Effects Of Isoproterenol Hydrochloride,Caffeic Acid And E-64D On The Growth And Apoptosis Of T Lymphocytes Under Microgravity

Objective:The splenocytes of KM mice were used as the research object to find the suitable concentration of ConA for T lymphocyte culture and the experimental method to obtain primary cells.On this basis,the effects of ISO,CA and E-64D on T lymphocytes in simulated microgravity were studied.On the basis of the previous research results,a new microgravity model was re-established on the spleen cells of C57/BL6J mice after adding microcarriers.The effects of Uridine and PMA on the number of T lymphocytes and cell division under simulated microgravity were used as positive control,and then the effects of ISO,E-64D and CA on the number of T lymphocytes under simulated microgravity were studied respectively,which were explained from the aspects of cell division,apoptosis and factor secretion.Methods:Using erythrocyte lysate to obtain primary cells from the spleen of healthy female K-M mice,the cells were activated and cultured with 2.5μg/ml and 5.0μg/ml ConA,respectively.The differences of cell proliferation under the action of the two concentrations of ConA were compared.Then the primary cells obtained from lymphocyte separation solution and erythrocyte lysate were activated and cultured respectively,and the changes of T lymphocyte number were compared by cell count and flow cytometry to detect the proportion of CD3~+cells.Splenocytes were cultured in simulated microgravity using a rotating incubator and ISO,CA and E-64D were added respectively before culture.After culture,the number of T lymphocytes was counted and the proportion of CD3~+cells was determined by flow cytometry to explore the effects of three drugs on T lymphocytes under simulated microgravity.When the spleen cells of C57/BL6J mice were cultured in a rotating incubator to simulate the microgravity environment,microcarriers were added to the mixed cells,so that the cells that needed adherent growth in the mixed cells could be attached to the carriers for microgravity culture.The new microgravity injury model was evaluated by the experimental results.Before normal gravity and simulated microgravity culture,Uridine and PMA,were added to study their effects on the number of T lymphocytes,and then cells were labeled with CFDA-SE before culture to detect their effects on cell division,and then to study the effects of ISO,E-64D and CA on the number of T lymphocytes under simulated microgravity.The better drugs were selected to continue the experiment,and CFDA-SE labeling was used to detect its effect on cell division,and then the cells cultured for 24 hours were stained with Annexin V/PI to detect the changes of apoptosis.finally,the contents of IL-4 and IFN-γin the culture medium after48 hours of culture were detected by ELISA.Results:The proliferation of cells stimulated by ConA at the final concentration of 2.5μg/ml was more obvious,and the proliferation of primary cells obtained from erythrocyte lysate was more obvious than that of lymphocyte separation after activation.The proliferation of splenocytes cultured in simulated microgravity was significantly lower than that in normal gravity,while the number of T lymphocytes in simulated microgravity could be increased by adding ISO,CA and E-64D respectively.In the new microgravity model,cells can adhere to the microcarrier,and the number of T lymphocytes is significantly inhibited in microgravity culture,and the addition of Uridine and PMA can increase the number of T lymphocytes after microgravity culture,and PMA is more effective in stimulating cell division.ISO and E-64D were more effective than CA in increasing the number of T lymphocytes under microgravity,further experiments showed that ISO could increase cell division under simulated microgravity,but E-64D could not.The detection of apoptosis showed that ISO only inhibited apoptosis under microgravity,while E-64D could inhibit apoptosis under both normal gravity and simulated microgravity.ISO can reduce the secretion of IFN-γand increase the secretion of IL-4;E-64D inhibited the secretion of IFN-γby cells in 1g gravity and simulated microgravity,but had no significant effect on the secretion of IL-4.Conclusions:When the primary cells obtained from erythrocyte lysate were stimulated by ConA at the final concentration of 2.5μg/ml,the cell proliferation was more obvious;simulated microgravity culture could inhibit T lymphocyte proliferation,and the use of ISO,CA and Eli 64D could effectively improve the inhibitory effect of simulated microgravity on T lymphocyte proliferation;the microgravity injury model of C57/BL6J mouse spleen cells was successfully established by adding microcarriers before microgravity culture.It was verified that Uridine and PMA increased the number of T lymphocytes under microgravity by stimulating cell division and providing metabolic materials for cells,respectively;ISO and Emur64D increased the number of cells under microgravity by stimulating cell division and inhibiting apoptosis,respectively;both ISO and Emur64D could reduce IFN-γsecretion,but ISO was only effective under microgravity;only ISO could increase cell secretion of IL-4.