重组质粒pEGFP-N2/Pim-3转染对胰腺细胞损伤的保护作用

Protective role of recombinant plasmid pEGFP-N2/Pim-3 transfecting in rat pancreatic acinar cells damaged by lipopolysaccharide

目的 :研究重组质粒p EGFP-N2/Pim-3转染对内毒素脂多糖(lipopolysaccharide,LPS)引起的大鼠胰腺AR4-2J细胞损伤的保护机制。方法:实验分为4组:A组(正常对照组),B组(5μg/m L LPS处理组),C组(空载质粒p EGFP-N2转染+5μg/m L LPS处理组),D组(重组质粒p EGFP-N2/Pim-3转染+5μg/m L LPS处理组)。流式细胞仪检测各组处理24 h后细胞凋亡情况。处理0、6、12、24 h后分别提取各组总RNA及蛋白,RT-PCR及Western blot分别检测Pim-3、细胞间黏附分子(intercellular cell adhesion molecule-1,ICAM-1)、紧密连接蛋白闭锁蛋白(Occludin)m RNA及蛋白的表达。结果 :处理24 h,各组细胞凋亡率分别为:A组(7.85±1.14)%、B组(53.13±5.73)%、C组(51.76±5.17)%、D组(21.13±4.15)%,D组细胞相对B、C组凋亡明显减少,差异有统计学意义(P<0.05);D组Pim-3表达与A、B、C组之间的差异有统计学意义(P<0.05);处理12 h后B、C、D组ICAM-1高表达,24 h达高峰,相对于A组,差异均有统计学意义(P<0.01),D组与B、C组差异有统计学意义(P<0.05);处理6 h后,B、C、D组Occludin高表达,12 h达高峰,相对于A组,差异均有统计学意义(P<0.01),D组与B、C组差异有统计学意义(P<0.05)。结论:丝/苏氨酸激酶Pim-3能够抑制胰腺细胞炎症反应和胰腺细胞凋亡,可能与上调闭锁蛋白和下调细胞间黏附分子的表达有关。

Objective：To explore the protective role of recombinant plasmid pEGFP-N2/Pim-3 transfecting in rat pancreatic acinar cells AR4-2J damaged by lipopolysaccharide （LPS）. Methods： AR4-2J cells were divided into 4 groups： Group A, as the normal control group; Group B, treated with 5 μg/mL of LPS; Group C, transfected with blank plasmid pEGFP-N2 and treated with 5 ？滋g/mL of LPS; Group D, transfected with recombinant plasmid pEGFP-N2 /Pim-3 and treated with 5 μg/mL of LPS. Flow cytometry examined the apoptosis of AR4-2J cells in four groups after 24 h. RT-PCR was performed to detect the mRNA expression of Pim-3, intercellular cell adhesion molecule-1 （ICAM-1）, and Occludin in AR4-2J cells of four groups treated in 0, 6, 12, 24 h, and Western blot was performed to detect the protein expression of Pim-3, ICAM-1, and Occludin in each group. Results： The apoptotic rates of Groups B and C were （53.13 ± 5.73）% and （51.76 ± 5.17）%, respectively, both significantly higher than that of Group D （21.13 ± 4.15）%（both P 〈 0.05）. The expressions of Pim-3 mRNA and protein in Groups A, B and C remained a low level. The Pim-3 expression of Group D was significantly higher than those in Groups A, B and C （both P 〈 0.05）. The ICAM-1 mRNA and protein expression levels of Groups B, C and D were constantly upregulated 6 h later, all significantly higher than those of Group A （all P 〈 0.01）, and the ICAM-1 expressions of Groups B and C were significantly higher than Group D （both P 〈 0.05）. The Occludin mRNA and protein expression levels of Groups B, C and D began to increase 6 h later, and peaked 12 h later, all significantly higher than those of Group A （all P 〈 0.01）, and the Occludin expressions of Groups B and C were significantly lower than that of Group D （both P 〈 0.05）. Conclusion： Pim-3 gene can inhibit the apoptosis of pancreatic acinar cell damaged by LPS, and also can upregulate the expression of Occludin and downregulate the expression of ICAM-1 to inhibit the pancreatic inflammatory reaction induced by LPS.