细胞质内高表达的细胞周期蛋白依赖性激酶抑制蛋白P~(21)抑制人支气管上皮细胞凋亡
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摘要
目的研究细胞周期蛋白依赖性激酶抑制蛋白P~(21)抑制人支气管上皮细胞凋亡的机制。方法 (1)表达细胞周期蛋白依赖性激酶抑制蛋白P~(21)的质粒P~(EGFP-N1-p21)转染人支气管上皮细胞系16HBE,研究P~(21)蛋白在细胞质、细胞核内的表达变化与质粒转染后细胞凋亡表现之间的相互关系。实验分组包括空白对照、空质粒及P~(EGFP-N1-p21)质粒组,应用Westen-blot方法检测质粒转染24 h后P~(21)蛋白在细胞质、细胞核内的表达变化相对值,流式细胞仪方法检测质粒转染24 h、48 h后的细胞凋亡率。(2)转化生长因子-β_1(TGF-β_1)刺激人支气管上皮细胞系16HBE,研究TGF-β_1刺激后P~(21)蛋白在细胞质、细胞核内的表达变化与支气管上皮细胞凋亡之间的相互关系。实验分组包括TGF-β_10 ng/mL、TGF-β_13 ng/mL及TGF-β_110 ng/mL组,应用Westen-blot方法检测TGF-β_1刺激24 h后P21蛋白在细胞质、细胞核内的表达变化,流式细胞仪方法检测TGF-β_1刺激12 h、24 h后的细胞凋亡率。结果质粒P~(EGFP-N1-p21)转染人支气管上皮细胞系16HBE 24 h后,P~(EGFP-N1-p21)质粒组的P~(21)蛋白在细胞质内表达水平高于空质粒组、空白对照组,并高于同组细胞核内,差异均有统计学意义(P<0.05);空质粒组、空白对照组的细胞质内P~(21)蛋白表达水平低于细胞核内,差异均有统计学意义(P<0.05)。P~(EGFP-N1-p21)质粒转染24 h、48 h后,P~(EGFP-N1-p21)质粒组凋亡率低于空质粒组及空白对照组,差异均有统计学意义(P<0.05)。P~(EGFP-N1-p21)质粒转染24 h后的16HBE细胞凋亡率高于转染48 h后,而空质粒组和空白对照组表现相反结果。P~(EGFP-N1-p21)质粒转染后,P~(21)蛋白在细胞质内表达与转染后的细胞凋亡呈负相关。TGF-β_1(0 ng/mL、3 ng/mL、10 ng/mL)作用于16HBE细胞24 h后,TGF-β_13 ng/mL及10 ng/mL组的细胞核、细胞质内P~(21)蛋白表达均高于TGF-β_10 ng/mL组,差异均具有统计学意义(P<0.05),TGF-β_13 ng/mL及10 ng/mL组的细胞质中P~(21)蛋白表达均高于细胞核内P~(21)蛋白(P均<0.05),10 ng/mL组胞质P~(21)蛋白表达量低于3 ng/mL组(P<0.05)。TGF-β_1(0 ng/mL、3 ng/mL、10 ng/mL)作用于16HBE细胞12h、24 h后,同一作用时间下,随着TGF-β_1刺激浓度增加,细胞凋亡率增加,TGF-β_1(3 ng/mL、10 ng/mL)作用于16HBE细胞12 h、24 h后,同一作用浓度下,随着刺激时间延长,细胞凋亡率增加(P均<0.05)。TGF-β_1作用于人支气管上皮细胞系16HBE后,细胞质内P~(21)蛋白增加与刺激后的细胞凋亡呈负相关。结论 P~(EGFP-N1-p21)质粒转染人支气管上皮细胞后,P~(21)蛋白表达于细胞质,抑制人支气管上皮细胞凋亡。TGF-β_1作用于人支气管上皮细胞系16HBE后,刺激细胞质内P~(21)蛋白表达增加,随着细胞质内P~(21)蛋白表达下降,细胞凋亡增加。细胞周期蛋白依赖性激酶抑制蛋白P~(21)可通过在细胞质内高表达这一机制抑制人支气管上皮细胞凋亡。
ObjectiveTo investigate the mechanisms of cyclin-dependent kinase inhibitor P~(21)inhibiting the apoptosis of human bronchial epithelial cells(HBECs).Methods The relationship of the expression of cytoplasm and nuclear cyclin-dependent kinase inhibitor P~(21)with the apoptosis of 16HBE cells was studied after the plasmid P~(EGFP-N1-p21)was transfected into the 16HBE cells,and the experiment included the blank,the empty plasmid and the P~(EGFP-N1-p21)group.The expression of P~(21)protein in cytoplasm and nucleus was detected by Western-blot after 24 hours of plasmid transfection.The apoptosis rate was detected by flow cytometry after 24 hours and 48 hours of plasmid transfection.The relationship of the expression of cytoplasmic and nuclear cyclin-dependent kinase inhibitor P~(21)with the apoptosis of 16HBE cells was studied after the stimuli of transforming growth factor-β_1(TGF-β_1)to 16HBE cells,and the experiment included TGF-β_10 ng/mL,TGF-β_13 ng/mL,and TGF-β_110 ng/mL group.The expression of P~(21)protein in cytoplasm and nucleus was detected by Western-blot after 24 hours of the stimuli of TGF-β_1.The apoptosis rate was detected by flow cytometry after 12 hours and 24 hours of the stimuli of TGF-β_1.Results After the plasmid P~(EGFP-N1-p21)was transfected into16HBE cells for 24 h,the cytoplasmic P~(21)expression in the P~(EGFP-N1-p21)group was higher than the nucleic P~(21)expression,which was also significantly higher than the cytoplasmic P~(21)expressions of the empty plasmid group and the blank group(P<0.05).Compared with the cytoplasmic P~(21)expression,the nucleic P~(21)expressions of the empty plasmid group and the blank group were higher(P<0.05).After the plasmid P~(EGFP-N1-p21)was transfected for 24 hours and 48 hours,the apoptosis rate of 16HBE cells were significantly lower than the blank plasmid group and the control group(P<0.05).The apoptosis rate of 16HBE cells of 24 