症状及临床表现不典型,约85%的患者初诊时已属疾病晚期。上皮性卵巢癌(Epithelial ovarian cancer, EOC),是卵巢癌中最常见类型,也是妇科肿瘤中死亡率最高的肿瘤类型,其死亡率高于其他所有妇科肿瘤之和。原因主要有早期筛查手段缺乏,早期诊断治疗对于延长患者生存时间是非常重要的一个环节,但是卵巢癌发生的分子生物学机制仍是未解之谜,给卵巢肿瘤的早期诊断带来困难和挑战。上皮性卵巢癌中又以卵巢高分化浆液性乳头状癌是常见的病理类型,普遍认为这种类型有部分起源于卵巢表面上皮细胞。然而,到目前为止,还没有任何直接实验证据证明卵巢表面上皮细胞能够形成高分化浆液性乳头状癌。研究表明,人卵巢恶性肿瘤的组织学类型分化是受多种因素影响的,包括上皮细胞一系列基因型和表现型的改变,可归纳为多种因素共同产生的生物学现象。探索卵巢癌的发生机制具有深远的理论和现实意义。HER2/neu基因是一种原癌基因,位于17号染色体长臂上,编码185 KD跨膜蛋白,具有酪氨酸激酶活性,属于人类表皮生长因子受体家族。以往对于HER2/neu基因的研究,主要集中于对乳腺癌的诊断、预后及靶向治疗的效果,而在上皮性卵巢癌中研究较少。研究表明,HER2/neu基因扩增及蛋白的过度表达与卵巢癌的预后及治疗有密切联系。25-30%的乳腺癌和卵巢癌中发现HER2/neu基因扩增及蛋白的过度表达,并被认为是与预后不良相关的因素,HER2/neu基因通过多种机制参与卵巢癌的演进,HER2/neu蛋白高表达与卵巢癌的多种临床特征相关,如临床分期较晚、手术中发现巨大肿块几率高、病理学分化差、较高复发率、生存时间短和对以铂类药为主的化疗不敏感。上皮-间质转化(epithelial-mesenchymal transition, EMT)是指上皮细胞在形态学上发生向成纤维细胞或间充质细胞表型的转变并获得迁移的能力。其主要特征为上皮表型缺失和间质表型获得。EMT在肿瘤发生与转移以及多种慢性疾病的发生发展过程中也起着重要的作用,研究发现,上皮-间质转化是细胞发生分子转换,失去上皮特性而获得间质特性,继而具备运动性以挣脱细胞间黏附,并侵犯到其他部位的过程。此外,肿瘤微环境通过特异因子的作用影响着肿瘤细胞的生物学特性。为了探讨HER2/neu基因过表达对卵巢上皮性肿瘤(Epithelial ovarian cancer,EOC)形成的作用,以及基因作用对上皮性卵巢癌生成的意义；进一步揭示肿瘤发生过程中上皮间质转化作用(Epithelial-mesenchymal Transition，EMT)以及肿瘤细胞生长微环境(Tumor Microenvironment)的影响。本文进行了以下几个方面的研究：1.建立遗传背景明确的过表达HER2/neu基因的T29Nt、T80Nt卵巢癌细胞模型。2.建立实验动物模型,揭示遗传成分与腹膜腔微环境共同诱导作用对正常人类卵巢表面上皮向卵巢恶性肿瘤转化过程的影响。3.观察并检测肿瘤生物学特性,为更多的卵巢肿瘤研究建立可靠稳定的肿瘤细胞模型,为临床卵巢恶性肿瘤的早期诊断提供实验基础和理论依据。第一部分通过逆转录病毒介导将HER2/neu基因导入永生化人类卵巢肿瘤前细胞系[研究目的]构建表达HER2/neu基因的逆转录病毒,将逆转录病毒感染两个永生化的人卵巢上皮肿瘤前细胞系T29、T80,经过药物筛选获得稳定过表达HER2/neu基因的T29N、T80N永生化细胞系。观察稳定过表达HER2/neu基因的T29N、T80N细胞株形态特征,并通过软琼脂实验检测细胞进行活体外探测评价和定量分析细胞纯系化集落生长的潜力和生物学特性。[研究方法]1.将带有HER2/neu基因的pBabe-HER2/neu-neomycin质粒转染Phoenix amphotropic包装细胞,获得带有目的基因的重组载体病毒上清。2.用带HER2/neu基因的逆转录病毒感染两个永生化的人类卵巢表面上皮肿瘤前细胞系T29和T80,T29和T80为本实验室通过将SV40 T/t抗原和hTERT转染至人类卵巢表皮细胞系IOSE29和IOSE80中而获得的永生化细胞系。感染细胞恢复后经新霉素筛选,分别得到稳定过表达HER2/neu基因的T29N和T80N两个永生化细胞系。3.倒置显微镜下观察T29、T80、T29N、T80N活细胞形态特征,并比较不同细胞系之间、子代细胞系与亲代细胞系之间的细胞表型差异。4.采用Western-blot印迹蛋白检测技术,验证HER2/neu蛋白在逆转录病毒感染后细胞系T29N、T80N与亲代细胞及对照细胞间表达的差异。5.应用软琼脂实验方法,取同代T29、T80、T29N、T80N细胞进行实验,进行活体外探测评价和定量分析细胞纯系化集落生长的潜力。[研究结果]1.带有HER2/neu基因质粒转染病毒包装细胞,产生病毒上清感染T29、T80细胞系,获得T29N、T80N经过pBabe-HER2/neu-neomycin质粒转染Phoenix amphotropic包装细胞,培养产病毒细胞系,获得带有HER2/neu基因的病毒上清。通过逆转录病毒介导,将HER2/neu基因导入到T29和T80中,用新霉素筛选获得含建立HER2/neu cDNA的纯化系,分别命名为T29N和T80N细胞系。2.亲代细胞与T29N、T80N细胞的形态学特征倒置显微镜下观察培养瓶内活细胞形态分别为：T29细胞与亲本细胞IOSE29一样具有间质细胞形态学特征,细胞呈现：细胞间隙略大,稍稍分离,细胞成短梭形类似成纤维细胞的间质细胞形态。而T80细胞与亲本细胞IOSE80一样具有上皮细胞形态学特征,显微镜下观察,细胞呈现：细胞核圆形或椭圆形,位于细胞中央或稍偏位,染色质细致均匀,为较典型的上皮细胞形态。T29N和T80N细胞都呈现上皮样细胞形态,细胞核圆形或椭圆形,位于细胞中央或稍偏位,染色质增粗不均匀。3.T29N和T80N细胞系中HER2/neu蛋白表达通过Western-blot蛋白印迹检测,以HER2/neu蛋白高表达的卵巢癌细胞SKOV3为对照,T29和T80细胞系为HER2/neu低表达,T29N和T80N细胞系获得稳定HER2/neu蛋白过表达。其中β-actin为内参照,比较加样总蛋白的量。4.体外细胞形成集落能力的差异取同代的过表达HER2/neu基因的T29N和T80N细胞与T29、T80细胞进行软琼脂实验,结果发现过表达HER2/neu的T29N、T80N细胞株比T29、T80细胞具有更强的集落形成能力,生成的集落数约为3倍。[结论]1.成功构建带有HER2/neu基因的逆转录病毒,并通过病毒感染获得稳定过表达HER2/neu的T29N、T80N永生化细胞株。2.T29N细胞呈现间质细胞形态,T80N细胞为上皮细胞形态。3.过表达HER2/neu的T29N、T80N株纯系化细胞比T29、T80细胞具有更强的集落形成能力。