中华消化杂志
中華消化雜誌
중화소화잡지
Chinese Journal of Digestion
2015年
1期
39-43
,共5页
刘晓%郭晓榕%张蓓蓓%李洁%吴敏%湛先保
劉曉%郭曉榕%張蓓蓓%李潔%吳敏%湛先保
류효%곽효용%장배배%리길%오민%담선보
胰腺炎,急性坏死性%自噬%胰蛋白酶原
胰腺炎,急性壞死性%自噬%胰蛋白酶原
이선염,급성배사성%자서%이단백매원
Pancreatitis acute necrotizing%Autophagy%Trypsinogen
目的 探讨自噬在实验性急性坏死性胰腺炎(ANP)大鼠发病中的变化及其意义.方法 按随机数字法将18只SD大鼠随机分为对照组、ANP组、ANP+雷帕霉素组.采用腹腔注射20％ L-精氨酸的方法制作大鼠ANP模型,造模后9h处死.ANP+雷帕霉素组于造模前30 min按大鼠体质量1.2 mg/kg腹腔注射雷帕霉素.对照组予腹腔注射等量0.9％ NaCl溶液.ELISA方法检测各组大鼠血清胰蛋白酶原活化肽(TAP)、IL-1、IL-6、TNFα水平,进行胰腺组织病理评分,电子显微镜下观察大鼠胰腺腺泡细胞自噬相关结构,实时定量-PCR、Western蛋白印迹法及免疫组织化学检测大鼠胰腺组织自噬标志物微管相关蛋白轻链3(LC3)-Ⅱ及Beclin-1 mRNA和蛋白的表达.组间均数比较采用单因素方差分析.结果 成功建立大鼠ANP模型,胰腺病理评分显示ANP+雷帕霉素组腺泡细胞坏死评分为2.19±1.38,高于ANP组(0.97±0.68),差异有统计学意义(F=33.75,P＜0.05).Western蛋白印迹法显示ANP组LC3-Ⅱ及Beclin-1蛋白表达(分别为35.25±2.68和49.40±5.28)明显高于对照组(分别为1.54±0.16和0.78±0.06);而ANP+雷帕霉素组LC3-Ⅱ及Beclin-1蛋白表达量(分别为123.53±3.21和76.41±3.80)比ANP组更高,差异均有统计学意义(F=2 045.54、326.87,P均＜0.01).免疫组织化学法显示ANP+雷帕霉素组LC3-Ⅱ及Beclin-1蛋白表达量(分别为7 570.63±4 357.67和3 418.09±2 035.78)明显高于ANP组(分别为1 926.53±1 414.44和536.11±403.10),差异均有统计学意义(F=39.83、41.58,P均＜0.01).Beelin-1 mRNA在ANP组的表达(107.12±29.10)较对照组(7.01±3.39)明显升高,差异有统计学意义(F=3.61,P＜0.05),但在ANP+雷帕霉素组的表达(97.63±65.38)与ANP组比较差异无统计学意义(P＞0.05).LC3-Ⅱ mRNA在ANP组的表达(1.76±1.59)与对照组(1.51±0.95)相比差异无统计学意义(P＞0.05),但在ANP+雷帕霉素组的表达(4.37±1.67)明显高于ANP组,差异有统计学意义(F=16.10,P＜0.05).电子显微镜显示ANP组自噬相关结构较对照组增加,ANP+雷帕霉素组则更多.ANP+雷帕霉素组血清TAP、IL-1、IL-6分别为(36.47±1.71) pmol/L、(122.88±26.67) pmol/L、(107.39±13.95) pg/mL,均明显高于ANP组(25.63±6.05) pg/mL、(98.06±9.29) pg/mL、(86.16±7.20) pg/mL,差异均有统计学意义(F=116.71、50.45、79.67,P均＜0.01);ANP+雷帕霉素组TNFα(140.80±60.82) pg/mL与ANP组(105.23±6.95) pg/mL相比,差异无统计学意义(F=14.76,P＞0.05).结论 ANP大鼠自噬增加,促进自噬可显著激活胰蛋白酶原,同时加重胰腺损伤和炎性反应,自噬可能通过激活胰蛋白酶原参与急性胰腺炎的发生.
