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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。
AIM: To investigate the protective mechanism of recombinant plasmid p EGFP-N2 / Pim-3 on the injury of rat AR4-2J cells induced by lipopolysaccharide (LPS). Methods: The experiment was divided into 4 groups: group A (normal control group), group B (5μg / m L LPS treatment group), group C (empty plasmid p EGFP-N2 transfection + 5μg / m L LPS treatment group) Group D (recombinant plasmid p EGFP-N2 / Pim-3 transfected + 5μg / m L LPS treatment group). Flow cytometry was used to detect the apoptosis of cells in each group for 24 h. The total RNA and protein of each group were extracted at 0, 6, 12 and 24 hours after treatment. The expressions of Pim-3, ICAM-1, ICAM-1 were detected by RT- Occludin m RNA and protein expression. Results: The apoptotic rate of each group was 7.85 ± 1.14% in group A, 53.13 ± 5.73% in group B, 51.76 ± 5.17% in group C, 21.13 ± 4.15% in group D, (P <0.05). There was significant difference between group D and group A, B and C (P <0.05) ). After treatment for 12 h, the expression of ICAM-1 in B, C and D groups reached a peak at 24 h, which was significantly higher than that in A group (P <0.01). There was statistical difference between D group and B and C groups (P <0.05). After 6 h of treatment, the expression of Occludin in groups B, C and D was high and reached a peak at 12 h, which was significantly different from that in group A (P <0.01) There was significant difference between group C and group C (P <0.05). CONCLUSION: Pim-3, a serine / threonine kinase, can inhibit pancreatic cell inflammatory response and pancreatic cell apoptosis, which may be related to the upregulation of laminin and down-regulation of the expression of intercellular adhesion molecule.