中华消化杂志
中華消化雜誌
중화소화잡지
Chinese Journal of Digestion
2014年
11期
752-755
,共4页
李洁%刘晓%吴敏%郭晓榕%湛先保
李潔%劉曉%吳敏%郭曉榕%湛先保
리길%류효%오민%곽효용%담선보
急性胰腺炎%自噬%胰蛋白酶原
急性胰腺炎%自噬%胰蛋白酶原
급성이선염%자서%이단백매원
Acute pancreatitis%Autophagy%Trypsinogen
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.
目的 觀察雨蛙肽誘導的實驗性急性胰腺炎(AP)細胞模型中酶噬的變化.方法 培養AR42J細胞株(胰腺外分泌細胞),將細胞接種于6孔闆培養至90％融閤,分為AP組與空白對照組,AP組加入終濃度為1×10-8 mol/L的雨蛙肽建立AP細胞模型,空白對照組加入1640培養液.在雨蛙肽處理後1、4、6、8、12、24 h收集細胞及培養上清液,以ELISA方法檢測上清液中炎性因子IL-1、TNFα、澱粉酶及胰蛋白酶原活化肽(TAP)水平.取各組細胞,採用RT-PCR和Western印跡法檢測LC3、Beclin-1mRNA和 LC3B蛋白的錶達.透射電子顯微鏡觀察各組細胞酶噬小體的變化.組間比較採用方差分析.結果 空白對照組上清液中的IL-1、TNFα、澱粉酶和TAP分彆為(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組在1h時即顯著升高至(62.13±11.25) pg/mL,(30.98±7.11) pg/mL、(25.06±6.82) U/L、(128.51±18.30) pg/mL,差異均有統計學意義(F=3.32、3.05、2.90、2.62,P＜0.05或0.01)在4或6h時達到高峰[IL-1在4h為(71.96±15.82) pg/mL,F=7.25,P＜0.01;TNFα在6h時為(39.92±8.94) pg/mL,F=4.93,P＜0.05;澱粉酶在4h時為(28.83±8.31) U/L,F=2.06,P＜0.05;TAP在4h時為(146.29±29.36) pg/mL,F=0.14,P＜0.01],之後逐漸下降趨于穩定,AP組細胞中第4、6h的LC3 mRNA含量為3.18±0.82、1.71±0.14,第1、4h時的Beclin-1 mRNA含量為2.44±0.34、4.13±0.30,較空白對照組(0.21±0.04、0.30±0.08)均明顯上升(LC3 mRNA,F=0.79、0.06 ;Beclin mRNA,F=2.31、0.36,P均＜0.05),其餘時間點差異均無統計學意義(P均＞0.05).電子顯微鏡下可見AP組自噬體和酶噬小體數目顯著多于空白對照組.結論 雨蛙肽誘導AP細胞模型時伴有酶噬的髮生,提示酶噬可能參與瞭AP的髮病機製.
목적 관찰우와태유도적실험성급성이선염(AP)세포모형중매서적변화.방법 배양AR42J세포주(이선외분비세포),장세포접충우6공판배양지90％융합,분위AP조여공백대조조,AP조가입종농도위1×10-8 mol/L적우와태건립AP세포모형,공백대조조가입1640배양액.재우와태처리후1、4、6、8、12、24 h수집세포급배양상청액,이ELISA방법검측상청액중염성인자IL-1、TNFα、정분매급이단백매원활화태(TAP)수평.취각조세포,채용RT-PCR화Western인적법검측LC3、Beclin-1mRNA화 LC3B단백적표체.투사전자현미경관찰각조세포매서소체적변화.조간비교채용방차분석.결과 공백대조조상청액중적IL-1、TNFα、정분매화TAP분별위(18.83±7.10) pg/mL、(14.20±3.79) pg/mL、(10.03±2.85)U/L、(39.48±8.62) pg/mL,AP조재1h시즉현저승고지(62.13±11.25) pg/mL,(30.98±7.11) pg/mL、(25.06±6.82) U/L、(128.51±18.30) pg/mL,차이균유통계학의의(F=3.32、3.05、2.90、2.62,P＜0.05혹0.01)재4혹6h시체도고봉[IL-1재4h위(71.96±15.82) pg/mL,F=7.25,P＜0.01;TNFα재6h시위(39.92±8.94) pg/mL,F=4.93,P＜0.05;정분매재4h시위(28.83±8.31) U/L,F=2.06,P＜0.05;TAP재4h시위(146.29±29.36) pg/mL,F=0.14,P＜0.01],지후축점하강추우은정,AP조세포중제4、6h적LC3 mRNA함량위3.18±0.82、1.71±0.14,제1、4h시적Beclin-1 mRNA함량위2.44±0.34、4.13±0.30,교공백대조조(0.21±0.04、0.30±0.08)균명현상승(LC3 mRNA,F=0.79、0.06 ;Beclin mRNA,F=2.31、0.36,P균＜0.05),기여시간점차이균무통계학의의(P균＞0.05).전자현미경하가견AP조자서체화매서소체수목현저다우공백대조조.결론 우와태유도AP세포모형시반유매서적발생,제시매서가능삼여료AP적발병궤제.
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 2.44±0.34 and 4.13±0.30,all of them were significantly increased compared with those of control group (0.21±0.04 and 0.30±0.08,LC3 mRNA F=0.79、0.06; Beclin mRNA F=2.31、0.36,all P＜ 0.05).There were no significant differences at other time points.The numbers of autophagosome and zymophagosome of AP group were significantly higher than those of control group under transmission electron microscopy.Conclusion Zymophagy occurred during AP cell model induced by caerulein,which suggested that zymophagy might involve in the mechanism of AP.
