【作者】 王晓翊；

【导师】 王泽华；

【作者基本信息】 华中科技大学 ， 妇产科学， 2008， 博士

【摘要】 第一部分卵巢癌相关成纤维细胞和正常卵巢成纤维细胞的分离培养鉴定及两者基因表达差异目的对卵巢癌相关成纤维细胞(carcinoma-associated fibroblasts,CAFs)及卵巢正常成纤维细胞(normal fibroblasts,NFs)进行分离、培养、纯化、并作初步的鉴定。分析CAFs与NFs中多种基因表达的差异。方法1.用组织块贴壁培养法获得原代卵巢癌CAFs和卵巢NFs,细胞铺满培养瓶后首次传代,通过胰酶消化法和反复传代法进行细胞的纯化;2.通过倒置显微镜观察细胞形态,透射电镜观察细胞内部结构和多种蛋白质的免疫细胞组化染色检测细胞膜波形蛋白(vimentin)、细胞角蛋白(keratin)和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA),及相应mRNA的半定量分析,对CAFs和NFs进行初步鉴定;3.应用RT-PCR的方法对CAFs和NFs中多种基因进行分析比较。结果1.获得了纯化的卵巢CAFs和NFs;2.卵巢CAFs呈长梭形,胞质突减少,电镜下可见粗面内质网扩张;3.卵巢CAFs的细胞免疫组化染色波形蛋白、α-SMA染色呈阳性,角蛋白呈阴性,卵巢NFs波形蛋白阳性,而α-SMA和细胞角蛋白阴性;4.卵巢CAFs与NFs中波形蛋白与α-SMA的mRNA发现均有表达,但CAFs中表达增加,差异有显著性(P﹤0.05);5.与NFs相比,CAFs中多种mRNA的表达增加,其中包括有生长因子,基质金属蛋白酶,血管生成因子,同时也发现MMP1的表达显著减少。结论与NFs相比, CAFs在形态结构、mRNA、蛋白表达等方面均有显著差异;卵巢癌中CAFs的直接分子表达的检测表明,其分子表达有显著变化,提示这些成纤维细胞可能为卵巢癌细胞的生长提供了最适的微环境。推测卵巢-宿主界面微环境在上皮性卵巢癌的发展中可能起重要作用。第二部分卵巢癌相关成纤维细胞对上皮性卵巢癌细胞增殖、凋亡、迁移和侵袭的生物学活性的影响目的采用Transwell体外间接共培养体系,研究直接分离自卵巢癌的癌相关成纤维细胞CAFs对卵巢癌细胞增殖、凋亡、迁移和侵袭活性的影响,探讨CAFs在上皮性卵巢癌发展中的作用。方法1.采用0.4μm孔径的Tranwell小室进行CAFs或者NFs与卵巢癌细胞CAOV3体外共培养,用RT-PCR的方法检测共培养之后的卵巢癌细胞CAOV3中增殖细胞核抗原(PCNA)表达的改变,以了解卵巢癌细胞增殖的变化;2.用流式细胞仪检测共培养之后CAOV3细胞的细胞周期和Annexin V-FITC/PI检测CAOV3细胞的凋亡情况;3.用8μm孔径的Transwell小室建立卵巢癌CAFs与CAOV3细胞的交互作用模型,观测CAFs对CAOV3细胞迁移特性的影响;4.以Matrigel模拟重建基底膜,用8μm孔径的Transwell小室建立卵巢癌CAFs与CAOV3细胞的交互作用模型,观测CAFs对CAOV3细胞侵袭特性的影响。结果1.与CAFs共培养之后卵巢癌细胞CAOV3的PCNA基因表达显著增高,并且随着CAFs细胞量的增加,CAOV3细胞增殖活性显著增强。同时CAFs细胞的PCNA基因表达下降,随着CAOV3细胞数量的增加,PCNA表达明显减少;2.采用流式细胞仪检测细胞周期的方法可以发现,与CAFs共培养之后CAOV3细胞的凋亡显著减少,而在与NFs共培养之后的CAOV3细胞中未发现这一现象;3.同时用Annexin V-FITC/PI的方法,用流式细胞仪进行检测,也可以证实与与CAFs共培养之后CAOV3细胞的凋亡显著减少,而与NFs共培养之后,CAOV3的凋亡没有明显变化;4.与对照组相比CAFs或者NFs对CAOV3作用8小时后,CAOV3迁移明显增加,但CAFs组的增加有极显著性(P﹤0.01);5.侵袭实验中发现,与对照组相比CAFs或者NFs对CAOV3作用24小时后,CAOV3侵袭明显增加,但CAFs组的增加有极显著性(P﹤0.01)。结论CAFs能促进卵巢癌CAOV3细胞的增殖,减少其凋亡,并且两种成纤维细胞对卵巢癌细胞均有促迁移和侵袭的作用,但是CAFs的作用更加显著。这些发现提示我们,在CAFs与卵巢癌CAOV3细胞的交互作用中,能够促进卵巢癌的进展。第三部分卵巢癌相关成纤维细胞与卵巢癌细胞CAOV3交互作用中基因表达的改变及机制目的探讨在卵巢癌相关成纤维细胞与卵巢癌细胞交互作用的过程中基因表达的改变,及其相应的机制,探讨在体内环境下卵巢癌相关成纤维细胞对卵巢癌的影响,寻找卵巢癌治疗的新靶点。方法1.采用逆转录聚合酶链反应(RT-PCR)的方法分别检测交互作用48小时后的卵巢癌相关成纤维细胞与卵巢癌细胞中基因表达的改变,CAFs及CAOV3细胞外信号调节激酶通路的ERK,PI3K基因表达的变化;2.与CAFs细胞共培养后,CAOV3细胞外信号调节激酶通路ERK1/2的蛋白表达变化采用Western-blot的方法检测;3.建立人卵巢癌CAOV3与CAFs1:1裸鼠荷瘤模型,观察荷瘤裸鼠生长状况的变化,称量其体重,测量其大小;用HE染色观察裸鼠荷瘤的一般情况;4.用免疫组织化学染色技术检测裸鼠荷瘤中Ⅷ因子相关抗原,PCNA等蛋白表达的变化,以单纯卵巢癌细胞株CAOV3裸鼠荷瘤模型最为对照。结果1.在卵巢癌细胞CAOV3中,FGF1、tTG、PDGF-A经CAFs作用后表达增加(P﹤0.05),而与NFs共同培养后无显著变化,在CAFs组中TGF-β2的表达显著减少(P﹤0.05)。基质金属蛋白酶中MMP1在NFs组中表达减少,MMP7在NFs组中表达显著增加(P﹤0.05),TIMP3在成纤维细胞CAFs和NFs作用后,均减少,并且随着CAFs的细胞数量增加,TIMP3减少越显著;2.与CAOV3细胞共同培养后,CAFs中的tTG、VEGF-C、MMP1、MMP2、MMP9、TIMP3均显著减少(P﹤0.05);3.卵巢癌细胞CAOV3与卵巢CAFs共培养之后,CAOV3和CAFs细胞中ERK2,PI3K的表达均增加(P﹤0.05);4.Western blot检测蛋白表达,与NFs共培养后,CAOV3中ERK2蛋白的表达显著减少(P=0.002),而在与CAFs共同培养时,ERK1/2蛋白的表达显著增加(P=0.018);5.CAFs使荷瘤裸鼠体重迅速减轻,CAFs使裸鼠荷瘤产生粗大丰富的血管,促进增殖,并且微血管密度增加。结论CAFs与CAOV3的交互作用致使两者的生长因子、基质金属蛋白酶等表达改变,并且CAFs对癌细胞ERK和PI3K通路有活化作用,CAFs在体内促进了肿瘤血管的生长,加速了肿瘤的进展。因此,针对CAFs的研究为卵巢癌的治疗策略提供了新的思路。更多还原

