肺组织粗提物影响人原发性肝癌细胞侵袭转移机制的初步探讨

Migration and Invasion of Human Hepatocellular Carcinoma Mediated by Lung Extracts

背景与目的:转移复发是原发性肝癌治疗失败的主要原因,而肺组织是原发性肝癌远处转移的好发部位。在小鼠不同组织粗提物诱导高转移潜能的人原发性肝癌细胞的体外趋化侵袭实验中,肺组织提取物具有最强的诱导能力。本研究旨在探讨小鼠肺组织粗提物促进人肝癌高转移细胞株移动侵袭的机制。方法:采用F型肌动蛋白聚合实验和流式细胞仪,分析C57BL/6小鼠肺组织粗提物诱导高转移潜能人肝癌细胞株MHCC97-H细胞骨架的变化。肺组织粗提物与MHCC97-H细胞孵育后,利用双重荧光染色分析F型肌动蛋白和基质金属蛋白酶-9(matrixmetalloproteinase-9,MMP-9)表达的关系。结果:经无血清培养液或脾组织粗提物孵育后,MHCC97-H细胞呈梭型或多边形。与肺组织粗提物孵育后,随时间的增加,MHCC97-H细胞片层样或丝状伪足明显增多,流式细胞仪分析显示F型肌动蛋白在30s内增加1.9倍,激光扫描共聚焦显微镜观察MHCC97-H细胞F型肌动蛋白从细胞外周浓染到重新分布于细胞的导引侧。无血清培养基孵育MHCC97-H细胞后,MMP-9和F型肌动蛋白主要位于细胞的核周池;经肺组织粗提物孵育后,MHCC97-H细胞MMP-9和F型肌动蛋白则定位于细胞伪足的前端。结论:肺组织粗提物可能通过诱导MHCC97-H细胞形成伪足和改变MMP-9运送方式,从而促进MHCC97-H细胞移动侵袭?

BACKGROUND &OBJECTIVE: Invasion and metastasis are obstacles to suc cessful treatment of hepatocellular carcinoma. Lung is the most common site of d istant metastasis from hepatocellular carcinoma. In in vitro chemoinvasion assay that different tissue extracts from mice were used to induce human hepatocellul ar carcinoma cells with different metastatic potentials, lung extracts show the strongest inducing activity. This study was designed to investigate the mechanis m of migration and invasion in human hepatocellular carcinoma with highly metast atic potential mediated by lung extracts from mice. METHODS:The changes of cytos keleton were tested using F-actin polymerization assay and flow cytometry (FCM) . Correlation between matrix metalloproteinas-9 and F-actin was analyzed by fl uorescence double staining in human hepatocellular carcinoma MHCC97-H cells ind uced by lung extracts. RESULTS: When MHCC97-H cells were incubated with serum- free medium or spleen extracts, the cells showed elongated or polygonal morpholo gy. When MHCC97-H cells were incubated with lung extracts, the formation of lam elliopodia or filopodia became more and more obvious and distinct as time increa sing. Results of FCM showed that 1.9-fold increase in intracellular F-actin wi thin 30s after MHCC97-H cells were incubated with lung extracts. Confocal laser scan microscopy of MHCC97-H cells stimulated in suspension showed intense F-a ctin staining in the periphery of the cells and redistribution of F-actin towar ds a leading edge. MMP-9 and F-actin were mainly localized in the perinuclear pool when the cells were incubated with serum-free medium. After stimulation wi th lung extracts, MMP-9 and F-actin were localized at the front of extending p seudopodia of MHCC97-H cells. CONCLUSION: The mechanism that lung extracts prom ote migration and invasion of MHCC97-H may correlate with the pseudopodia forma tion and reorganization of MMP-9. Lung extracts may contribute to organ-specif ic metastasis of hepatocellular carcinoma.