CAJ | 학술논문

目的探索环孢菌素A抑制胰岛素分泌的分子机制方法经胆管不离体灌注胶原酶消化分离Wistar大鼠胰岛,环孢菌素A(0.5、1.0、2.5、5.0、10.0 μg/ml)干预不同时间后,吖啶橙/碘化丙锭染色,未经环孢菌素A处理的胰岛作为对照组,荧光显微镜下观察腆岛细胞存活变化.对照组和1.0 μg/ml环孢菌素A干预组中的胰岛分别孵育24 h后,进行高糖刺激胰岛素分泌实验;通过实时定量PCR方法探测P糖蛋白基因(abcb1b)、凋亡基因(casp3、Bcl-2)、胰岛素合成相关基因(pdx1、ins1、ins2)和胰高血糖素基因(glucagon)表达量的变化;通过罗丹明细胞内聚集实验评估P糖蛋白外排泵功能.结果通过胰岛存活状态的观察,确定1.0 μg/ml环孢菌素A为合适的干预浓度.环孢菌素A 干预对体外胰岛高糖刺激下的第一时相分泌无抑制,而对第二时相抑制作用显著(P＜0.01) 环孢菌素A干预24 h后,abcb1b、ins1、ins2、pdx1、glucagon、casp3表达量均无变化;Bcl-2基因较对照组下调明显(P＜0.01)；环孢菌素A干预24 b后胰岛细胞内罗丹明聚集量的显著增加(P＜0.01) 结论环孢菌索A 干预抑制胰岛素第二相分泌,凋亡基因Bcl-2表达的变化是关键因素之一.胰岛素分泌的变化与P糖蛋白的表达、胰岛素合成及胰高血糖素无关,但是环孢菌素A对P糖蛋白功能的影响非常显著,不能排除P糖蛋白调节胰岛素分泌的可能性.
목적 탐색배포균소A억제이도소분비적분자궤제 방법 경담관불리체관주효원매소화분리Wistar대서이도,배포균소A(0.5、1.0、2.5、5.0、10.0 μg/ml)간예불동시간후,아정등/전화병정염색,미경배포균소A처리적이도작위대조조,형광현미경하관찰전도세포존활변화.대조조화1.0 μg/ml배포균소A간예조중적이도분별부육24 h후,진행고당자격이도소분비실험;통과실시정량PCR방법탐측P당단백기인(abcb1b)、조망기인(casp3、Bcl-2)、이도소합성상관기인(pdx1、ins1、ins2)화이고혈당소기인(glucagon)표체량적변화;통과라단명세포내취집실험평고P당단백외배빙공능.결과 통과이도존활상태적관찰,학정1.0 μg/ml배포균소A위합괄적간예농도.배포균소A 간예대체외이도고당자격하적제일시상분비무억제,이대제이시상억제작용현저(P＜0.01) 배포균소A간예24 h후,abcb1b、ins1、ins2、pdx1、glucagon、casp3표체량균무변화;Bcl-2기인교대조조하조명현(P＜0.01)；배포균소A간예24 b후이도세포내라단명취집량적현저증가(P＜0.01) 결론 배포균색A 간예억제이도소제이상분비,조망기인Bcl-2표체적변화시관건인소지일.이도소분비적변화여P당단백적표체、이도소합성급이고혈당소무관,단시배포균소A대P당단백공능적영향비상현저,불능배제P당단백조절이도소분비적가능성.
Objective To explore the underlying mechanisms of inhibiting insulin secretion of rat islets by cyclosporine A in vitro.Methods Rat islets were isolated from pancreas by collagenase digestion.The islets were stained by acridine orange/propidium iodide and evaluated under fluorescence microscope after cyclosporine A were inoculated (0.5,1.0,2.5,5.0,and 10.0 μg/ml) over different periods (6,24,and 48 hours).The islets treated only with the vehicle were served as control.After inoculation of 1 μg/ml cyclosporine A or the vehicle for 24 hours,insulin secretion of the islets was determined by radioimmunol assay(RIA).The expressions of abcb1 b,pdx1,ins1,ins2,glucagon,casp3,and Bcl-2 were evaluated by realtime fluorescence quantitative PCR after inoculations of cyclosporine A for 24 hours.A rhodamine 123 uptake measurement was used to analyze P-glycoprotein efflux pump function.Results Inoculation of 1.0 μg/ml cyclosporine A for 24 hours did not affect islet survival significantly.Only the second phase of insulin secretion was inhibited by the cyclosporine A inoculation (P＜0.01),but not the first phase.Compared to the control group,the expressions of abcb1b,ins1,ins2,pdx1,glucagon,casp3 did not show any difference in the cyclosporine A inoculated group.But the expression of Bcl-2 was down-regulated significantly in the cyclosporine A inoculated group (P＜0.01).The efflux pump function of P-glycoprotein was inhibited by the cyclosporine A inoculation (P＜0.01).Conclusions Inhibitory effects of cyclosporine A on the second phase of insulin secretion may be through apoptosis pathway.Cyclosporine A did not influence biogenesis of insulin or glucagon.Even though cyclosporine A did not reduce the expression of P-glycoprotein,its specific inhibitory effect on P-glycoprotein in impairing insulin secretion could not be excluded.The underlying mechanism needs to be further investigated.