Effects Of Temperature On The Viability And Function Of Rat Peritoneal Mast Cells And RBL-2H3 Cells

Mast cells are generally considered to be the key effector cells in IgE-mediated allergic diseases.Recent studies show that mast cells play a detrimental role in the regulation of innate and adaptive immune responses, autoimmunity, graft rejection and transplantation tolerance, and cancer suppression or promotion. Mast cells can synthesize and secrete a variety of cytokines and biologically active substances, including histamine, heparin, tumor necrosis factor, platelet activating factor, and interleukin etc. In addition, the mast cell granules contain tryptase, chymotryptic protease and other important proteolytic enzymes. Exogenous or endogenous stimuli evokes the above bioactive substances released from mast cells, which drive allergic reactions, induce fibroblast proliferation, promote collagen synthesis, or participate in pathological fibrosis, also may cause inflammation. Mast cells are usually located in connective tissue, mucous layer of skin, respiratory and gastrointestinal tract and other organs. The skin temperature is 34℃ and the body core temperature is 37℃, while the temperature in the place where mast cells located is generally between 34℃ and 37℃. Therefore, different temperatures may change the viability and function of mast cells, cause mast cells degranulation, and result in a series of reactions. RBL-2H3 cell line is an important tool to study the function of mast cells in vitro. Nevertheless, there are some studies showed that the RBL-2H3 cells are different from mast cells. So, we investigate the effects of different temperatures on viability and function of rat peritoneal mast cells, and compare the differences between the RBL-2H3 cells and rat peritoneal mast cells in vitro.Objective:Explore the effects of different temperature on the viability and function of rat peritoneal mast cells and RBL-2H3 cellsMethods:1. Isolated and Purified peritoneal mast cells from 220-250 g SD male rats with Percoll Density Gradient Centrifugation Technique. Using toluidine blue staining, and flow cytometry with c-kit antibody labeling to identify cell purity.2. The rat peritoneal mast cells and RBL-2H3 cells were cultured at 35℃ and 37 ℃ for 2 h,4 h,6 h,12 h,24 h,48 h,72 h, CCK8 assay was used to detect cell proliferation activity.3. Cultured rat peritoneal mast cells and RBL-2H3 cells at 35℃ and 37℃ exogenous stimulus C48/80 and IgE were used to stimulate cell degranulation, and mast cell stabilizers cromolyn sodium used to inhibit cell degranulation.4. β-hexosaminidase release rate of peritoneal mast cells and RBL-2H3 cells was determinated by chromogenic substrate method after treated with exogenous stimuli C48/80 and IgE.5.ELISA method for determining histamine release rate of peritoneal mast cells and RBL-2H3 cells after treated with exogenous stimuli C48/80 and IgE.Results:1.Rat peritoneal mast cells were obtained and purified by an improved method of Percoll Density Gradient Centrifugation Technique, the purity of mast cells were measured by toluidine blue stain and flow cytometry with c-kit antibody labeling.The results showed that the purity of mast cells reached 95%, and had good cell activity.2. The proliferation activity had no significant difference between peritoneal mast cells cultured at 35℃ and 37℃ for 2 h. After 2 h, the proliferation activity of peritoneal mast cells cultured at 35℃ was significantly weaker than 37℃ (P< 0.05), and the difference gradually increased along with the time development; the p-hexosaminidase and histamine release rate of peritoneal mast cells cultured at 35℃ was significantly higher than at 37℃ without external stimuli; the P-hexosaminidase and histamine release rate were significantly increased at 35℃, and significantly higher than at 37℃ after C48/80 or IgE stimulation.3. The proliferation activity of RBL-2H3 cell is higher than the peritoneal mast cells either at 35℃ or 37℃; without external stimuli, the β-hexosaminidase release rate of peritoneal mast cells was significantly higher than that of RBL-2H3 cells, but the histamine release rate had fluctuations; after C48/80 stimulation, the P-hexosaminidase and histamine release rate were increased in both cells, the cell degranulation intensified, and the release rate of P-hexosaminidase and histamine of peritoneal mast cells were both significantly higher than that of RBL-2H3 cells, after IgE stimulation, the β-hexosaminidase release rate was significantly higher than that that of RBL-2H3 cells; histamine release rate was also increased in peritoneal mast cells, and was higher at 35℃ but lower at 37℃ compared with the RBL-2H3 cells.Conclusion:1. Establish and improve the extraction of rat peritoneal mast cells culture system.2. The proliferation activity of peritoneal mast cells and RBL-2H3 cell cultured at 35℃was significantly weaker than at 37℃, and the difference gradually increased along with the time development.The severity of mast cell degranulation of peritoneal mast cells cultured at 35℃ was significantly higher than 37℃, and more vulnerable to external stimuli.3. Under the condition of same temperature, the proliferation activity of RBL-2H3 cell is higher than the peritoneal mast cells; For different stimuli, the β-hexosaminidase and histamine release rate of the rat peritoneal mast cells are different from RBL-2H3 cells.4. The viability and function of peritoneal mast cells and RBL-2H3 cells cultured at 35℃ or 37℃ are different, and different stimuli can prompt different response on these two cell types, RBL-2H3 cells can not completely replace mast cells in vitro studies.Features and innovations in this study:1. Confirmed the effect of temperature on the vitality and function of mast cell, and provided certain theoretical basis to explore the pathogenesis of mast cells located in different position.2. Revealed the peritoneal mast cells and RBL-2H3 cells have different reaction when cultured at different temperature respectively.