型机械敏感性离子通道在软骨细胞凋亡中的作用机制  

The study of the mechanism of the Piezol protein - induced apoptosis of the ostearthritis chondrocytes

作　　者：王天宝[1] 李晓飞[1] 冷萍[2] 荣春[3] 张钊 李晓东[1] 张海宁[1] 

机构地区：[1]青岛大学附属医院关节外科,266003 [2]青岛大学附属医院药剂科,266003 [3]青岛大学附属医院手术室,266003 [4]青岛海慈医院关节外科,266003

出　　处：《中华实验外科杂志》2017年第11期1820-1823,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81171774、81272056）

摘　　要：目的观察新型机械敏感性离子通道压电蛋白1（Piezo1）在骨性关节炎患者软骨细胞中的表达并探讨其在软骨细胞凋亡中的作用机制。方法体外培养骨性关节炎患者退变软骨细胞，利用细胞体外加力实验系统（FX-5000 Tension System）处理细胞，根据预实验结果分成0 h加力组（空白组）、2 h加力组、12 h加力组、24 h加力组、48 h加力组和相应的抑制剂红玫瑰毛蜘蛛来源刮刀样机械敏感毒剂4型（GsMTx4）组、抑制剂氟甲基酮（Z-ATAD-FMK）组。利用反转录-聚合酶链反应（RT-PCR）检测不同力学条件刺激下各组细胞的Piezo1、半胱氨酰天冬氨酸特异性蛋白酶-12（Caspase-12）、蛋白激酶R样内质网激酶（PERK）、葡萄糖调节蛋白78（GRP78）、激活因子4（ATF4）和C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白（CHOP）的表达量。钙离子荧光探针（Fluo3-AM）试剂盒检测空白组，加力组和抑制剂组细胞胞质内的钙离子含量。膜联蛋白V-碘化丙锭（Annexin V-PI）凋亡试剂盒检测各分组的凋亡。结果（1）Pizeo1 mRNA在骨性关节炎退变软骨中有少量表达，且12 h加力组表达量增加，为51.21±7.42（采用2－ΔΔCt的方法无计量单位），24 h加力组达到高峰，为153.98±10.34，48 h加力组为73.98±14.01，与24 h加力组比较，Piezo1 mRNA的表达出现下降（P＝0.013）。内质网应激信号分子Caspase-12、PERK、GRP78、ATF4和CHOP的mRNA的表达也出现相同趋势。（2）2 h加力组软骨细胞胞质中的钙离子较空白组有所增加，钙离子荧光强度由（226±25） nm增加到（481±19） nm，P＝0.032，12 h加力组钙离子浓度升高，荧光强度为（612±21） nm，24 h加力组钙离子浓度达峰值，荧光强度为（1 009±29） nm，48 h加力组钙离子荧光强度为[（609±91） nm]，48 h加力组钙离子浓度较24 h加力组有所降低（P＝0.017）。（3）在牵张应力作用下，2 h加力�Objective To study whether the new mechanically - activated cation channel Piezol protein can cause the apoptosis of the human chondrocytes under compressive loading, using a Flexercell u- nit by activating estrogen receptor （ER） stress signal pathway. Methods Primary human chondrocytes were isolated, cultured, and then subjected to the static compressive loading for 0, 2, 12, 24 and 48 h, respectively. The expressions of Piezol and the apoptosis related protein Caspase - 12, protein kinase R - like ER kinase （ PERK）, glucose - regulated protein78 （ GRP78 ）, activating transcription factor 4 （ATF4） and C/EBP homologous protein （CHOP） were assessed by reverse transcription -polymerase chain reaction （PT - PCR）. In addition, Piezol inhibitor, grammostola sptulata mechanotoxin 4 （GsMTx4）, was used to block the MA cation channel Piezol, served as a inhibitor control. As well as the Z - ATAD - FMK, which is the inhibitor of the Caspase - 12. The calcium fluorescence probe （ Fluo3 - AM） kit was used to test the calcium influx in the cytoplasm of the chondrocytes. Annexin V - ropidium iodide （PI） was used to detect the apoptosis of the osteoarthritis （OA） chondrocytes. Results （1） The Piezol could be tested in the OA chondrocytes, and the 12 h group was 51.21 ± 7.42, which was increased signif-icantly, the 24 h group was 153.98 ± 10. 34, which was the highest expression while the expression of the 48 h group was lower than the 24 h group （P = 0. 013）, as well as the caspase - 12, PERK, GRP78, ATFd and CHOP. （2） The calcium in the cytoplasm was increasing from the 2 h group, from （226 ± 25） nm to （481 ± 19） nm （P =0. 032）, the calcium in the 12 h group was （612 ±21 ） nm, the calcium in the 24 h group was （1 009 ±29） nm and the 48 h group was （609 ±91 ） nm so the calcium in the 48 h group was lower than the 24 h group （P =0. 017）. The result of AV -PI had shown that the early stage of apoptosis in the 2 h group was （0. 214 ±0. 091 �

关 键 词：新型机械敏感性离子通道压电蛋白1 软骨细胞 骨性关节炎 内质网应激 凋亡 机械牵张 

分 类 号：R684.3[医药卫生—骨科学]