Translocation and Cellular Entering Mechanisms of Oil Body Fusion Protein

• Main Authors: Chia-Pei Wu, 吳佳霈
• Other Authors: Chung-Jen Chiang
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/322ukw

碩士 === 中國醫藥大學 === 醫學檢驗生物技術學系碩士班 === 102 === Nanocarriers have been widely studied for their use in cancer therapy. In this study, we used nanoscale oil bodies as carriers. In this study, Ole was fused with apoptin (VP3) by genetic engineering. The fusion protein (Ole-VP3) was mass-produced in Escherichia coli and assembled artificial oil bodies (AOBs) by ultrasonic transducer. VP3, a protein derived from chicken anemia virus (CAV), can induce apoptosis in transformed cells or cancer cells but not in non-transformed cells or normal cells. Therefore, we used nanoscale oil bodies as carriers to investigate how VP3 enter the cells. The size, stability, and electronegative repulsion of AOBs were measured by in vitro test. We observed Ole-VP3 using in vivo fluorescence microscope and found it can combine with cancer cells but not showing apoptosis. Therefore, we used three kinds of chemical inhibitors, chlorpromazine, amiloride and filipin to inhibit clathrin-mediated endocytosis , macropinocytosis and caveolin-dependent endocytosis, respectively. After adding inhibitors, we found that the quantities of Ole-VP3 significantly decreased in the cells. At the same time, we deleted different sequences of VP3 by genetic engineering to see if it would affect the quantities of Ole-VP3 entering cancer cells. Finally, we found the quantities of Ole-VP3 entering cancer cells are different while in different conditions.