CAJ | 학술논문

目的分析煤焦沥青烟提取物诱导人支气管上皮细胞BEAS-2B恶变过程中细胞内中心体及其调控蛋白的变化,探讨中心体在煤焦沥青烟提取物致肺癌中的作用及机制.方法用中温煤焦沥青烟提取物作为诱导剂,建立永生化人支气管上皮细胞BEAS-2B的恶性转化模型,作为煤焦沥青烟提取物染毒组(煤焦沥青组),设溶剂对照组和正常对照组平行传代培养,用细胞爬片间接免疫荧光法检测中心体改变,用实时定量反转录聚合酶链式反应(RT-PCR)法检测相关基因的mRNA表达;细胞爬片免疫组化半定量法检测相关蛋白表达.结果煤焦沥青组第20代细胞中心体总异常率为6.56％±1.01％,明显高于正常对照组(3.40％±0.86％)和溶剂对照组(3.14％±0.59％),差异有统计学意义(P＜0.05).与正常对照组(4.34％±1.04％)和溶剂对照组(4.33％±1.20％)比较,煤焦沥青组第30代细胞中心体总异常比例(22.39％±9.5％)明显增大,差异有统计学意义(P＜0.05).煤焦沥青组第20、30代细胞的P53、P21基因和蛋白表达明显降低,Cyclin E基因和蛋白表达明显升高,与正常对照组相和溶剂对照组比较,差异均有统计学意义(P＜0.05).结论在煤焦沥青烟提取物诱导BEAS-2B细胞恶性转化过程中,中心体异常出现在细胞恶性改变前.中心体异常可能是通过P53/P2 1/CyclinE通路异常而引起.
목적 분석매초력청연제취물유도인지기관상피세포BEAS-2B악변과정중세포내중심체급기조공단백적변화,탐토중심체재매초력청연제취물치폐암중적작용급궤제.방법 용중온매초력청연제취물작위유도제,건립영생화인지기관상피세포BEAS-2B적악성전화모형,작위매초력청연제취물염독조(매초력청조),설용제대조조화정상대조조평행전대배양,용세포파편간접면역형광법검측중심체개변,용실시정량반전록취합매련식반응(RT-PCR)법검측상관기인적mRNA표체;세포파편면역조화반정량법검측상관단백표체.결과 매초력청조제20대세포중심체총이상솔위6.56％±1.01％,명현고우정상대조조(3.40％±0.86％)화용제대조조(3.14％±0.59％),차이유통계학의의(P＜0.05).여정상대조조(4.34％±1.04％)화용제대조조(4.33％±1.20％)비교,매초력청조제30대세포중심체총이상비례(22.39％±9.5％)명현증대,차이유통계학의의(P＜0.05).매초력청조제20、30대세포적P53、P21기인화단백표체명현강저,Cyclin E기인화단백표체명현승고,여정상대조조상화용제대조조비교,차이균유통계학의의(P＜0.05).결론 재매초력청연제취물유도BEAS-2B세포악성전화과정중,중심체이상출현재세포악성개변전.중심체이상가능시통과P53/P2 1/CyclinE통로이상이인기.
Objective To analyze the centrosome abnormalities in the malignant transformation of human bronchial epithelial cells (BEAS-2B) induced by coal tar pitch smoke extracts and to investigate the role and action mechanism of centrosome in the lung cancer induced by coal tar pitch.Methods Mediumtemperature coal tar pitch smoke extracts were used to treat immortalized human bronchial epithelial cells (BEAS-2B) and establish a malignant transformation model.The treated BEAS-2B cells were used as exposure group,and solvent control group and normal control group were also set for passage culture.The changes of centrosome in BEAS-2B cells seeded on coverslips were evaluated by indirect immunofluorescence assay.The mRNA expression of p53,p21,and cyclin E in BEAS-2B cells was measured by real-time quantitative RT-PCR,and their protein levels in BEAS-2B cells seeded on coverslips were measured by semiquantitative immunohistochemical analysis.Results The overall rate of centrosome abnormalities in BEAS-2B cells at passage 20 was 6.56±1.01％ in the exposure group,significantly higher than those in the normal control group (3.40±0.86％) and solvent control group (3.14±0.59％) (P＜0.05).In addition,the exposure group had a significantly higher overall rate of centrosome abnormalities in BEAS-2B cells at passage 30 compared with the normal control group and solvent control group (22.39±9.5％ vs 4.34±1.04％,P＜0.05; 22.39±9.5％ vs 4.33± 1.20％,P＜0.05).Compared with the normal control group and solvent control group,the exposure group had significantly decreased mRNA and protein expression of p53 and significantly increased mRNA and protein expression of cyclin E in BEAS-2B cells at passages 20 and 30 (P＜0.05).Conclusion Centrosome abnormalities occur before the malignant transformation in BEAS-2B cells treated with coal tar pitch smoke extracts,and they may be mediated by the p53/p21/cyclin E signaling pathway.