第二部分永生化细胞系T29N、T80N及移植瘤细胞系T29Nt、T80Nt的体内外实验研究[研究目的]分别将T29. T80、T29N、T80N细胞接种到裸鼠的皮下及腹膜腔内,比较这些细胞系在异种动物体内成瘤性及肿瘤生长速度。观察移植瘤细胞T29Nt和T80Nt的形态学特征,并将移植瘤细胞T29Nt和T80Nt再次接种到裸鼠皮下和腹膜腔内。观察生成的移植瘤的病理学特征,并检测新生肿瘤相关蛋白的表达。[研究方法]1.裸鼠致瘤实验,取对数生长期的T29, T80, T29N, T80N细胞用无血清培养基配制,分别取5×106个T29, T80, T29N, T80N细胞接种于4-6周龄雌性裸鼠皮下,每只两点,每2天观察一次裸鼠,观察肿瘤出现的时间和肿瘤的大小,测量肿瘤体积,绘出细胞生长曲线。皮下移植瘤直径达到1.5cm时,处死裸鼠,切取肿瘤组织,继续培养移植瘤细胞T29Nt和T80Nt,剩余组织以中性福尔马林固定,石蜡包埋,切片,HE染色。2.分别取5×106个T29, T80, T29N, T80N细胞接种于4-6周龄雌性裸鼠腹膜腔内接种。腹膜腔内细胞移植的裸鼠观察6个月后处理。3.取对数生长期的细胞用无血清培养基配制,取5×106个T29Nt, T80Nt细胞分别接种于4-6周龄雌性裸鼠皮下,每只两点；取5×106个T29Nt, T80Nt细胞分别接种于4-6周龄雌性裸鼠腹膜腔内。观察6个月后,处死实验裸鼠后,切取皮下移植瘤及腹腔移植瘤组织以中性福尔马林固定,石蜡包埋,切片,HE染色,观察组织形态。4.肿瘤组织石蜡切片用免疫组织化学染色方法检测各种蛋白表达,主要检测指标有HER2/neu、SV40大T抗原、p53、泛-细胞角蛋白、CA125、WT-1。用相应二抗进行反应。[研究结果]1.T29N、T80N细胞系裸鼠皮下移植瘤模型建立及皮下移植瘤生长曲线在异种动物接种试验中,T29N细胞经历4个月缓慢的生长在4只裸鼠皮下形成移植瘤,T80N细胞经历4个月缓慢的生长在9只裸鼠皮下形成移植瘤。所形成的移植瘤组织学为低分化肿瘤。对照组T29、T80细胞在裸鼠皮下没有形成移植瘤。2. T29N、T80N细胞系裸鼠腹膜腔内移植瘤实验情况T29N、T80N在腹腔腔内接种后观察6个月,没有观察到肿瘤生长,对照组T29、T80的腹腔内接种也没有任何肿瘤形成。3.移植瘤活细胞T29Nt和T80Nt细胞与其亲代细胞的形态学特征。T29N和T80N在裸鼠皮下形成的移植瘤细胞继续培养,肿瘤细胞命名为T29Nt和T80Nt。倒置显微镜下观察均呈上皮细胞形态4.T29Nt、T80Nt肿瘤细胞的裸鼠移植瘤模型建立T29Nt细胞经历6个月在3只裸鼠皮下形成移植瘤,T80Nt细胞经历6个月在3只裸鼠皮下形成移植瘤,生成低分化肿瘤T29Nt与T80Nt。而T29Nt在腹膜腔内接种观察6个月后有3只裸鼠生成高分化乳头状癌,并有大网膜、肝脏等多器官肿瘤转移。T80Nt在腹膜腔内接种观察6个月后有3只裸鼠形成转移肿瘤,且都伴有多器官转移,但是不同的是肿瘤组织为低分化卵巢癌。5. T29Nt、T80Nt移植瘤的HER2/neu等蛋白表达T29Nt和T80Nt肿瘤细胞在裸鼠腹膜腔内生成的肿瘤,HER2/neu蛋白、证明细胞起源的SV40大T抗原、p53、证明细胞上皮起源的泛-细胞角蛋白、人类卵巢癌常用标志物CA125、WT-1的免疫组化染色结果均为阳性。[结论]1.成功地将T29N和T80N在皮下形成的低分化移植瘤并获得T29Nt和T80Nt移植瘤细胞系。2.在裸鼠腹膜腔内,移植瘤细胞T29Nt可以生成类似于人类浆液性乳头状癌的高分化乳头状恶性肿瘤。3. HER2/neu蛋白的过度表达可能与卵巢浆液性乳头状癌的生成密切相关。更多还原

【Abstract】 BackgroundOvarian cancer has the highest mortality of all the gynecologic malignancies worldwide. With no adequate screening tests, detection at an early stage remains the most significant prognostic factor. There are no gold standard screening methods for the early detection of ovarian cancer exist. Approximately 85% of patients with ovarian cancer are diagnosed at a late stage of the dieases. Epithelial ovarian cancer (EOC), the most common subgroup of ovarian cancer, is the deadliest gynecological cancer, accounting for more deaths than all other gynecological cancers combined. The high mortality rate for EOC is a result of technical obstacles to early detection of the disease and a high prevalence of distal metastasis at late stages of the disease, seventy percent of the cases occur distal metastasis. It is important for enlonging the survival time to have the early detection. However,the explaination of the etiology of epithelial ovarian cancer, is still doubted. The development of specific histotypes of human ovarian cancer is thought to be influenced by multiple factors, including genetic and epigenetic changes in epithelial cells.HER2/neu gene is an oncogene, which encodes a 185 kd trans-membrane protein partially homologous to epidermal growth factor receptor with intrinsic tyrosine kinase activity. This oncogene has been studied mainly in breast