胆囊癌相关成纤维细胞促进胆囊癌血管新生及其分子机制和干预研究

On the basis of our previous studies, gallbladder cancer-associated fibroblasts (GCAFs) successfully cultured in gallbladder carcinoma tissues and new angiogenic genes ANGPTL4 and NOX1 found in GCAFs, our project was to explore the effects of GCAFs on angiogenesis and vasculogenic mimicry (VM) of gallbladder cancer cells (GBC-SD, SGC-996, ECV-SD) in vitro and xenografts in nude mice in vivo, their molecular mechanisms, related-signaling pathways mediated by ANGPTL4/NOX1, and the anti-angiogenic or anti-VM activity of norcantharidin (NCTD) combined with bevacizumab for angiogenesis and VM of gallbladder cancers promoted by GCAFs and their molecular interventions, through culture and purification of GCAFs, tumor-mesenchymal cells-/Matrigel co-culture and siRNA technology, establishment of the cell models in three-dimensional co-culture and xenograft models in nude mice of gallbladder cancer GBC-SD, SGC-996 and ECV-SD cells/GCAFs (including ANGPTL4/NOX1 silencing cells and nude mice models), and by the assays of proliferation, apoptosis, migration, invasion, tube formation and hemodynamics, immunohistochemistry and immunofluorescence, Western blot, RT-PCR, light/electron microscopy, Affymetrix gene chip/miRNA microarray analysis and computer aided image detection; at the same time, the project was also to clinically determine GCAFs-related proteins and tumor angiogenesis-related genes in gallbladder cancer specimens, in order to expand for understanding the development mechanism and the unique malignant biological behavior of gallbladder cancers, for finding new targets for diagnosis, monitoring, and treatment of gallbladder cancers. So, it is an innovative and feasible project, with important scientific significance and potential application value.

该项目旨在前期研究和成功培养胆囊癌相关成纤维细胞（GCAFs）并发现GCAFs源性血管新生ANGPTL4、NOX1新基因基础上，通过培养纯化GCAFs、肿瘤-间质细胞-Matrigel共培养和siRNA技术，建立胆囊癌/GCAFs细胞和裸鼠移植瘤模型（包括ANGPTL4/NOX1沉默细胞裸鼠模型），应用增殖凋亡、迁移侵袭、管道形成和血流动力学测定，免疫组化+荧光、Western blot、RT-PCR、光电镜、基因芯片和计算机辅助图像定量检测，探讨GCAFs对胆囊癌GBC-SD、SGC-996、ECV-SD细胞和裸鼠移植瘤血管新生的影响及其分子调控机制和去甲斑蝥素干预GCAFs促肿瘤血管新生作用及机制，并临床检测胆囊癌标本GCAFs和肿瘤血管新生相关蛋白，以拓展对胆囊癌发生发展机制及特有恶性生物学行为了解，为胆囊癌诊断监测、治疗寻找新靶点。故有创新、可行性，重大科学意义和潜在临床应用价值。

目的 探讨胆囊癌相关成纤维细胞（GCAFs）促进胆囊癌血管新生及其分子机制和干预。方法 通过细胞和动物实验建立GCAFs、TJ-GBC2、胆囊癌细胞+ GCAFs共培养细胞模型和裸鼠荷瘤血管新生和血管生成拟态（VM）模型；应用Affymetrix芯片GCAFs/NFs和lncRNAs芯片VM(+)/VM(-)差异表达基因谱分析、Western blot、qRT-PCR、免疫组化/荧光检测、光电镜和计算机辅助图像定量检测，增殖、凋亡、迁移、侵袭、管道形成、VM、裸鼠荷瘤测定，验证GCAFs源性血管新生基因，探讨GCAFs能否促进胆囊癌血管新生、VM形成和裸鼠移植瘤生长及其潜在分子机制，以及小分子纳米去甲斑蝥素（NCTD）的抗胆囊癌血管新生、VM和裸鼠移植瘤生长及其机制。结果 ①成功构建新胆囊癌细胞系TJ-GBC2，符合高侵袭胆囊癌细胞结构和表型特征；②自建的GCAFs符合癌相关成纤维细胞（CAFs）结构和表型特征，抑制胆囊癌细胞凋亡，促进其细胞增殖、侵袭、迁移、管道和VM形成及裸鼠荷瘤生长；③GCAFs有16个肿瘤血管新生基因，ANGPTL4、NOX1是新发现的GCAFs源性血管新生关键基因；胆囊癌VM基因有154个，STAT3、NOX-4为胆囊癌VM关键基因；GCAFs通过旁分泌IL-6介导IL-6-JAK-STAT3-NOX-4促进胆囊癌VM形成；④NCTD抑制GCAFs触发的胆囊癌细胞增殖、侵袭、迁移、管道和VM形成及裸鼠荷瘤生长，抑制GCAFs源性ANGPTL4、NOX1、NOX4和GCAFs旁分泌IL-6，通过阻止JAK-STAT3-NOX-4信号通路抑制胆囊癌VM形成；⑤临床研究表明，胆囊癌组织和间质HIF-1α、α-SMA、ANGPTL4、NOX1、NOX4高表达，且与临床病理预后相关，是胆囊癌病人不良预后的独立因素。结论 GCAFs可通过旁分泌IL-6介导IL-6-JAK-STAT3-NOX-4促进胆囊癌血管新生和VM形成；NCTD通过阻止IL-6-JAK-STAT3-NOX-4信号通路抑制GCAFs触发的胆囊癌血管新生和VM形成，可能是新的抗胆囊癌血管生成和抗VM治疗靶。