下调PKR基因对胰腺细胞AR42J增殖、凋亡的影响及机制

Effect of down-regulating the PKR gene on the proliferation and apoptosis of pancreatic AR42J cells and its mechanism

摘要:

目的：探讨下调PKR基因对大鼠胰腺腺泡细胞AR42J增殖、凋亡的影响及机制。方法：利用雨蛙素作用AR42J细胞建立急性胰腺炎细胞模型，在细胞模型中通过慢病毒载体下调PKR基因为实验组，对照病毒转染组为对照组。平板克隆形成和CCK-8法检测细胞增殖情况，流式细胞术检测细胞凋亡情况，qRT-PCR及流式细胞术分别检测核因子-κB（nuclear factor-κB，NF-κB）通路关键基因表达变化。结果：相较于对照组，实验组中NF-κB通路关键基因NF-κB、肿瘤坏死因了-α（tumor necrosis factor-α，TNF-α）、白介素-6（interleukin-6，IL-6）、iNOS相对表达量为0.45±0.08、0.26±0.05、0.51±0.25、0.38±0.04，相对表达下降（t=7.64，t=16.41，t=6.67，t=10.15，均P＜0.05）；实验组细胞克隆数为（275.33±7.02）个，对照组克隆数为（36.00±4.00）个，克隆形成能力明显增强（t=51.29，P=0.00）；CCK-8数据：在24 h、48 h、72 h，实验组细胞增殖率为（25.00±3.00）%、（50.33±4.04）%、（62.33±4.51）%，对照组为（12.33±1.53）%、（17.00±1.00）%、（24.00±2.00）%，实验组增殖能力明显增强（t=6.52，t=13.87，t=13.46，均P＜0.05）。实验组和对照组中细胞早期凋亡率分别为（6.2±0.6）%和（13.4±0.9）%，实验组凋亡明显减少（t=11.53，P=0.00）；晚期凋亡率分别为（4.1±0.2）%和（12.2±0.5）%，实验组凋亡明显减少（t=26.05，P=0.00）。结论：下调PKR基因可以抑制NF-κB通路减缓细胞炎症反应，致AR42J细胞凋亡减少、增殖能力增强。

Abstract:

Objective：To investigate the effect of down-regulating the PKR gene on the proliferation and apoptosis of pancreatic AR42J cells and its mechanism. Methods：AR42J cells were treated with cerulein to establish an acute pancreatitis model，in which the cells with the down-regulated PKR gene mediated by a lentiviral vector were taken as experimental group，and the cells trans－fected with control virus were taken as control group. The cells were tested for proliferation by cell counting kit（CCK-8） and colony formation assay and for apoptosis by flow cytometry（FCM）；the changes in the expression of key genes of the nuclear factor-κB（NF-κB） pathway were measured by both qRT-PCR and FCM. Results：Compared with the control group，the experimental group had significantly reduced relative expression of key genes[NF-κB，tumor necrosis factor-α（TNF-α），interleukin-6（IL-6），and iNOS] of the NF-κB pathway（0.45±0.08，0.26±0.05，0.51±0.25，and 0.38±0.04，respectively，t=7.64，16.41，6.67，and 10.15，respectively，all P<0.05）；the experimental group had significantly enhanced colony formation ability than the control group（colony count：275.33±7.02 vs. 36.00±4.00，t=51.29，P=0.00）；the result of CCK-8 assay showed a significantly enhanced cell proliferation ability in the experimental group compared with the control group，with the cell proliferation rates at 24 h，48 h，and 72 h as （25.00±3.00）%，（50.33±4.04），and （62.33±4.51）%，respectively，for the experimental group versus（12.33±1.53）%，（17.00±1.00）%，and （24.00±2.00）%，respectively，for the control group（t=6.52，13.87，and 13.46，respectively，all P<0.05）. Compared with the control group，the experimental group had significantly reduced values in both early apoptosis rate[（13.4±0.9）% vs. （6.2±0.6）%，t=11.53，P=0.00] and late apoptosis rate[（12.2±0.5）% vs. （4.1±0.2）％，t=26.05，P=0.00]. Conclusion：Down-regulating the PKR gene can slow down the cellular inflammatory response by suppressing the NF-κB pathway，leading to reduced apoptosis and enhanced proliferation of AR42J cells.