CAJ | 학술논문

目的探讨糖原合酶激酶3β(GSK3β)在轻型/重症急性胰腺炎发病中的表达及作用方法采用单独雨蛙素、雨蛙素联合脂多糖刺激分别构建轻型急性胰腺炎(MAP)和重症急性胰腺炎(SAP)的细胞模型,分别在2、4、8、12、24 h收集细胞培养基上清,酶联免疫吸附测定(ELISA)检测淀粉酶、脂肪酶、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的表达变化,流式细胞术检测细胞凋亡率及坏死率,实时荧光定量聚合酶链反应(FQ-PCR)检测GSK3β及半胱氨酸天冬氨酸蛋白酶(Caspase)-3、-8、-9 mRNA表达变化.结果雨蛙素组和雨蛙素+脂多糖组各时间点淀粉酶、脂肪酶、TNF-α、IL-6较对照组均增高,且在大多数时间点,雨蛙素+脂多糖组组比雨蛙素组水平更高,开始上调时间相同或更早(P＜0.05).刺激24 h后,雨蛙素组凋亡率[(19.2±0.9)％]显著高于雨蛙素+脂多糖组[(10.2±0.5)％,P ＜0.05].雨蛙素组坏死率[(5.8±0.3)％]显著低于雨蛙素+脂多糖组[(13.6±0.7)％,P＜0.05].SAP组的坏死/凋亡比值是MAP组的5倍.GSK3β的mRNA水平在雨蛙素组中是2h短暂下调[相对表达量为(0.5±0.1)倍]后于8h显著上升[相对表达量为(3.0±1.1)倍,P ＜0.05],然后又逐步下降;而在雨蛙素+脂多糖组中则是是8h才缓慢上调[相对表达量为(1.8±0.6)倍,P＜0.05],至24 h达到高峰[相对表达量为(3.6±0.3)倍,P＜0.05].GSK3β和Caspase-3 mRNA水平均有升高,但差异无统计学意义(P＞0.05).结论采用单独雨蛙素、雨蛙素联合脂多糖刺激分别建立MAP和SAP细胞模型可行;GSK3β在MAP和SAP中可能通过促进腺泡细胞凋亡来起作用.
목적 탐토당원합매격매3β(GSK3β)재경형/중증급성이선염발병중적표체급작용방법 채용단독우와소、우와소연합지다당자격분별구건경형급성이선염(MAP)화중증급성이선염(SAP)적세포모형,분별재2、4、8、12、24 h수집세포배양기상청,매련면역흡부측정(ELISA)검측정분매、지방매、종류배사인자-α(TNF-α)、백세포개소-6(IL-6)적표체변화,류식세포술검측세포조망솔급배사솔,실시형광정량취합매련반응(FQ-PCR)검측GSK3β급반광안산천동안산단백매(Caspase)-3、-8、-9 mRNA표체변화.결과 우와소조화우와소+지다당조각시간점정분매、지방매、TNF-α、IL-6교대조조균증고,차재대다수시간점,우와소+지다당조조비우와소조수평경고,개시상조시간상동혹경조(P＜0.05).자격24 h후,우와소조조망솔[(19.2±0.9)％]현저고우우와소+지다당조[(10.2±0.5)％,P ＜0.05].우와소조배사솔[(5.8±0.3)％]현저저우우와소+지다당조[(13.6±0.7)％,P＜0.05].SAP조적배사/조망비치시MAP조적5배.GSK3β적mRNA수평재우와소조중시2h단잠하조[상대표체량위(0.5±0.1)배]후우8h현저상승[상대표체량위(3.0±1.1)배,P ＜0.05],연후우축보하강;이재우와소+지다당조중칙시시8h재완만상조[상대표체량위(1.8±0.6)배,P＜0.05],지24 h체도고봉[상대표체량위(3.6±0.3)배,P＜0.05].GSK3β화Caspase-3 mRNA수평균유승고,단차이무통계학의의(P＞0.05).결론 채용단독우와소、우와소연합지다당자격분별건립MAP화SAP세포모형가행;GSK3β재MAP화SAP중가능통과촉진선포세포조망래기작용.
Objective To investigate the expression and role of glycogen synthase kinase-3β (GSK3β) in mild/severe acute pancreatitis.Methods Cell models of mild acute pancreatitis (MAP) and severe acute pancreatitis(SAP) were constructed with using cerulein solely or cerulein plus lipopolysaccharide.The cells were collected at 2,4,8,12,24 h.The expression of amyase,lipase,tumor necrosis factor (TNF)-α and interleukin (IL)-6 were examined by enzyme-linked immune sorbent assay (ELISA) and the apoptosis/necrosis rate was detected by flow cytometric analysis.Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was performed to detect the expression changes of GSK3β and Caspase-3,-8,-9 mRNA.Results Compared with control,the amyase,lipase,TNF-α and IL-6 in cerulein and cerulein plus lipopolysaccharide group were elevated at every time point.Moreover,in most time point these levels in cerulein plus lipopolysaccharide group were higher than cerulein group,and were up-regulated at the same time or earlier (P ＜ 0.05).After 24 h,the apoptosis ratio in cerulein group [(19.2 ± 0.9) ％] was higher than cerulein plus lipopolysaccharide group [(10.2 ±0.5) ％,P ＜ 0.05] while the necrosis ratio in cerulein group [(5.8 ± 0.3) ％] was lower than cerulein plus lipopolysaccharide group [(13.6 ± 0.7) ％,P ＜ 0.05),and the ratio of necrosis/apoptosis of cerulein plus lipopolysaccharide group was five times larger than that of cerulein group.The mRNA expression of GSK3β in cerulein group dropped briefly at 2 hours (relative ratio =0.5 ±0.1),but it rose again and reached the peak at 8 hours (relative ratio =3.0 ± 1.1,P ＜ 0.05),and then it declined again.On the other hand,the expression of GSK3β in cerulein plus lipopolysaccharide group wasn' t up-regulated until 8 hours (relative ratio =1.8 ± 0.6,P ＜ 0.05) and reached a climax at 24 hours (relative ratio =3.6 ± 0.3,P ＜ 0.05).Both the level of GSK3β and Caspase-3 mRNA were elevated,but the positive correlation didn' t reach statistical significance.Conclusion It is technically feasible to construct cell models of MAP and SAP with using cerulein solely or cerulean plus lipopolysaccharide; GSK3β might play a role in MAP and SAP by promoting acinous cell apoptosis.