煤焦沥青烟提取物致BEAS-2B恶性转化细胞中心体及其调控蛋白的变化

Changes centrosome and related protein in malignant transformation of BEAS-2B cell induced by coal tar pitch smoke extracts

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.

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.