Piezo1蛋白经MAPK／ERK5信号通路介导软骨细胞凋亡的机制研究  

Mechanism of Piezol protein induced apoptosis of osteoarthritis chondrocytes through MAPK/ERK5 signal pathway

作　　者：李晓飞[1] 张钊[1] 王天宝[1] 王兰凤[1] 张海宁[1] 

机构地区：[1]青岛大学附属医院关节外科,266000

出　　处：《中华骨科杂志》2016年第12期795-803,共9页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金（81171774,81272056）

摘　　要：目的探讨骨关节炎患者软骨细胞中机械敏感性离子通道Piezol蛋白经MAPK／ERK5信号通路介导软骨细胞凋亡的机制。方法体外培养骨关节炎患者软骨细胞，应用细胞牵张应力加载系统对软骨细胞分别加载0h、2h、12h、24h和48h的周期性牵张应力，在各加力组加入Piez01的特异性拮抗剂GsMTx4和ERK5的特异性抑制剂BIX02188作为抑制剂组。用免疫荧光激光共聚焦显微镜观察定位Piezol蛋白在软骨细胞中的表达位置；用RT-PCR检测细胞Piez01、ERK5、凋亡相关基因Bcl-2、Bcl-2相关x蛋白（Bcl．associatedXprotein，Bax）及Bcl-2相关促凋亡蛋白（Bcl咀ssociateddeathpromoter，BAD）的mRNA相对表达量；用AV-PI凋亡试剂盒检测细胞凋亡情况。结果激光共聚焦显微镜观察Piez01蛋白可以在细胞核和细胞质表达。RT-PCR检测PizeolmRNA在骨关节炎退变软骨中有少量表达，12h加力组Pize01、ERK5、BAD和BaxmRNA的相对表达量增加，24h达到峰值，48h表达量下降。而Bcl-2mRNA的相对表达量自2h开始下降，12h降到最低值，24h开始升高。Piezol蛋白特异性抑制剂GsMTx4组Pizeol、ERK5、BAD、BaxmRNA的相对表达量均下降，Bcl-2表达量升高。ERK5特异性抑制剂BIX02188组ERK5、BAD、BaxmRNA的相对表达量均下降，Bcl．2表达量升高，Piezol表达量不变。2h加力组细胞凋亡率于早期开始增加，晚期凋亡率无明显增加；12h加力组调亡率于晚期开始增加；24h加力组晚期细胞凋亡率最高；48h加力组晚期凋亡率较24h加力组降低。RT-PCR检测结果显示Piez01、ERK5、Bcl-2、BAD和BaxmRNA的表达量与晚期凋亡具有相似的表达趋势。结论Piezol参与骨关节炎的软骨细胞晚期凋亡过程，且通过MAPK／ERK5信号通路启动细胞凋亡。Objective To investigate whether the new mechanically-activated （MA） cation channel Piezol protein can cause the apoptosis of human chondrocytes under compressive loading, using a Flexercell unit by activating MAPK/ERK5 signal pathway. Methods Primary human chondrocytes were isolated, cultured, and then subjected to the static compressive loading for 2 h,12 h, 24 h and 48 h, respectively. The GsMTx4, which is the special inhibitor of the Piezol protein and the BIX02188, which is the inhibitor of the ERK5 served as the inhibitor group. The immunofluorescence was used to locate the expression of the Piezo 1 protein. The expressions of Piezol and ERK5 were assessed by reverse transcription-polymerase chain reaction （RT-PCR）, as well as the apoptosis gene B cell lymphoma/leukemia-2 （Bcl-2）, Bcl-associated X protein （Bax） and Bcl-associated death promoter （BAD）. In addition, Piezol inhibitor, GsMTx4, was used to block the MA cation channel Piezol, served as the inhibitor group. AV- PI was used to detect the apoptosis of the OA chondrocytes. Results The location of the Piezo 1 was expressed in nucleus and cy- toplasm of chondrocytes. The expression of the Piezol, ERKS, BAD, and Bax mRNA in the OA chondrocytes is weak. The 12 h stretch force group was significant increased, and the 24 h stretch force group was the highest expression. However, the expression of the 48 h group was decreasing. The expression of the Bcl-2 in the 12 h group was decreasing, and the 12 h stretch force group was the lowest expression while the 24 h stretch force group was increasing. In the GsMTx4 group, the expression of the Piezol, ERKS, BAD, Bax was decreasing while the Bcl-2 was increasing. In the BIX02188 group, the expression of the ERKS, BAD, Bax was increasing while the Bcl-2 was decreasing, while the expression of the Piezol was not change. The result of AV-PI shown that the 2 h stretch force group increased early stage of apoptosis. The 12 h stretch force group increased late stage of apoptosis, and the 24 h stretc

关 键 词：离子通道 软骨细胞 骨关节炎 MAP激酶信号系统 细胞凋亡 

分 类 号：R684.3[医药卫生—骨科学]