蛋白激酶Cα-胞外调节蛋白激酶1／2与人气道平滑肌细胞中周期蛋白D1及P21^cip1表达的关系  

PKCα-ERK1/2 cascade is involved in up-regulation of cyclinD1 and P21eip1 in human airway smooth muscle cells sensitized by sera from atopic asthmatics

作　　者：杜春玲[1] 徐永健[1] 刘先胜[1] 谢俊刚[1] 张珍祥[1] 张建[1] 乔礼芬[1] 倪望[1] 陈士新 

机构地区：[1]华中科技大学同济医学院附属同济医院呼吸内科卫生部呼吸疾病重点实验室,武汉430030

出　　处：《中华结核和呼吸杂志》2008年第12期915-920,共6页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：国家自然科学基金资助项目（30670925）

摘　　要：目的探究蛋白激酶C（PKC）α-胞外调节蛋白激酶（ERK）1／2级联对支气管哮喘（简称哮喘）患者血清被动致敏的人气道平滑肌细胞（HASMC）周期蛋白D1（cyclinD1）、周期蛋白依赖性激酶（CDK）抑制剂P21^cip1的调节作用及细胞增殖的影响。方法用含10％哮喘患者血清的DMEM培养基被动致敏HASMC后，按随机数字表法分为对照组、12-肉豆蔻酰-13-乙酸佛波酯（PMA）处理组、PMA±PKCα错配寡核苷酸组、PMA±PKCα反义寡核苷酸组以及PMA±U0126组，每组4个样本。用Westernblot方法检测各组细胞磷酸化PKCα（p-PKCα）、ERK1／2、磷酸化ERK1／2（p-ERK1／2）、cyclinDl和P21eipI的蛋白表达水平，用流式细胞术和四甲基偶氮唑盐（MTT）法检测HASMC增殖。结果PMA处理组p-PKCα水平、ERK1／2和p-ERKl／2蛋白水平高于对照组，cyclinD1、P21^rip1表达明显强于对照组（相对于各对照组的A值分别为2．10±0．29、1．67±0．19、2．20±0．27、1．99±0．22和3．11±0．29，均P〈0．05），HASMC的增殖[S期细胞比例为（30．3±2．4）％，A490值为0．80±0．06]高于对照组[S期细胞比例为（13．9±2．6）％，A490值为0．41±0．04]，均P〈0．05；PMA±PKCct反义寡核苷酸组中p-PKCot水平低于PMA处理组，ERK1／2和p-ERK1／2表达明显弱于PMA处理组，cyclinD1和P21cipl表达也明显低于PMA处理组（相对于各对照组的A值分别为1．23±0．19、1．34±0．18、1．52±0．20、1．45±0．18和1．49±0．18，均P〈0．05），HASMC的增殖显著下降[S期细胞比例为（21．2±2．8）％，A490值为0．51±0．04；g值分别为6．07，12．63；均P〈0．05]；同样与PMA处理组相比，PMA±U0126组中P-PKCα水平无明显改变（A值为1．99±0．18，g=0．94，P〉0．05），但ERK1／2、P—ERKI／2的表达，cyclinD1和P21^eip1的表达明显低于PMA处理组（A值分别为0．95±0．21、1．15±0．19、1．37±0．15和1．96±0．21，均P〈0�Objective To explore the role of PKCα-ERK1/2 cascade in PMA induced up-regulation of cyclinD1 and P21^eip1 in human airway smooth muscle cells （HASMCs） sensitized by sera from atopic asthmatics. Methods HASMCs in cultures were passively sensitized by 10% serum from asthmatic patients and were randomly divided into five groups： the control group, PMA treated group, PMA and PKCα mismatched Oligodeoxynucleotides （PKCα-mmODN） treated group, PMA and PKCα antisense Oligodeoxynucleotides （PKCα-asODN） treated group, PMA and U0126 （ MAP Kinase Kinase inhibitor ） treated group. The expression of p-PKCα, ERK1/2, p-ERK1/2, cyclinD1 and P21^eip1 protein were determined by western blotting. The proliferation of HASMC was examined by cell cycle analysis and MTI＇ colorimetric assay. Results Compared with the control group, the expression of p-PKCα and ERK1/2, p-ERK1/2 protein increased, the expression of cyclinD1, P21^eip1 protein increased correspondingly （the A value % control was 2. 10 ± 0.29, 1.67 ± 0. 19, 2. 20 ± 0. 27, 1.99 ± 0. 22 and 3.11 ± 0. 29 respectively ; q value was 9. 87, 7.06, 10. 57, 11.10 and 20. 33 respectively; all P 〈 0. 05） in PMA treated group, and ceils proliferation [ the percentage of cells in S phase was （ 30. 3 ± 2.4 ） % , A490 value was 0. 80 ± 0. 06 ] enhanced significantly compared with those [ the percentage of cells in S phase was （ 13.9 ± 2. 6） %, A490 value was 0. 41 ±0. 04]of the control group（q =6.07, 12. 63; all P 〈0. 05）. In PMA and PKCα-asODN treated group, the level of p-PKCα decreased, the expression of ERK1/2, p-ERK1/2 and the expression of cyclinD1, P21 eipl decreased correspondingly（the A value % control was 1.23 ± 0. 19, 1.34 ± 0. 18, 1.52 ± 0.20, 1.45 ±0.18 and 1.49 ±0.18 respectively; q value was 7.49, 3.58, 5.97, 6.06 and 15.65 respectively; all P 〈 0. 05 ）, and cells proliferation reduced significantly [ the percentage of cells in S phase was （21.2 ± 2. 8） %, A49o value was 0. 51± 0. 04 ; q = 6. 07, 12. 6

关 键 词：蛋白激酶CΑ 细胞外信号调节MAP激酶类 细胞周期蛋白D1 肌细胞 平滑肌 

分 类 号：R72[医药卫生—儿科] R737.902[医药卫生—临床医学]