| Bronchial asthma is a chronic airway inflammatory disease characterized by the infiltration of airway T cells, CD(+) (T helper) cells, mast cells, basophils, macrophages, and eosinophils(EOS).EOS play a major role in the onset and maintenance of bronchial inflammation and tissue injury in asthma,and eosinophil-mediated damage to the respiratory epithelium is a major pathogenetic mechanism in asthma. EOS release highly toxic products and cytokines which may influence the immune system, amplify the inflammatory response and participate in damaging and remodeling processes that occur in the airway mucosa. Saito has proved that the calcium-activated potassium channels ( Kca) could cause the secretion of granular proteins from human EOS,but it is still debated the mechanism and function of EOS Kca in asthma. The experiment researchs changes of EOS Kca in asthma and effects of doxofylline on Kca of EOS from asthma patients in acute attack stage to study the effect of Kca of human peripheral blood EOS in bronchial asthma.The experiment was divided into three parts.Firstly, isolation of EOS from peripheral blood by means of discontinuouspercoU density gradients centrifugation to be improved and recording the electric currents in the single Ca2+-activated K+ channels using patch-clamp technique with cell attached configuration and identification of Kca.The Result: The purity and the ratio of viability and recovery of EOS from healthy donors were (90.5 ±3.6)%, over 99% and (48. 2 + 6.9)% respectively. Stimulation of EOS with 0.2 microM platelet-activating factor (PAF) caused activation of single channels as recorded by the cell-attached patch-clamp technique. These channels were selectively permeable to K+ because the reversal potential was close to the equilibrium potential for K+. However, the channels were not permeable to Na+ or CI- as demonstrated by ion substitution experiments. The calcium ionophore A-23187, at 1 microM, increased the K+ channel activity in the presence of Ca2+ in the external perfusate. These results indicate the channels activated by PAF is Kca channels.Secondly,compared the reconding of EOS Kca channels from asthma patients in acute attack stage and asthma patients in stablestage and healthy individuals.The experiments record the currents of EOS Kca of human peripheral blood in asthma patients in acute attack stage and asthma patients in stablestage and healthy individuals,and compare the changes of the conductances and the open probability and the open time and closed time.The Result: we record the currents of healthy individuals(17 cells,8 persons) and asthma patients in stablestage (11 cells,5 patients)and asthma patients in acute attack stage(15 cells,8 patients). In healthy individuals, the conductances are 9.2+ l.lpS and 22.1±2.4pS,and the open probability is 0.04976±0.015,and the open time and closed time are 1. 32+0.4ms and 1.25+0.5ms. In asthma patients in stablestage, the conductances are 14.9 + 3.9pS and 26.9 ± 2.8pS,and the open probability is 0.05232 ± 0.018,and the open time and closed time are 1.46+0.3msand 1.26+0.4ms. In asthma patients in acute attack stage, the conductances are 15.3 ±3.5pS and 35.1±3.8pS,and the open probability is 0.1623+0.023,and the open time and closed time are 5.73±0.7ms and 2.31±0.6ms. Three groups of the large-conductances are compared significantly one another (P<0.05 ) . The small-conductances of healthy individuals and asthma patients in stablestage, healthy individuals and asthma patients in acute attack stage are compared significantly each other (P<0.05) . The open probability of healthy individuals and asthma patients in stablestage don't have significantly defference (P>0.05) ,yet the open probability of asthma patients in stablestage and asthma patients in acute attack stage is compared significantly (P<0.05 ) . The open time and closed time of asthma patients in stablestage and asthma patients in acute attack stage is compared significantly (P<0.05) .Compared with the open probability of three groups at — 40mv^ — 20mv> 0mv\ 20mv> 40mv holding potential respectively, the open probability of every group don't has significantly defference at different holding potential (P>0.05 ) .The EOS Kca don't have volatage dependence.Conclusion: In asthma patients the conductances of EOS Kca are larger than in healthy individuals,and the open probability of EOS Kca is increased and the open time is prolonged in acute attack stage asthma patients.The EOS Kca activity increased in asthma which maybe cause eosinophils to secrete and cause bronchial inflammation constantly.Thirdly, the effects of doxofylline on Ca2+-activated K+ (KCa) channels of human peripheral blood EOS in bronchial asthma in acute attack stage.Result: The data were recorded using cell-attached configuration of patch-clamp technique and the kinetic changes of Kca channels be activated by 0.2umol ? L"1 PAF were compared before and after lOOumol ? L"1 doxofylline incubated EOS for 30 minutes. Before and after lOOumol ? L"1 doxofylline ,theopen probabihty of the Kca channels decreased from (0.1347+0.021) to (0.04352 ±0.018) (n =12, P<0.05) , open time from (5.75+0.4) ms to (2.39±0.13) ms, close time raised from (2.17±0.5) ms to (23.73+2.5) ms (n= 11, P<0.05).Conclusion: Doxofylline could greatly decrease the open probability of the channels as a result of both the shorten of open period and prolongation of close time, which maybe inhibit the EOS infiltration within airway walls . |
PDF Full Text Request