hours transfection in the plasmid P~(EGFP-N1-p21)group was significantly higher than that of 48hours transfection(P<0.05),while the apoptosis rates of 16HBE cells of 24 hours transfection in the empty plasmid group and the blank group were significantly lower than those of 48 hours transfection(P<0.05).After the plasmid P~(EGFP-N1-p21)transfected into 16HBE cells,the cytoplasmic P~(21)expressions were negatively related with the apoptosis of the transfected 16HBE cells.After the stimuli with TGF-β_1(3 ng/mL,10 ng/mL)for 24 hours,both the cytoplasmic P~(21)expressions and the nucleic P~(21)expressions increased compared with the TGF-β_10 ng/mL group(P<0.05),while the cytoplasmic P~(21)expressions was higher than the nucleic P~(21)expressions(P<0.05).After the stimuli with TGF-β_1(3 ng/mL,10 ng/mL)for 24 hours,the cytoplasmic P~(21)expressions of the 3 ng/mL group were higher than that of 10 ng/mL group(P<0.05).After the stimuli with TGF-β_1(0 ng/mL,3 ng/mL,10 ng/mL)for 12 hours and 24 hours,the apoptosis of16HBE cells increased with the growing concentrations of the stimuli of TGF-β_1at the same timeof stimuli(P<0.05);the apoptosis of 16HBE cells increased with the growing time of the stimuli of TGF-β_1at the same concentrations of stimuli of TGF-β_1(P<0.05).After the stimuli with TGF-β_1,the cytoplasmic P~(21)expressions were negatively related with the apoptosis of the transfected 16HBE cells.Conclusion After the plasmid P~(EGFP-N1-p21)was transfected into 16HBE cells,the P~(21)expression of the plasmid P~(EGFP-N1-p21)was in the cytoplasm.As the times of the transfection growing,the apoptosis of 16HBE cells decreased.The cytoplasmic P~(21)expressions inhibited the apoptosis of HBECs.After the stimuli with TGF-β_1,the cytoplasmic P~(21)expressions decreased withthe concentration of the stimuli of TGF-β_1,but the apoptosis of HBECs increased.The cytoplasmic P~(21)expression inhibited the apoptosis of HBECs
ObjectiveTo investigate the mechanisms of cyclin-dependent kinase inhibitor P~(21)inhibiting the apoptosis of human bronchial epithelial cells(HBECs).Methods The relationship of the expression of cytoplasm and nuclear cyclin-dependent kinase inhibitor P~(21)with the apoptosis of 16HBE cells was studied after the plasmid P~(EGFP-N1-p21)was transfected into the 16HBE cells,and the experiment included the blank,the empty plasmid and the P~(EGFP-N1-p21)group.The expression of P~(21)protein in cytoplasm and nucleus was detected by Western-blot after 24 hours of plasmid transfection.The apoptosis rate was detected by flow cytometry after 24 hours and 48 hours of plasmid transfection.The relationship of the expression of cytoplasmic and nuclear cyclin-dependent kinase inhibitor P~(21)with the apoptosis of 16HBE cells was studied after the stimuli of transforming growth factor-β_1(TGF-β_1)to 16HBE cells,and the experiment included TGF-β_10 ng/mL,TGF-β_13 ng/mL,and TGF-β_110 ng/mL group.The expression of P~(21)protein in cytoplasm and nucleus was detected by Western-blot after 24 hours of the stimuli of TGF-β_1.The apoptosis rate was detected by flow cytometry after 12 hours and 24 hours of the stimuli of TGF-β_1.Results After the plasmid P~(EGFP-N1-p21)was transfected into16HBE cells for 24 h,the cytoplasmic P~(21)expression in the P~(EGFP-N1-p21)group was higher than the nucleic P~(21)expression,which was also significantly higher than the cytoplasmic P~(21)expressions of the empty plasmid group and the blank group(P<0.05).Compared with the cytoplasmic