目的 探討自噬在實驗性急性壞死性胰腺炎(ANP)大鼠髮病中的變化及其意義.方法 按隨機數字法將18隻SD大鼠隨機分為對照組、ANP組、ANP+雷帕黴素組.採用腹腔註射20％ L-精氨痠的方法製作大鼠ANP模型,造模後9h處死.ANP+雷帕黴素組于造模前30 min按大鼠體質量1.2 mg/kg腹腔註射雷帕黴素.對照組予腹腔註射等量0.9％ NaCl溶液.ELISA方法檢測各組大鼠血清胰蛋白酶原活化肽(TAP)、IL-1、IL-6、TNFα水平,進行胰腺組織病理評分,電子顯微鏡下觀察大鼠胰腺腺泡細胞自噬相關結構,實時定量-PCR、Western蛋白印跡法及免疫組織化學檢測大鼠胰腺組織自噬標誌物微管相關蛋白輕鏈3(LC3)-Ⅱ及Beclin-1 mRNA和蛋白的錶達.組間均數比較採用單因素方差分析.結果 成功建立大鼠ANP模型,胰腺病理評分顯示ANP+雷帕黴素組腺泡細胞壞死評分為2.19±1.38,高于ANP組(0.97±0.68),差異有統計學意義(F=33.75,P＜0.05).Western蛋白印跡法顯示ANP組LC3-Ⅱ及Beclin-1蛋白錶達(分彆為35.25±2.68和49.40±5.28)明顯高于對照組(分彆為1.54±0.16和0.78±0.06);而ANP+雷帕黴素組LC3-Ⅱ及Beclin-1蛋白錶達量(分彆為123.53±3.21和76.41±3.80)比ANP組更高,差異均有統計學意義(F=2 045.54、326.87,P均＜0.01).免疫組織化學法顯示ANP+雷帕黴素組LC3-Ⅱ及Beclin-1蛋白錶達量(分彆為7 570.63±4 357.67和3 418.09±2 035.78)明顯高于ANP組(分彆為1 926.53±1 414.44和536.11±403.10),差異均有統計學意義(F=39.83、41.58,P均＜0.01).Beelin-1 mRNA在ANP組的錶達(107.12±29.10)較對照組(7.01±3.39)明顯升高,差異有統計學意義(F=3.61,P＜0.05),但在ANP+雷帕黴素組的錶達(97.63±65.38)與ANP組比較差異無統計學意義(P＞0.05).LC3-Ⅱ mRNA在ANP組的錶達(1.76±1.59)與對照組(1.51±0.95)相比差異無統計學意義(P＞0.05),但在ANP+雷帕黴素組的錶達(4.37±1.67)明顯高于ANP組,差異有統計學意義(F=16.10,P＜0.05).電子顯微鏡顯示ANP組自噬相關結構較對照組增加,ANP+雷帕黴素組則更多.ANP+雷帕黴素組血清TAP、IL-1、IL-6分彆為(36.47±1.71) pmol/L、(122.88±26.67) pmol/L、(107.39±13.95) pg/mL,均明顯高于ANP組(25.63±6.05) pg/mL、(98.06±9.29) pg/mL、(86.16±7.20) pg/mL,差異均有統計學意義(F=116.71、50.45、79.67,P均＜0.01);ANP+雷帕黴素組TNFα(140.80±60.82) pg/mL與ANP組(105.23±6.95) pg/mL相比,差異無統計學意義(F=14.76,P＞0.05).結論 ANP大鼠自噬增加,促進自噬可顯著激活胰蛋白酶原,同時加重胰腺損傷和炎性反應,自噬可能通過激活胰蛋白酶原參與急性胰腺炎的髮生.
목적 탐토자서재실험성급성배사성이선염(ANP)대서발병중적변화급기의의.방법 안수궤수자법장18지SD대서수궤분위대조조、ANP조、ANP+뢰파매소조.채용복강주사20％ L-정안산적방법제작대서ANP모형,조모후9h처사.ANP+뢰파매소조우조모전30 min안대서체질량1.2 mg/kg복강주사뢰파매소.대조조여복강주사등량0.9％ NaCl용액.ELISA방법검측각조대서혈청이단백매원활화태(TAP)、IL-1、IL-6、TNFα수평,진행이선조직병리평분,전자현미경하관찰대서이선선포세포자서상관결구,실시정량-PCR、Western단백인적법급면역조직화학검측대서이선조직자서표지물미관상관단백경련3(LC3)-Ⅱ급Beclin-1 mRNA화단백적표체.조간균수비교채용단인소방차분석.결과 성공건립대서ANP모형,이선병리평분현시ANP+뢰파매소조선포세포배사평분위2.19±1.38,고우ANP조(0.97±0.68),차이유통계학의의(F=33.75,P＜0.05).Western단백인적법현시ANP조LC3-Ⅱ급Beclin-1단백표체(분별위35.25±2.68화49.40±5.28)명현고우대조조(분별위1.54±0.16화0.78±0.06);이ANP+뢰파매소조LC3-Ⅱ급Beclin-1단백표체량(분별위123.53±3.21화76.41±3.80)비ANP조경고,차이균유통계학의의(F=2 045.54、326.87,P균＜0.01).면역조직화학법현시ANP+뢰파매소조LC3-Ⅱ급Beclin-1단백표체량(분별위7 570.63±4 357.67화3 418.09±2 035.78)명현고우ANP조(분별위1 926.53±1 414.44화536.11±403.10),차이균유통계학의의(F=39.83、41.58,P균＜0.01).Beelin-1 mRNA재ANP조적표체(107.12±29.10)교대조조(7.01±3.39)명현승고,차이유통계학의의(F=3.61,P＜0.05),단재ANP+뢰파매소조적표체(97.63±65.38)여ANP조비교차이무통계학의의(P＞0.05).LC3-Ⅱ mRNA재ANP조적표체(1.76±1.59)여대조조(1.51±0.95)상비차이무통계학의의(P＞0.05),단재ANP+뢰파매소조적표체(4.37±1.67)명현고우ANP조,차이유통계학의의(F=16.10,P＜0.05).전자현미경현시ANP조자서상관결구교대조조증가,ANP+뢰파매소조칙경다.ANP+뢰파매소조혈청TAP、IL-1、IL-6분별위(36.47±1.71) pmol/L、(122.88±26.67) pmol/L、(107.39±13.95) pg/mL,균명현고우ANP조(25.63±6.05) pg/mL、(98.06±9.29) pg/mL、(86.16±7.20) pg/mL,차이균유통계학의의(F=116.71、50.45、79.67,P균＜0.01);ANP+뢰파매소조TNFα(140.80±60.82) pg/mL여ANP조(105.23±6.95) pg/mL상비,차이무통계학의의(F=14.76,P＞0.05).결론 ANP대서자서증가,촉진자서가현저격활이단백매원,동시가중이선손상화염성반응,자서가능통과격활이단백매원삼여급성이선염적발생.