cancer where it has prognostic, predictive and therapeutic target value. The expression of HER2/neu in epithelial ovarian cancer has been less studied. Amplication of this gene has been found in 25-30% breast and ovarian cancer and correlate with poor prognosis. HER2/neu has been shown to be involved in the progression of ovarian cancer through multiple mechanisms. High expression of HER2/neu is associated with advanced cancer stages, higher frequency of surgical residual larger tumor, poor histological grade, higher recurrence frequency, shorter survival time and lower sensitivity to platinumbased chemotherapy.In this study, we generated T29Nt and T80Nt cell models using genetic modification of human ovarian surface epithelial cell lines in combination with a peritoneal microenvironment to describe the development of a novel murine model of high-grade papillary serous carcinoma. This murine model should be useful for studying the mechanisms involved in the development and histologic differentiation of human ovarian carcinoma. It may also help us to generate other ovarian epithelial cancer models with additional or different genetic modifications. This model should be helpful for defining pathways and identifying novel targets to improve the treatment and early detection of ovarian cancer. ObjectiveTo harvest the retrovirus-containing HER2/neu and infect two ovarian epithelial cell lines, T29 and T80, and select the cells. To design the resulting cells overexpressing HER2/neu as T29N and T80N cells. To observe the characters of theT29N and T80N cell lines and test if HER2/neu overexpression can increase the number of anchorage independent colonies that grew in soft agar in both the T29N and T80N cell lines.Methods1. HER2/neu was transfected into the Phoenix amphotropic cell line, and the retrovirus-containing HER2/neu cells were harvested.2. After a 24-h recovery from virus infection, the cells were selected by growing them in a medium containing neomycin then grown in the medium without neomycin,and obtained the T29N and T80N cells.3. Observe the T29, T29Nt, T80 and T80Nt cells by phase-contrast microscopy to compare the morphologic characteristics.4. HER2/neu expression was detected using Western blot analysis.5. We use Anchorage-independent cell growth assay to compare the cells growth character in vitro. Cells were suspended in 2 mL of growth medium with 0.35% agarose (Invitrogen), and the suspension was placed on 5 mL of solidified 0.7% agarose. Triplicate cultures of each cell type were maintained for 14 days at 37℃in a 5% CO2 atmosphere, and fresh medium was added at 7 days. The number of colonies that were larger than 50μm in diameter was counted on the 14th day. These experiments were repeated twice.Results1. We Harvested the retrovirus-containing HER2/neu cells were harvested for the experiments.2. We infected the retrovirus-containing HER2/neu into two immortalized