构建微流控芯片仿生模型研究转移相关蛋白在肝癌侵袭转移中的作用及机制

Invasion and metastasis are the main challenges in the treatment of primary liver cancer. It has been approved that metastasis-associated proteins may be involved in the changes of the hepatic tumor microenvironment, which are closely related to invasion and metastasis of liver cancer. In our previous research project "Study on Proteomic Modification of Human Liver (863 Plan)", we identified a number of specific metastasis-associated proteins by using quantitative proteomic technique. The research is conducted through a large scale data analysis and has high credibility. However, how these specific metastasis-associated proteins correlate the microenvironment of primary liver cancer and what effect of these proteins are on the process of invasion and metastasis remain unclear. Therefore, the purpose of our new study is to explore the relationship between specific metastasis-associated proteins and the microenvironment of primary liver cancer and to analyze the possible effects of the proteins on the process of invasion and metastasis. Our study is designed on the basis of the principles of fluid mechanics and the characteristics of the interaction between cells and cells, as well as the interaction between cells and microenvironment. Through tissue engineering technique, we will design and establish a multi-unit integrated, multi-channel connected, and high-throughput bionic chip liver cancer model to simulate the real condition of liver cancer cells. Then we may be able to watch the process of invasion and metastasis in a dynamic and real-time way. We will further screen and verify the specificity and function of metastasis-associated proteins of liver cancer in the process. Our research will establish a new method for analyzing invasion and metastasis of primary liver cancer. It will be also likely to provide a unique way for understanding the mechanism of invasion and metastasis on molecular level, and give us the predictive indicators for diagnosis and the useful information for the molecularly targeted therapy.

肝癌侵袭转移是肝癌治疗的难点和"瓶颈"，转移相关蛋白参与的微环境变化与肝癌侵袭转移密切相关，本课题组在前期合作项目"人类肝脏蛋白质组修饰谱研究"（863计划）中，利用定量蛋白质组学技术规模化、高可信度的筛选出多个与肝癌侵袭转移相关的特异性蛋白，但其作用的确切机理尚不清楚。本研究拟利用微流控芯片组织工程学技术，依据体内细胞与细胞、细胞与微环境相互作用的特性以及流体学原理，设计和构建一个多单元集成、多通道连接的高通量的仿生芯片肝癌模型，模拟体内肝癌细胞的真实状况，动态实时的观察肝癌细胞侵袭转移的全过程，进一步筛选与验证肝癌侵袭转移相关蛋白的特异性及功能，探讨肝癌侵袭转移过程中相关特异性蛋白与肝癌微环境的关系及其在肝癌侵袭转移中的作用机制。旨在建立肝癌细胞侵袭转移研究的新方法，并探讨肝癌侵袭转移的分子机制，寻找肝癌侵袭转移的预测指标和干预治疗的靶点。

肝癌的侵袭转移以及多药耐药是肝癌治疗的难点和"瓶颈"，转移和耐药相关蛋白参与的微环境变化与肝癌发生发展密切相关，本课题组在前期合作项目"人类肝脏蛋白质组修饰谱研究"（863计划）中，利用定量蛋白质组学技术规模化、高可信度地筛选出多个与肝癌侵袭转移以及耐药相关的特异性蛋白，本研究利用微流控芯片组织工程学技术，依据体内细胞与细胞、细胞与微环境相互作用的特性以及流体学原理，设计和构建一个多单元集成、多通道连接的高通量的仿生芯片肝癌模型，模拟体内肝癌细胞的真实状况，动态实时的观察肝癌细胞侵袭转移以及耐药的全过程，进一步筛选与验证肝癌侵袭转移和耐药相关蛋白的特异性及功能，探讨肝癌侵袭转移和耐药过程中相关特异性蛋白与肝癌微环境的关系及其作用机制。 . 我们首先总结了现有的微流控仿生模型的国内外研究进展，重点关注了不同微流控仿生模型在各自领域的应用研究（RSC Adv., 2016），以此为基础构建并优化了我们的肝癌微流控仿生芯片。同时，我们对筛选的系列肝癌转移和耐药相关蛋白（如：bFGF，Hippo信号通路，Pinin，COX-2，NgBR蛋白等）先后进行了验证，并进行了深入研究发现：二甲双胍能够通过Akt/GSK3β/Twist通路抑制bFGF诱导的肝癌侵袭与转移，通过AMPK/YAP信号途径促进肝癌化疗敏感性（Oncotarget，2016），并能逆转bFGF诱导的HCC细胞中的上皮-间质转化（Oncotarget，2017）；NgBR能够影响p53泛素化，通过调控细胞进展影响肝癌细胞化疗药物耐药性（Oncotarget，2016），能通过Akt信号途径促进人肝细胞癌细胞进一步生长（J Cell Biochem，2018）；Pinin蛋白可以调节ERK磷酸化水平参与肝癌细胞糖耐受水平（Oncotarget，2016）。褪黑素能够通过AKT / p27介导的细胞周期阻滞在肝细胞肝癌中增强索拉非尼的抗肿瘤作用（RSC Adv.，2017）。COX-2可以用作荧光探针对肝细胞癌进行荧光成像（RSC Adv.，2018）。我们的工作不仅为进一步寻找肝癌预测转移及耐药的潜在生物标记物和干预治疗的靶分子提供了理论基础，同时也对二甲双胍、褪黑素以及荧光探针在肝癌临床诊疗中的应用具有重要的理论指导意义。