Objective To investigate the changes and significance of autophagy in rats with experimental acute necrosis pancreatitis (ANP).Methods According to method of random number,18 rats were randomly divided into control group,ANP group,ANP+rapamycin (RAP) group.The ANP rat model was established by intraperitoneal injection of 20％ L-arginine.The rats of ANP+RAP group were intraperitoneal injected with RAP 1.2 mg/kg at 30 minutes before modeling.The rats of control group were intraperitoneal injected with 0.9％ NaCl solution.The blood was drawed from the hearts nine hours after modeling for subsequent experiments.Serum levels of trypsinogen activation peptide (TAP),interleukin (IL-1),IL-6 and tumor necrosis factor (TNF) α were measured with enzyme-linked immunosorbent assay.The pancreatic tissues were pathologically scored.Autophagy-related structures in rat pancreatic acinar cells were observed by transmition electron microscopy.The expression of autophagy marker microtuble assciated protein 1 light chain 3 (LC3)-Ⅱ and Beclin-1 at mRNA and protein level were measured by quantitative real-time polymerase chain reaction (qRT-PCR),Western bloting and immunohistochemistry.The single factor analysis of variance was used for mean comparison among groups.Results A rat model of ANP was successfully established.Histopathological score of pancreas acinar cell necrosis of ANP+RAP group (2.19±1.38) was higher than that of ANP group (0.97±0.68),and the difference was statistically significant(F=33.75,P＜0.05).The results of Western blotting indicated that the protein expression of LC3-Ⅱ and Beclin-1 in ANP group (35.25±2.68 and 49.40±5.28)were higher than those in control group (1.54±0.16 and 0.78±0.06),furthermore the expressions in ANP+RAP group(123.53±3.21 and 76.41±3.80) were higher than those in ANP group,and the differences were statistically significant(F=2 045.54,326.87,both P＜0.01).Immunohistochemistry results also indicated that the LC3Ⅱ and Beclin-1 expression at protein level of ANP+RAP group (7 570.63±4 357.67 and 3 418.09±2 035.78) were higher than those of ANP group (1 926.53±1 414.44 and 536.11±403.10),and the differences were statistically significant (F=39.83,41.58,both P＜0.01).The expression of Beclin-1 at mRNA level of ANP group (107.12±29.10) was statistically higher than that of control group(7.01 ±3.39),and the difference was statistically significant (F=3.61,P＜0.05),but the expression of ANP+RAP group (97.63 ± 65.38)was no significant difference compared with ANP group.However,the expression of LC3-Ⅱ at mRNA level of ANP+ RAP group (4.37 ± 1.67) was statistically higher than that of ANP group (1.76 ± 1.59),and the difference was statistically significant(F=16.10,P＜0.05),but the expression of ANP group was no significant difference compared with control group (1.51 ±0.95).The result of electron microscopy showed that autophagy related structures increased in ANP group compared with that of control group,which of ANP+RAP group was more.The serum levels of TAP,IL-1 and IL-6 of ANP + RAP group were (36.47 ± 1.71) pmol/L,(122.88± 26.67) pg/mL and (107.39±13.95) pg/mL,which were all higher than those of ANP group ((25.63 ± 6.05) pmol/L,(98.06 ±9.29) pg/mL and (86.16± 7.20) pg/mL),and the differences were statistically significant (F=116.71,50.45,79.67; all P＜0.01).There was no significant difference in TNFα between ANP+ RAP group ((140.80±60.82) pg/mL) and ANP group ((105.23±6.95) pg/mL,F=14.76,P＞0.05).Conclusions Autophagy increased in rats with ANP.Promoting autophagy could significantly activate trypsinogen,aggravate pancreatic injury and increase inflammation reaction,which indicated that autophagy might involve in the pathogenesis of ANP through trypsinogen activation.
