摘要
目的 探讨石英诱导的人胚肺成纤维细胞（HELF）中丝裂素活化蛋白激酶（MAPK）／细胞周期蛋白和细胞周期蛋白依赖激酶（cyclinD1-CDK4）信号转导通路的活化。方法 两种处理方式：（1）石英刺激细胞2h后，收获细胞；（2）石英长时间（2个月）作用于细胞，使细胞具有部分转化细胞的特征（S-HELF），检测信号蛋白因子和细胞周期的变化。分别采用Westernblot、免疫细胞化学和流式细胞术方法检测细胞内信号蛋白和细胞周期的改变。选用特异化学抑制剂或分子抑制剂抑制上游激酶，检测下游激酶的变化。结果 HELF暴露于石英粉尘2h后，可以导致MAPK家族中ERK1/2、p38和JNK1／2 3个亚家族的磷酸化水平升高。在SHELF中，只有细胞外调节蛋白激酶（ERK）和C4un氨基末端激酶[JNK1（p46）]较未处理的HELF磷酸化水平增高。而JNK2（p54）的磷酸化水平没有变化，p38的磷酸化水平反而下降。cyclinD1和CDK4蛋白在SHELF中较HELF中表达增多。抑制ERK和JNK活化或者抑制核转录因子活化蛋白1（AP-1）的活化后，S-HELF中cyclin D1和CDK4蛋白过表达得到控制。而抑制p38的活性不能改变cyclinD1和CDK4蛋白过表达。结论 石英刺激HELF2h后可诱导ERK、JNK和p38的活化，而SHELF中ERK、JNK活化，p38没有被活化。SHELF中，cyclinD1和CDK4蛋白质过表达与ERK、JNK和AP-1活化有关。
Objective To study the phosphorylation level ofmitogen activated protein kinase （MAPK） in human embryonic lungfibroblasts （HELF） , and the expression level ofcyclin D1-CDK4 protein in SHELF and whether the expression level of cyclin D1-CDK4 protein mediated by MAPK/AP-1 signaling pathway in SHELF. Methods Two kinds of treatment： （1） Cells were harvested after stimulation 2 h for the detection of cytokines. （2） Cells were stimulated by quartz for a long time （2 months） for transformation characters（S-HELF）. The MAP kinase was detected by western blot. Cyclin D1 and CDK4 （cyclin dependent kinase 4） proteins were measured by immunocytochemistry. Flow cytometry was used to evaluate the alternation of cell cycle. Results Crystalline quartz could cause the phosphorylation level of ERKs, p38K, and JNKs in HELF increase. However, activated levels of ERKs and p46 of JNKs increased in SHELF, and p38K activation decreased, and no effect on activation of p54 of JNKs, as compared with those in parental HELF. Cyclin D1 and CDK4 protein expression levels increased in S- HELF as compared with parental HELF. Inhibition of ERKs activation by AG126, AP-1 by curcumin, and JNKs by SP600125 could reduce the induction of cyclin D1 and CDK4, whereas inhibition of p38K by SB203580 did not show any inhibitory effects on SHELF. Conclusion The phosphorylation levels ofERK1/2, JNK1/2, and p38 in- crease in HELF exposed to quartr. The phosphorylation levels of ERK1/2 and JNK1 increase, but the phosphorylation level ofp38 decreases in SHELF. The expression level of cyclin D1-CDK4 protein decreases in SHELF. Overexpression of cyclin D1-CDK4 is due to the activation ofERKs, JNKs/AP-1 signaling pathway in S-HELF.
出处
《中华劳动卫生职业病杂志》
CAS
CSCD
北大核心
2007年第1期5-10,共6页
Chinese Journal of Industrial Hygiene and Occupational Diseases
基金
国家自然科学基金资助项目（30028019,30371206）,国家973计划项目（2002cB512905）及中科院百人计划和美国Philip Morris USA Ine and Philip Morris International资助
