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磷脂酰肌醇3-激酶γ对急性胰腺炎小鼠胰腺腺泡细胞自噬作用的影响

Influence of PI3K gamma on pancreatic acinar cells autophagy in experimental acute pancreatitis in mice

摘要:

目的 探讨磷脂酰肌醇3-激酶γ(PI3Kγ)对小鼠实验性急性胰腺炎胰腺腺泡细胞自噬作用的影响,并探讨其意义.方法 野生型C57 BL/6小鼠和PI3Kγ基因敲除小鼠各18只,按数字表法随机分为对照组(6只)和急性胰腺炎(AP)组(12只),采用蛙皮素50μg/kg体重腹腔内注射7次、每次间隔1h的方法制备AP模型.首次注射后7h处死小鼠,光镜下观察胰腺病理学变化,免疫荧光检测自噬泡主要组成蛋白LC3颗粒,荧光分光光度计测定胰蛋白酶活性,蛋白质印迹法检测自噬相关蛋白beclin1、LC3-Ⅱ和p62的表达.结果 AP组野生型小鼠和PI3Kγ基因敲除小鼠的胰腺自噬空泡数量分别为(5.14±0.85)、(2.25±0.54)个/每高倍视野(HPF),LC3荧光免疫颗粒数量分别为(78.6±9.38)、(26.4±4.21)个/HPF,胰蛋白酶活性分别为(0.827±0.126)、(0.358±0.098) pmol/mg蛋白,各组间差异均具有统计学意义(P值均<0.05).野生型小鼠的p62蛋白表达较PI3Kγ基因敲除小鼠显著减弱(0.11比0.92,P<0.05),面LC3 -Ⅱ、beclin1蛋白表达较PI3Kγ基因敲除小鼠显著增强(1.82比0.93,1.43比1.05,P值均<0.05).结论 AP时PI3Kγ可能通过增强小鼠胰腺腺泡细胞的自噬作用,促进胰蛋白酶原的活化及诱导腺泡细胞坏死.

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Objective To investigate the influence of phosphoinositide 3-Kinase-C2-gamma (PI3Kγ)on pancreas acinar cells autophagy in experimental acute pancreatitis in mice and explore its significance.Methods Eighteen C57BL/6 wild type (WT) and eighteen PI3Kγ knockout (KO) mice were randomly divided into control group (n =6) and acute panereatitis (AP) group (n =12),respectively.AP models were induced by intraperitoneal injection of 50 μg cerulein/kg body weight,once the other hour for seven times.The mice were sacrificed 7 hours after model induction.The pathological changes of the pancreas were observed through microscope,LC3 dots were determined by immunofluorescence,the trypsin activity was measured by fluorescence spectrophotometer,and the expression of autophagy related protein beclin1,p62 and LC3- Ⅱ were measured by Western blot.Results The autophagy vacuoles counts in pancreatic tissue of WT mice and KO mice were (5.14 ±0.85),(2.25 ±0.54)/HPF,the LC3 immunofluorescence dots counts were (78.6 ±9.38),( 26.4 ± 4.21 )/HPF,the trypsin activities were ( 0.827 ± 0.126 ),( 0.358 ± 0.098 ) pmol/mg protein,the difference between the two groups was statistically significant ( P < 0.05 ).The p62 protein expression was greatly decreased in WT mice compared with their KO counterpart (0.11 vs 0.92,P < 0.05 ),while the expressions of LC3 Ⅱ,beclin1 were greatly increased in WT mice compared with their KO counterpart ( 1.82 vs 0.93,1.43 vs 1.05,P < 0.05 ).Conclusions PI 3 Kγmay up- regulate autophagy of pancreatic acinar cells during acute pancreatitis in mice,then promote trypsinogen activation and necrosis of acinar cells.

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