nontumorigenic human ovarian surface epithelial cell lines T29 and T80. The cells were selected in medium containing neomycin (1 mg/ml) after recovery from virus infection.3. The resulted cells increased expression of the HER2/neu protein.4. The T29N cells have mesenchymal morphologic characteristics, and the T80N cells have an epithelial phenotype.5. HER2/neu overexpression increased the number of anchorage independent colonies that grew in soft agar in both the T29N and T80N cell lines.Conclusion1. The introduction of HER2/neu into two immortalized nontumorigenic human ovarian surface epithelial cell lines (T29 and T80) resulted in increased expression of the HER2/neu protein.2. The T29N cells showed mesenchymal morphologic characteristics, and the T80N cells have an epithelial phenotype.3. The HER2/neu overexpression T29N and T80N cell lines grew more anchorage independent colonies in soft agar than T29 and T80 cell lines. ObjectiveMice subcutaneously or intraperitoneally injected with T29, T80, T29N, T80N cells to compare the tumor development abilities of these defferent cell lines. To observe the characters of theT29Nt and T80Nt cell lines. Mice subcutaneously or intraperitoneally injected with T29Nt, T80Nt cells to compare the tumor development abilities of these defferent cell lines. To do the histopathologic examination and immunohistochemical analysis with the tumors grown.Methods1. Harvested, washed 5 x 10^6 cells from T29, T80, T29N, T80N cell lines twice and resuspended in 0.1 mL of saline. The T29, T80 and the T29-and T80-control cell suspensions were injected subcutaneously into 4-to 6-week-old BALB/c athymic nude mice. The mice were kept in a pathogen-free environment and checked every 2 days. The date on which grossly visible tumors first appeared and the size of the tumors were recorded. The mice were killed when tumors reached 1.5 cm in diameter. T29Nt and T80Nt cells were isolated from the T29N-and T80N-derived xenografts and cultured for the further experiments.2. Harvested, washed 5 x 10^6 cells from T29, T80, T29N, T80N cell lines twice and resuspended in 0.1 mL of saline. The T29, T80 and the T29-and T80-control cell suspensions were injected intro-peritoneally into 4-to 6-week-old BALB/c athymic nude mice. The mice were kept in a pathogen-free environment and checked every 2 days. The mice were observed for increased abdominal size, lethargy and jaundice and were killed when these signs occurred.3. Harvested, washed 5 x 10^6 cells from T29Nt, T80Nt cell lines twice and resuspended in 0.1 mL of saline. Each cell line was injected subcutaneously and introperitoneally into six 4-to 6-week-old BALB/c athymic nude mice individually. The mice were kept in a pathogen-free environment and checked every 2 days for 6 months. The date on which grossly visible tumors first appeared and the size of the tumors were recorded. The mice were killed when tumors