P~(21)expression,the nucleic P~(21)expressions of the empty plasmid group and the blank group were higher(P<0.05).After the plasmid P~(EGFP-N1-p21)was transfected for 24 hours and 48 hours,the apoptosis rate of 16HBE cells were significantly lower than the blank plasmid group and the control group(P<0.05).The apoptosis rate of 16HBE cells of 24 hours transfection in the plasmid P~(EGFP-N1-p21)group was significantly higher than that of 48hours transfection(P<0.05),while the apoptosis rates of 16HBE cells of 24 hours transfection in the empty plasmid group and the blank group were significantly lower than those of 48 hours transfection(P<0.05).After the plasmid P~(EGFP-N1-p21)transfected into 16HBE cells,the cytoplasmic P~(21)expressions were negatively related with the apoptosis of the transfected 16HBE cells.After the stimuli with TGF-β_1(3 ng/mL,10 ng/mL)for 24 hours,both the cytoplasmic P~(21)expressions and the nucleic P~(21)expressions increased compared with the TGF-β_10 ng/mL group(P<0.05),while the cytoplasmic P~(21)expressions was higher than the nucleic P~(21)expressions(P<0.05).After the stimuli with TGF-β_1(3 ng/mL,10 ng/mL)for 24 hours,the cytoplasmic P~(21)expressions of the 3 ng/mL group were higher than that of 10 ng/mL group(P<0.05).After the stimuli with TGF-β_1(0 ng/mL,3 ng/mL,10 ng/mL)for 12 hours and 24 hours,the apoptosis of16HBE cells increased with the growing concentrations of the stimuli of TGF-β_1at the same timeof stimuli(P<0.05);the apoptosis of 16HBE cells increased with the growing time of the stimuli of TGF-β_1at the same concentrations of stimuli of TGF-β_1(P<0.05).After the stimuli with TGF-β_1,the cytoplasmic P~(21)expressions were negatively related with the apoptosis of the transfected 16HBE cells.Conclusion After the plasmid P~(EGFP-N1-p21)was transfected into 16HBE cells,the P~(21)expression of the plasmid P~(EGFP-N1-p21)was in the cytoplasm.As the times of the transfection growing,the apoptosis of 16HBE cells decreased.The cytoplasmic P~(21)expressions inhibited the apoptosis of HBECs.After the stimuli with TGF-β_1,the cytoplasmic P~(21)expressions decreased withthe concentration of the stimuli of TGF-β_1,but the apoptosis of HBECs increased.The cytoplasmic P~(21)expression inhibited the apoptosis of HBECs
引文
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[2]Yao H,Sundar I,Gorbunova V,et al.p21-PRAP-1 pathway is involved in cigarette smoke-induced lung DNA damage and cellular senescence[J].PLoS One,2013,8(11):e80007.
[3]Yamasaki M,Kang H,Homer R,et al.p21 regulates tgf-β1-induced pulmonary responses via a TNF-αsignaling pathway[J].American Journal of Respiratory Cell and Molecular Biology,2008,38(3):346-353.
[4]Tomita K,Caramori G,Lim S,et al.Increased p21cip1/waf1 and b cell lymphoma leukemia-xl expression and reduced apoptosis in alveolar macrophages from smokers[J].American Journal of Respiratory and Critical Care Medicine,2002,166(5):724-731.
[5]Puddicombe S,Carlos T,Richter A.Increased expression of p21waf cyclin-dependent kinase inhibitor in asthmatic bronchial epithelium[J].American Journal of Respiratory Cell and Molecular Biology,2003,28(5):61-68.
[6]Child E,Mann D.The intricacies of phosphorylation:protein/protein interactions sub-cellular localization and stability[J].Cell Cycle,2006,5(12):1313-1319.
[7]Chen A,Huang X,Xue Z,et al.The role of p21 in apoptosis,proliferation,cell cycle arrest,and antioxidant activity in UVB-irradiated human HaCaT keratinocytes[J].Med Sci Monit Basic Res,2015,21(4):86-95.