胰腺癌分泌的exosomes教育BMDCs致预转移小生境在胰腺癌肝转移作用中的分子机制

The liver metastasis is an important factor affecting the prognosis of patientswith pancreatic cancer. Exosomes are vesicles secreted by a variety of livingcells. They can horizontal transfer a variety of biologically active molecules like proteins to the receptor cells and play a biological role.Exosomes are currently considered as one of the main contributors to tumor progression and metastasis.In this research,we study the pancreatic cancer derived exosomes horizontal transfer of liver metastasis-associated protein/oncoprotein educate and mobilize bone marrow-derived cells (BMDCs) to form the pre-metastatic niche.We ues the low temperature ultra-high-speed centrifugation and density gradient centrifugation to extract the the pancreatic cancer derived exosomes from the same parent, same genetic background but with different metastatic potential of the mouse pancreatic cancer cell lines.Then we use the iTRAQ quantitative proteomics. analysis of two pancreatic cancer cells derived exosomes.And we can acquire the liver metastasis-associated protein/oncoprotein.Through the surgical orthotopic transplantation tumor metastasis model and various means of molecular biology, we can clarify the cellular and molecular mechanisms of directed liver metastases by pancreatic cancer cells derived exosomes which cause the formation of the pre-metastatic niche. So we can provides a new target for the treatment of pancreatic cancer liver metastases through intervention exosomes secreted by tumor cells and host interactions.Then the prevention and treatment of pancreatic cancer liver metastases will become possible.

胰腺癌肝转移是影响胰腺癌患者预后的重要因素。Exosomes是多种活细胞分泌的小囊泡体, 并能将其携带的与来源细胞功能相关的蛋白质等生物活性分子传递给受体细胞而发挥生物学作用。目前认为exosomes是影响肿瘤进展和转移的主要原因之一。本研究从胰腺癌细胞分泌的exosomes传递肝转移相关蛋白/癌蛋白“教育”并动员骨髓衍生细胞（BMDCs）形成“预转移小生境”角度入手，提取不同肝转移潜能的小鼠胰腺癌细胞株分泌的exosomes，并采用iTRAQ定量蛋白质组学对exosomes进行比较蛋白质组学分析，筛选获得肝转移相关蛋白/癌蛋白。通过建立小鼠胰腺癌原位移植瘤转移模型并采用各种分子生物学手段，阐明胰腺癌分泌的exosomes致预转移小生境形成并引起定向肝转移发生的细胞及分子机制。为通过干预胰腺癌细胞分泌的exosomes来治疗胰腺癌肝转移提供新的靶点，使防治胰腺癌肝转移成为可能。

胰腺癌肝转移是影响胰腺癌患者预后的重要因素。外泌体是多种活细胞分泌的小囊泡体, 并能将其携带的与来源细胞功能相关的蛋白质等生物活性分子传递给受体细胞而发挥生物学作用。目前认为外泌体是影响肿瘤进展和转移的主要原因之一。本研究成功从胰腺癌细胞培养液上清中提取并纯化外泌体， Western Blot实验验证了两种外泌体均表达外泌体结构蛋白CD9, MHC-I, TSG101。Panc02-H7 EXO蛋白浓度高于Panc02 EXO；体外实验结果表明Panc02-H7 EXO容易被Panc02细胞所摄取，并能显著降低Panc02细胞的粘附特性，增加其迁移和侵袭能力，Panc02-H7 EXO可以增加Panc02细胞MMP-9, CXCR4的表达；尾静脉注射24 h后外泌体主要积聚在肺脏、肝脏以及脾脏，较少的分布在脑组织和骨髓中；Panc02-H7 EXO与Panc02 EXO可以动员并募集CD11b+和CD45+造血前体细胞至肝脏微环境，激活α-SMA+的肝星状细胞，诱导肝脏组织Fibronection, S100A8和S100A9表达上调产生肝脏预转移小生境；Panc02-H7 EXO与Panc02 EXO在体内促进Panc02细胞的侵袭与转移，募集F4/80+的巨噬细胞、α-SMA+的肝星状细胞以及中细粒细胞积聚在肝脏微环境，并诱导炎症因子Fibronection, S100A8和S100A9在肝脏表达上调及胶原纤维在肝脏组织中沉积；通过iTRAQ定量蛋白质组学分析我们共鉴定出4517个外泌体蛋白质，筛选出79个差异蛋白，其中33个蛋白表达上调，46的蛋白表达下调。这些差异蛋白所涉及的分子功能和信号通路可能在胰腺癌的侵袭与转移过程中发挥重要作用，为确定胰腺癌定向肝转移的预测指标和新的治疗靶点提供理论和实践基础。