reached 1.5 cm in diameter. The mice given intraperitoneal injections were observed for increased abdominal size, lethargy and jaundice and were killed when these signs occurred. Tumors from mice were fixed in 10% formalin and stained with hematoxylin and eosin. The histopathologic characteristics were examined by microscopy.4. The following antibodies were used for immunohistochemical analysis: monoclonal anti-HER2/neu, monoclonal anti-SV40 T antigen, monoclonal anti-p53 antigen, monoclonal anti-pan cytokeratin antigen, and mouse monoclonal anti-CA125, anti-WT-1 antigen. Secondary antibodies against each host were biotinylated.Results1. Four of the nine mice injected with the T29N cells developed subcutaneous tumors. Nine of the fourteen mice subcutaneously injected with the T80N cells developed subcutaneous tumors. Histopathologic examination of the tumors showed undifferentiated carcinomas.2. There were no tumors revealed in other mice either injected subcutaneously or intraperitoneally.3. The T29Nt and T80Nt both had an epithelial phenotype. Three of the six mice subcutaneously injected with the T29Nt and T80Nt cell lines respectively developed subcutaneous tumors. Histopathologic examination of these tumors revealed undifferentiated carcinomas. Three of the ten mice injected intraperitoneally with T29Nt and five of the nine mice injected with T80Nt developed tumors. Intraperitoneal inoculation of mice with T29Nt cells resulted in extensive tumor growth along the omentum. Interestingly, histologic examination revealed papillary carcinoma that was indistinguishable from human papillary serous ovarian carcinoma. These tumors also invaded the liver and pancreas. Tumors derived from the T80Nt cells grew along the peritoneal fat and the pancreas but remained undifferentiated.4. Immunohistochemical staining for large T antigen was positive, demonstrating that the tumors were derived from the immortalized T29 and T80 cells rather than having developed spontaneously in the mice. The tumor cells were also positive for cytokeratin, verifying their epithelial cell origin, and positive for p53, demonstrating that their p53 stability was increased and its function was disabled due to its binding by T antigen. The cells were also positive for CA125, WT-1, which are common markers expressed in human ovarian cancer.Conclusion1. We successfully isolated T29N-and T80N-derived xenografts and named them T29Nt and T80Nt, which have epithelial phenotype.2. we also found that the peritoneal microenvironment and the genetic background of the cells play a critical role in the differentiation of papillary carcinoma, genetic modifications alone are not sufficient for development of this carcinoma; rather, a specific ovarian epithelial cell type and an appropriate tumor microenvironment are required.3. HER2/neu can lead to papillary differentiation of immortalized human ovarian epithelial cells.更多还原