三维数控肿瘤细胞EMT微流控芯片的构建及其初步应用

Cancer is considered as "the second killer" that has threatened human lives. One significant sign of cancer malignancy is cell invasion and metastasis, and the precursor of metastasis,however,is highly associated with Epithelial-Mesenchymal Transition (EMT).Right now the means of EMT research in carcinoma remains limited, microfluidic chip then can compensate for this limitation with its merits of CNC, trace, three-dimention and real-time cell culture,etc.Our program is aimed to construct and optimize the microfluidic chip which integrating with numerical control on-line transfection module for EMT studying in tumors; With gastric cancer cells as cell models,we will use the co-cultured mesenchymal cells and vascular endothelial cells in three-dimemtional (3D) to induce EMT of these model cells; The EMT canonical pathway in gastric cancer cells will be blocked via numerical control on-line transfection of Smad4 RNA interfering fragments; Furthermore,monoclonal antibody against TGF-β and targeting drug,Sunitinib,will be performed to intervene and therefore inhibit the process of EMT in gastric cancer cells in reverse; Eventually, a comprehensive evaluation of this chip's performance would be provided, and it would provided solid technical support for next-step investigation of the EMT mechnisms in tumors. In conclusion,the successful development of this chip will offers us an effective method to study EMT for valuable and fresh tumor tissues and cells.

癌症是威胁人类生命的"二号杀手"，其中肿瘤细胞侵袭转移是病情恶化的重要标志，而上皮间质转化（EMT）是其转移的前兆。传统研究肿瘤细胞EMT的方法存在一定的局限性，而微流控芯片技术具有数控、微量、三维、实时培养细胞等优点，能弥补其不足。本项目拟构建并优化可用于研究肿瘤细胞EMT并集成数控在线转染模块的微流控芯片；以胃癌细胞株为模型，利用三维共培养的间质细胞和血管内皮细胞诱导胃癌细胞发生EMT；通过数控在线转染针对Smad4的RNA干扰片段，对胃癌细胞EMT经典通路实施阻抑；并用TGF-β单克隆抗体和靶向药舒尼替尼进行干预，反向抑制胃癌细胞发生EMT；最后，综合考察该芯片的性能，为下一步肿瘤细胞EMT机制研究提供可靠的技术支撑。该芯片的成功研制，将为临床弥足珍贵的新鲜肿瘤组织细胞进行EMT研究提供有效手段。

肿瘤细胞侵袭转移是癌症病情恶化的重要标志，而上皮间质转化（EMT）是其转移的前兆。肿瘤细胞EMT的发生与肿瘤微环境和恶性肿瘤细胞相互作用密切相关。传统研究肿瘤细胞微环境和EMT的方法存在一定的局限性，而微流控芯片技术具有数控、微量、三维、实时培养细胞等优点，能弥补其不足。本项目构建并优化肿瘤细胞在线转染和EMT微流控芯片，以具有不同转移能力的胃癌细胞株AGS、SGC-7901和MKN-1为模型，通过在线转染针对Smad4的RNA 干扰片段对胃癌细胞EMT经典通路实施阻抑，采用FITC标记的RNA片段观察转染效率，效率可达80%以上；采用细胞免疫荧光法检测EMT相关蛋白表达，Snail、SMA表达下调，而E-cadherin表达上调，符合预期，并和在芯片外培养皿中转染、western blot检测蛋白表达结果一致。利用微流控芯片上三维共培养的成纤维细胞（血管内皮细胞）诱导胃癌细胞发生EMT，观察胃癌细胞株AGS、SGC-7901和MKN-1的迁移能力，迁移能力有所增加。使用TGF-β单克隆抗体SB431542反向抑制胃癌细胞发生EMT，胃癌细胞迁移得到抑制。鉴于此肿瘤细胞转染和共培养EMT 研究微流控芯片平台具有很大程度上的普适性，本项目也将为其它肿瘤微环境研究提供一种新的技术手段。