机械激活离子通道压力蛋白与细胞骨架的相关性研究  

Study on connection between tensile force-activated ion channel and F-actin of cytoskeleton

作　　者：李晓飞 孙一[2] 张钊[3] 张海宁[2] Li Xiaofei;Sun Yi;Zhang Zhao;Zhang Haining(Department of Orthopaedics, The Afflicated Jinhua Hospital of Zhejiang University, Jinhua 321000, China;Department of Joint Surgery,Affiliated Hospital of Qingdao University,Qingdao 266000,China;Hand and Foot Surgery, Affiliated Hospital of Qingdao University, Qingdao 266000, China)

机构地区：[1]浙江大学附属金华医院(金华市中心医院)骨一科,金华321000 [2]青岛大学附属医院关节外科,266000 [3]青岛大学附属医院手足外科,266000

出　　处：《中华关节外科杂志（电子版）》2020年第6期691-697,共7页Chinese Journal of Joint Surgery(Electronic Edition)

基　　金：国家自然科学基金(81672197);浙江省医药卫生科技项目(2020KY343);浙江省自然科学基金(Q20H060058)。

摘　　要：目的探讨周期性机械牵张应力作用下,人软骨细胞中新型机械激活离子通道压力蛋白(Piezo1)与细胞骨架的关系。方法体外培养骨关节炎病人软骨细胞,然后利用多通道细胞牵张应力加载系统处理细胞,根据预实验结果分成无牵张应力组(空白组),2 h牵张应力组,12 h牵张应力组,24 h牵张应力组,48 h牵张应力组和相应的抑制剂浓度为0.275 mmol/L的蜘蛛毒液肽(GsMTx4)组以及浓度为0.573 mmol/L细胞松弛素D组。免疫组化鉴定软骨细胞,荧光定量聚合酶链式反应(RT-qPCR)检测不同力学条件刺激下各组细胞的Piezo1的表达量。以及细胞松弛素D作用下Piezo1的表达量。免疫荧光定位人软骨细胞Piezo1蛋白的表达,激光共聚焦显微镜观察各组细胞的细胞骨架以及Piezo1蛋白共染相关性。RT-q PCR结果比较采用单因素方差分析,两两组间比较采用q检验。结果RT-q PCR检测结果显示,2 h牵张应力组比空白组Piezo1的表达量有所增加,但差异无统计学意义(P>0.05)。12 h牵张应力组比空白组相比有统计学意义(F=9.785,P<0.05)。24 h牵张应力组表达量最多,48 h牵张应力组表达量有所降低。经GsMTx4处理后的各组Piezo1的表达量均出现明显降低(F=13.907,P<0.001)。在牵张应力作用下,2 h牵张应力组至24 h牵张应力组细胞骨架呈渐进性增粗、浓集现象,但是48 h牵张应力组软骨细胞却出现了细胞骨架的松散与降解。相应的RT-qPCR结果显示Piezo1 mRNA表达量与细胞骨架的浓集与松散情况具有相同的趋势。GsMTx4处理后的各加力组表达均下降。结论机械敏感性离子通道Piezo1的开放和关闭是与细胞骨架微丝F-actin肌动蛋白密切相关,由细胞骨架F-actin形变激活Piezo1蛋白通道,并与力学加载呈时间依赖性。Objective To study the connection between the new mechanically-activated(MA)cation channel protein(Piezo1)and the cytoskeleton of the human osteoarthritis(OA)chondrocytes under mechanical tensile force by using a Flexercell unit.Methods The primary human chondrocytes were isolated,cultured,and then subjected to the mechanical tensile force for zero,two,12,24 and 48 h,respectively.The expressions of Piezo1 in each group were assessed by reverse transcription-polymerase chain reaction(RT-qPCR).The immunofluorescence was used to prove that the Piezo1 could be expressed in the human chondrocytes,and the location of the Piezo1 protein.The Piezo1 inhibitor GsMTx4 was used to block the cation channel of Piezo1,served as a positive control.The relative expression levels of the RT-qPCR were compared by one-way ANOVA,and the comparison between the two groups was performed by q test.Results The results of the RT-qPCR showed that the relative mRNA expression of Piezo1 in the two-hour group was higher than that in the blank group without statistical significance(P>0.05).The 12 h group was significantly higher than the blank group(F=9.785,P<0.05).The expression level of the 24 h group was the highest,and that of the 48 h group was decreased.After GsMTx4 treatment,the expression of the Piezo1 significantly decreased in all the groups(F=13.907,P<0.001).The results of laser scanning confocal microscopy showed that the cytoskeleton of chondrocytes gradually thickened and concentrated from the two-hour group to the 24 h group,but the cytoskeleton of chondrocytes in 48 h group was loose and degraded.The corresponding RT-qPCR results showed that the expression of Piezo1 mRNA had the same trend with the concentration and looseness of cytoskeleton.The expression of GsMTx4 decreased in all the groups.Conclusion The expression of the new mechanical cation channel Piezo1 protein shows that it is time-dependence under the tensile force in the human chondrocytes and the activation of the Piezo1 protein could be triggered by the change of th

关 键 词：离子通道 软骨细胞 肌动蛋白类 应力 机械性 

分 类 号：R684.3[医药卫生—骨科学]