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。
Objective:To investigate the effect of down-regulating the PKR gene on the proliferation and apoptosis of pancreatic AR42 J cells and its mechanism.Methods:AR42 J 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 transfected 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 AR42 J cells.
作者
李勇
权刚
李孝琼
肖云峰
吴妮莎
徐黎明
周国俊
李建水
冷政伟
Li Yong;Quan Gang;Li Xiaoqiong;Xiao Yunfeng;Wu Nisha;Xu Liming;Zhou Guojun;Li Jianshui;Leng Zhengwei(Department of Hepatobiliary SurgeryⅡ,Affiliated Hospital of North Sichuan Medical College)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2019年第6期734-739,共6页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:81402444)
四川省科技厅资助项目(编号:2017JY0170、2018JY0489)