【Abstract】 Part I The Differential Gene Expression between Cancer-Associated Fibroblasts and Normal Fibroblasts separated、cultured and identified from Human Ovarian CarcinamasObjective: To separate, cultivate, purify and identify ovarian carcinoma-associated fibroblasts (CAFs) and ovarian normal fibroblasts (NFs) preliminarily. Analyze the distinct molecular expression profiles between CAFs and NFs.Methods:1. The primary ovarian CAFs and NFs of ovarian were obtained by tissue culture. The second passage was obtained after the cell overgrew the bottom of the culture flask and purified by trypsinization and adherence repeatedly methods. 2. The identity of the CAFs and NFs was through the observation of the shape of the cell with inverted microscope and the cellular organ with transmission electron microscope, the protein expression of vimentin, keratin andα-SMA in the cellular membrane with immunocytochemistry and mRNA expression of vimentin andα-SMA with RT-PCR. 3. Analyze the distinct molecular expression profiles between CAFs and NFs with RT-PCR.Results:1. The purified ovarian CAFs and NFs were maintained. 2. The characteristics of shape and growth of the ovarian CAFs changed significantly comparing to the NFs. They are in long-fusiform shape, cytoplasmic reduction, and expansion of rough endoplasmic reticulum. 3. The ovarian CAFs showed negative staining for cytokeratin , and positive staining for vimentin andα-SMA, while NFs showed negative staining for cytokeratin andα-SMA,positive staining for vimentin. 4.α-SMA was found mRNA expression in both CAFs and NFs, but the expression increased significantly in CAFs (P﹤0.05). 5. Control with the NFs, molecular expression profiles increased in CAFs, including growth factors, MMPs, angiogenesis factors, at the same time, MMP1 was found decreased.Conclusions:There are obvious differences of the morphological characteristics and expression of certain mRNA and proteins between the CAFs and NFs. The distinct molecular expression profiles of CAFs in ovarian cancer support the notion that these fibroblasts form a favorable microenvironment for cancer cells. The microecology of the oral tumor-host interface might be one of the most important factors affecting the progression of epithelial ovarian cancer.Part II The effect of Ovarian Carcinoma-associated Fibroblasts on the Proliferation, Apoptosis, Migration and Invasion of Epithelial Ovarian Carcinoma CellObjective: To observe the effects of CAFs on the proliferation, apoptosis, migration and invasion of ovarian caicinoma cell line CAOV3 cells, and to elucidate the role of CAFs in ovarian carcinoma progression.Methods:1. The interaction model between primary ovarian CAFs,NFs and ovarian carcinoma cell line CAOV3 was established by Transwell chamber of 0.4μm diameter. In order to find out the effect of proliferation of CAFs to CAOV3, the RT-PCR assay was used to observe the changing of PCNA mRNA expression. 2. Cell cycle examination and Annexin V-FITC/PI assays to verify the apoptosis of CAOV3 after the co-culture with CAFs or NFs with flow cytometry. 3. The interaction model between primary ovarian CAFs,NFs and ovarian carcinoma cell line CAOV3 was established by Transwell chamber of 8μm pore to observe the effect of migration of CAOV3. 4. Matrigel was used to remodel the basement membrane, and the interaction model between primary ovarian CAFs,NFs and ovarian carcinoma cell line CAOV3 was established by Transwell chamber of 8μm diameter to observe the effect of invasion of CAOV3.Results:1. The expression of PCNA mRNA in CAOV3 was significantly increased after the co-culture with CAFs, and with the number of CAFs increased, the motility of proliferation in CAOV3 was significantly increased. 