Making Gastric Mucus Cell Suspension And Generally Analysing Its Proteins Expression

Objective: To find an method which can remove the gastric mucus gel and make gastric mucus cell suspension , which the single cell proteins could be investigated by Flow cytometry et al , to be prepared for cytomics project. the control is Molt-4 cultured in vitro.Methods: We use enzyme to solube the mucus gel and another enzyme to isolate mucous layer from the residual tissue , and then make cell suspension with machinery methods. We use DNA histogram to analyse the cell cycle and Western methods to show cyclins comparing to the mucous layer tissue which is not dealt whith pepsin , the Major cyclins expressed in mucous layer cells were analyzed by FCM and Confocal.Results: The 0.05-0.1% concentration of pepsin can successfully dissolve the gel and the mucous layer can completely be separated from other tissue after using the 1.2-2.4u/ml concentration of dispase. The positive rates and the levels of cyclins were expressed from the cell suspension that we successfully acquired from mucus layer. While cyclins D3, B1 were obviously positive, Cyclins D1,D2,A,E were not, which were also confirmed by Western and Confocal.Conclusion: After using pepsin and dispase, the cell suspension from mucous layer can be easily obtained, also the cytometry results indicated that the Cyclins Schedule expression in vivo was different from Cyclins Schedule expression in vitro. Objective: We use enzyme to solube the mucus gel and make cell suspension with machinery methods. Then membrane proteins were extracted from the cell suspension compared to the gastric mucus tissue which is not dealt with enzyme, the control is Hela cultured in vitro. to provide the membrane protomics support for gastric cytomics project.Methods: We use 0.1% concentration of pepsin to solue the mucus gel and the 1.2-2.4u/ml concentration of dispase isolate mucous layer from the residual tissue , and then make cell suspension with machinery methods. The membrane proteins and cytosol proteins were divided by Triton-114,The membrane proteins were confired by Westernblot, also the SDS-PAGE was done.Results: Membrane proteins could be acquired from the cell suspension that we can successfully acquired from mucus layer after using the 0.1% concentration of pepsin and the 1.2-2.4u/ml concentration of dispase. The membrae proteins (Na+/K+ATPase, caveolin,et al) extracted through Triton-114 were enriched in the Westenblot results and the SDS-PAGE show the same.Conclusion: After using pepsin and dispase, the cell suspension from mucous layer could be easily obtained, also we can use Triton-114 to acquired membrane proteins which could further be analysed by membrane protomics methods.