Study The Expression And Distribution Of Membrane Proteins On Living Cells Using Single Molecular Force Measurement Technology

The membrane protein takes an important part in membrane recognition, signal transmittion, enzyme activation and substantial transportation. It is important to study the expression and distribution of protein on living cell membrane. It is suitable to measure the interaction between biomolecules in physiological environment by single molecular force measurement (SMFM) based on atomic force microscopy (AFM). The interactions between protein and antibody or aptamer were studied in this thesis by SMFM. Furthermore, the expression and distribution of protein on living cell membrane were also detected in nanoscale. These were in favor of understanding the properties of membrane protein on living cell. And had good reference for studying biological process which induced by membrane protein. The main work finished in this thesis including:1. Studied the expression of multidrug resistance associate protein 1(MRP1) on living cell membrane by SMFM. The interaction force between MRP1 and anti-MRP1 was measured using SMFM. And the express differentia of MRP1 on human tongue cancer cell before or after repeating treat with high dose of bleomycin(BLM) were detected. The results showed that there was special interaction between MRP1 and anti-MRP1. when the loading rate was 2.5μm/s, the force was about 182±35 pN. The expression of MRP1 on human tongue cancer cell before repeating treat with high dose of BLM was much lower than that after repeating treat with high dose of BLM. These results are significant in studying the single molecular properties of MRP1 on living cell level. They can also provide reference for studying multidug resistance (MDR) engender and better therapy.2. Studied the expression and distribution of tenascin-C protein on living mammary cancer cell membrane by SMFM and confocal. The interaction force between tenascin-C and GBI-10 aptamer was measured in this work by SMFM. The different expression and distribution of tenascin-C on mammary cell and mammary cancer cell membrane were also detected by combination AFM with confocal. The results showed that there was special interaction between tenascin-C and GBI-10, and the force was about 190±15 pN at 2μm/s loading rate. The expression of tanascin-C on mammary cancer cell membrane was electropositive, but negative on mammary cell membrane. These have important signification in understanding the interaction between aptamer and protein. Also provided reference for mammary cancer early diagnosis.3. Studied the expression and distribution of tenascin-C protein on living liver cell membrane by SMFM. The tenascin-C may express different on different types of tissues which related to many cancers. The interaction forces between tanascin-C and aptamer on normal liver cell and three different kinds of liver cancer cell lines were measured in his work by SMFM in basis of previous work. The different expression and distribution of tenascin-C on liver cell and liver cancer cell membrane were also detected by combination AFM with confocal. The results showed that there was special interaction between tenascin-C and GBI-10 on different kinds of liver cancer cells. And the forces was the same as measured on mammary cancer cell which was about 190±15 pN at 2μm/s loading rate. The expression of tanascin-C on liver cancer cell membrane were electropositive, but negative on normal liver cell membrane. These are good for study the interaction between protein and aptamer by combination SMFM with confocal .