2. By flow cytometry of the cell cycle, the apoptosis of CAOV3 was found significantly decreased after the co-culture with CAFs, while it was not found in CAOV3 co-cultured with NFs. 3. Meanwhile, the decreasing apoptosis of CAOV3 was observed with flow cytometry in Annexin V-FITC/PI assay, but the apoptosis of CAOV3 did not change significantly after the co-culture with NFs. 4. When contrast with the control group, the migration of CAOV3 was significantly increased after the co-culture with CAFs or NFs for 8hours, moreover, there was an extremely significant increase in CAFs group. 5. In the invasion experiment, when CAOV3 was co-cultured with CAFs or NFs for 24hours, the invasion of CAOV3 was significantly increased, especially in CAFs group.Conclusions:CAFs can promote the proliferation and decrease the apoptosis of ovarian carcinoma CAOV3 cells, both CAFs and NFs can promote the migration and invasion of CAOV3, but it was much more obvious in CAFs. All the findings suggest, during the interaction between CAFs and ovarian carcinoma CAOV3 cells, the signaling can promote the progression of ovarian carcinoma. Part III The cross-talk of the change of genes expression between CAFs and ovarian cancer cell CAOV3 co-culture and the possible mechanismsObjective:To investigate the change of genes expression between CAFs and ovarian cancer cell CAOV3 co-culture and the possible mechanisms,and the effects of ovarian CAFs on ovarian cancer,new targets were to be found.Methods:1. To explore the reciprocal effects of host-tumor interaction, we developed a system to assess the gene expression patterns and the diversity of gene expression of ERK and PI3K pathway in CAOV3 human ovarian cancer cells and primary carcinoma-associated fibroblasts cocultured with each other for 48 hours with RT-PCR. 2. Western blot was used to detect the expression of extracellular signal-regulated kinase1/2 prtotein in ovarian cancer cell CAOV3 after the co-culture with CAFs. 3. We established the model of human ovarian carcinoma with human ovarian carcinoma cell CAOV3 and CAFs 1:1 of nude mice to observe the weight and volume of those carcinomas in nude mice,and hematoxylin and eosine staining of tumor-bearing nude mice in general. 4. Immunohistochemistry was used to determine the expression ofⅧfactors antigen and PCNA protein in those carcinomas in nude mice,compared with the carcinomas of CAOV3 in nude mice.Results:1. After the co-culture with CAFs, the expression of FGF1、tTG、PDGF-A were significantly increased(P﹤0.05).2. The expression of TGF-β2 was significantly decreased(P﹤0.05). In MMPs,the expression of MMP1 was decreased when co-cultured with NFs,while MMP7 was increased. But TIMP3 both were decreased after the co-culture with CAFs and NFs. 2. After the co-culture with CAOV3, the expression of tTG、VEGF-C、MMP1、MMP9 and TIMP3 were significantly decreased. 3. The expression of ERK2、PI3K mRNA and ERK1/2 protein in CAOV3 were increased after the co-culture with CAFs. 4. The weight of treated nude mice was decreased fast by CAFs, and a large number of vessels were generated、the proliferation inceased and the MVD raised.Conclusions:The expression of several growth factors and MMPs changed in CAFs and CAOV3 after the co-culture of them,and CAFs have the activation effect on ERK and PI3K pathway in CAOV3 cells. The cancer angiogenesis and progression were stimulated by CAFs in vivo. Therefore,the study targeting CAFs would afford us a new thincking of therapeutic strategies.更多还原