Anaphylaxis C5a Stimulates Mast Cell Degranulation To Promote Hepatocyte Injury

Objective Discuss the method for establishing CD88 phenotype mast cell for studying the effect of fusion protein C5a on mast cell degranulation and using cobalt chloride to stimulate hepatic parenchymal cells, the model of hepatic parenchymal cells was established, to observe the effect of degranulation of mast cells on damaged liver parenchyma cells by co culture of AML12 and mast cells.Methods 1) extraction of bone marrow mast cells and skin derived mast cells, flow cytometry and toluidine blue was used to identify the phenotype of mast cells; 2) PMA or ionomycin treatment of bone marrow-derived mast cells and P815 cells, the expression of CD88 was analyzed by flow cytometry, and find PMA and ionomycin stimulated mast cells express CD88 appropriate treatment time and dose;3) Analysis of the expression of CD88 and beta -actin protein in mast cells by Western blot; 4) C5a fusion protein was used to stimulate the mast cells of CD88 phenotype, and the degranulation of mast cells was detected by the method of beta lactamase; 5)The establishment of liver hypoxia model by using cobalt chloride to treat AML12; 6) the degranulation of mast cells and the cultured AML12 cells were used to observe the effect of mast cell degranulation on the damaged hepatocytes.Results 1) A large number of mast cells can be obtained after a period of cytokine induced differentiation, and the mast cells can not be obtained from the skin; 2) Bone marrow-derived mast cells after PMA or ionomycin stimulation, CD88 expression increased significantly tested by Flow cytometry and found in the contrast test of dose and time, the optimum time of PMA and ionomycin stimulated mast cell expression of CD88 is 24h,the most suitable concentration were 50ng/ml and 1000ng/ml while there was no obvious CD88 expression in P815 mast cells; 3) The results of Western blotting showed that the expression of CD88 protein was significantly increased in the mast cells stimulated by PMA or ionomycin,compared with the mast cells without stimulation; 4) fusion protein C5a stimulated CD88 phenotype of mast cells after application of beta lactamase method to measure the degranulation of mast cells,compared with the control group without C5a, its degranulation increased significantly, and increased with the increase of C5a fusion protein was added, shown in a concentration dependent manner; 5)According to the literature report, after the experiment, we can find the appropriate concentration of cobalt chloride to establish the hypoxia injury of liver parenchyma cells; 6) hepatocyte damage model was established by cobalt chloride and found that in mild hepatic parenchymal cell damage, mast cell degranulation can stimulate hepatocytes reactive hyperplasia, while in severe cases of impaired hepatic parenchymal cells, mast cell degranulation can further promote the occurrence of injury, inhibit the proliferation of hepatic the role of parenchymal cellsConclusion: A large number of high purity mast cells can be obtained through cytokines and after PMA or ionomycin stimulation, the surface expression of CD88 increased significantly. By means of C5a stimulation,mast cell degranulation and showed concentration dependent .Mast cell degranulation can inhibit cell proliferation in severe damage model of the liver parenchyma cells...