B细胞淋巴瘤腺病毒干扰蛋白3/凋亡诱导因子信号通路介导的营养剥夺条件下髓核细胞凋亡的研究

The B-cell lymphoma 2/adenovirus E1B 19000-inter-acting protein 3/apoptosis induced factorsignalpathwayinduces apoptosis of nucleus pulposus cells during nutrient deprivation

目的探讨通过营养剥夺模拟体内退变环境下,髓核细胞凋亡的信号转导机制。方法体外培养大鼠尾椎间盘髓核细胞,传代至P1代后将细胞分为正常对照组:杜尔贝科改良培养基(10 mg葡萄糖/L)、10%胎牛血清、21%O2;实验组:杜尔伯科改良伊格尔(DMEM)无糖培养基、无血清、1%O2;分别将两组细胞培养0、24、48、72 h后,流式细胞仪检测细胞凋亡率及线粒体膜电位,蛋白质印迹法(Western blot)分别检测B细胞淋巴瘤腺病毒干扰蛋白3(BNIP3)、凋亡诱导因子(AIF)蛋白表达并定位,并分别通过过表达和抑制BNIP3蛋白后Western blot再次检测BNIP3、AIF蛋白表达及髓核细胞凋亡率。实验结果采用均值±标准差(Mean±SD)表示,应用SPSS 22.0软件单因素方差分析(One-way ANOVA)评估组间差异。结果实验组髓核细胞培养24 h后BNIP3蛋白表达明显增高,并同时伴有AIF蛋白核转位和细胞凋亡率增高[(2.07±0.23)%比(12.51±2.33)%、(32.33±4.12)%、(48.15±3.67)%,F=141.082,P<0.05],此外,过表达BNIP3蛋白后可明显加速AIF蛋白核转位和细胞凋亡率增高[(2.07±0.23)%比(50.67±3.01)%、(93.52±2.41)%、(95.83±1.20)%,F=1488.638,P<0.05],而抑制BNIP3蛋白和AIF蛋白表达后可明显降低髓核细胞凋亡率[(50.67±3.01)%、(93.52±2.41)%、(95.83±1.20)%比(8.63±0.67)%、(23.84±3.11)%、(34.22±2.92)%,F=559.631,P<0.05]。结论BNIP3/AIF信号转导通路介导了营养剥夺环境下髓核细胞凋亡。

Objective To detect the mechanism in apoptosis of nucleus pulposus cells induced by nutrient deprivation(ND).Methods Ratcaudal disc nucleus pulposus cells(NPs)were passaged to P1,divided into control group[culturedin dulbecco’s modified eagle medium(DMEM),21%O2,10%fatal bovine serum]and experiment group(culturedin DMEM with no glucose,noserum,1%O2).After cells were cultured for 0,24,48,72 h,apoptosis,mitochondrial membrane potential(Δψm),expression and location of B-cell lymphoma 2(bcl-2)/adenovirus E1B 19000-inter acting protein 3(BNIP3)and apoptosis induced factor(AIF)were detected.Meanwhile,we over expression and knock down the BNIP3 and AIF,then detected the apoptosis,Δψm,expression and location of BNIP3 and AIF.Results Western blotting demonstrated BNIP3 expression was significantly upregulated in NPs subjected to ND,which coincided with AIF translocation to nucleus and alterations in apoptosis[(2.07±0.23)%vs.(12.51±2.33)%,(32.33±4.12)%,(48.15±3.67)%,F=141.082,P<0.05].Furthermore,BNIP3 overexpression increased ND-induced NPs apoptosis[(2.07±0.23)%vs.(50.67±3.01)%,(93.52±2.41)%,(95.83±1.20%,F=1488.638,P<0.05],whereas knockdown of BNIP3 or AIF abolished ND-induced NPs apoptosis[(50.67±3.01)%,(93.52±2.41)%,(95.83±1.20)%vs.(8.63±0.67)%,(23.84±3.11)%,(34.22±2.92)%,F=559.631,P<0.05].Conclusion The apoptosis of NPs induced by ND via activation of the BNIP3/AIF signal pathway.