雨蛙肽诱导的实验性急性胰腺炎细胞模型中酶噬的变化  

Changes of zymophagy during cell model of caerulein-induced experimental acute pancreatitis

作　　者：李洁[1] 刘晓[1] 吴敏[1] 郭晓榕[1] 湛先保[1] 

机构地区：[1]长海医院消化内科,上海200433

出　　处：《中华消化杂志》2014年第11期752-755,共4页Chinese Journal of Digestion

基　　金：国家自然科学基金（81170434,81370571）

摘　　要：目的 观察雨蛙肽诱导的实验性急性胰腺炎（AP）细胞模型中酶噬的变化.方法 培养AR42J细胞株（胰腺外分泌细胞）,将细胞接种于6孔板培养至90％融合,分为AP组与空白对照组,AP组加入终浓度为1×10-8 mol/L的雨蛙肽建立AP细胞模型,空白对照组加入1640培养液.在雨蛙肽处理后1、4、6、8、12、24 h收集细胞及培养上清液,以ELISA方法检测上清液中炎性因子IL-1、TNFα、淀粉酶及胰蛋白酶原活化肽（TAP）水平.取各组细胞,采用RT-PCR和Western印迹法检测LC3、Beclin-1mRNA和 LC3B蛋白的表达.透射电子显微镜观察各组细胞酶噬小体的变化.组间比较采用方差分析.结果 空白对照组上清液中的IL-1、TNFα、淀粉酶和TAP分别为（18.83±7.10） pg/mL、（14.20±3.79） pg/mL、（10.03±2.85）U/L、（39.48±8.62） pg/mL,AP组在1h时即显著升高至（62.13±11.25） pg/mL,（30.98±7.11） pg/mL、（25.06±6.82） U/L、（128.51±18.30） pg/mL,差异均有统计学意义（F=3.32、3.05、2.90、2.62,P＜0.05或0.01）在4或6h时达到高峰[IL-1在4h为（71.96±15.82） pg/mL,F=7.25,P＜0.01;TNFα在6h时为（39.92±8.94） pg/mL,F=4.93,P＜0.05;淀粉酶在4h时为（28.83±8.31） U/L,F=2.06,P＜0.05;TAP在4h时为（146.29±29.36） pg/mL,F=0.14,P＜0.01],之后逐渐下降趋于稳定,AP组细胞中第4、6h的LC3 mRNA含量为3.18±0.82、1.71±0.14,第1、4h时的Beclin-1 mRNA含量为2.44±0.34、4.13±0.30,较空白对照组（0.21±0.04、0.30±0.08）均明显上升（LC3 mRNA,F=0.79、0.06 ;Beclin mRNA,F=2.31、0.36,P均＜0.05）,其余时间点差异均无统计学意义（P均＞0.05）.电子显微镜下可见AP组自噬体和酶噬小体数目显著多于空白对照组.结论 雨蛙肽诱导AP细胞模型时伴有酶噬的发生,提示酶噬可能参与了AP的发病机制.Objective To observe the changes of zymophagy during experimental acute pancreatitis （AP） induced by caerulein.Methods Pancreatic acinar cell line AR42J cells were cultured in 6-well plates till 90％ confluent and then divided into AP group and control group.Caerulein （1 × 10-8 mol/L） was added into AP group to establish AP cell model,and 1640 cell culture medium was added into control group.After caerulein treated for one,four,six,eight,12 and 24 hours,cells and cell culture supernatant were collected.The levels of cytokine interleukin （IL）-1,tumor necrosis factor （TNF）α,trypsinogen activation （TAP） and amylase were measured with enzyme-linked immunosorbent assay （ELISA） method.The expression of LC3 and Beclin1 at mRNA of each group were detected by reverse transcription-polymerase chain reaction （RT-PCR）.The LC3B protein level of each group were detected by Western blotting.The changes of autophagosome and zymophagosome were observed by transmission electron microscopy.The difference between AP group and control group was analyzed by analysis of variance.Results The level of IL-1,TNFα,amylase and TAP in cell culture supernatant of control group was （18.83±7.10） pg/mL,（14.20±3.79） pg/mL,（10.03±2.85） U/L and （39.48±8.62） pg/mL,respectively.Those of AP group significantly increased at first hour （（62.13±11.25） pg/mL,F=3.32,P＜0.01 ; （30.98±7.11） pg/mL,F=3.05,P＜0.05; （25.06±6.82） U/L,F=2.90,P＜0.05 and （128.51± 18.30） pg/mL）,F=2.62,P＜0.01,at fourth or sixth hour reached peak （IL-1 at fourth hour：（71.96± 15.82） pg/mL,F=7.25,P＜0.01;TNFα at sixth hour：（39.92±8.94） pg/mL,F=4.93,P＜0.05; amylase at fourth hour：（28.83 ± 8.31） U/L,F=2.06,P＜0.05; TAP at fourth hour：（146.29± 29.36） pg/mL,F=0.14,P＜0.01） and then gradually decreased.At fourth and sixth hour,the expression of LC3 at mRNA level in AP group was 3.18±0.82,1.71±0.14,respectively,while the expression of Beclin-1 rnRNA at first,fourth hour was

关 键 词：急性胰腺炎 自噬 胰蛋白酶原 

分 类 号：R576[医药卫生—消化系统]