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新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 Piezo1 protein-induced apoptosis of the ostearthritis chondrocytes

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0h加力组(空白组)、2h加力组、12h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12h加力组表达量增加,为51.21 ±7.42(采用2-△△Ct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)2h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 =0.032,12h加力组钙离子浓度升高,荧光强度为(612±21) nm,24h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24h加力组有所降低(P=0.017).(3)在牵张应力作用下,2h加力组早期细胞凋亡率为(0.214±0.091)%,较空白组开始增加(P=0.044),12h加力组晚期凋亡率为(0.258±0.115)%,较空白组开始增加(P=0.028),24 h加力组的晚期细胞凋亡率达峰值,为(0.587 ±0.214)%,但48 h加力组凋亡率为(0.231 ±0.218)%,较24h加力组有所降低(P=0.033).结论 机械敏感性离子通道Piezo1参与骨性关节炎的软骨细胞晚期凋亡过程,并且是以钙离子为第二信使,通过内质网应激通路启动细胞的凋亡过程.

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abstracts:

Objective To study whether the new mechanically-activated cation channel Piezo1 protein can cause the apoptosis of the human chondrocytes under compressive loading,using a Flexercell unit 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 Piezo1 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,Piezo1 inhibitor,grammostola sptulata mechanotoxin 4 (GsMTx4),was used to block the MA cation channel Piezo1,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 significantly,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,ATF4 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 the48 h group was (609 ±91) nm so the calcium in the48 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) (P=0.044).The late stage of apoptosis in 12 h group was (0.258-±0.115)(P=0.028),and the 24 h group was (0.587 ±0.214),the 48 h group was (0.231 ±0.218).So the 24 h group's apoptotic rate was highest while the apoptotic rate of the 48 h group was lower than the 24 h group (P =0.033).Conclusion Piezo1 plays an important role in the apoptosis of the human chondrocyte through the ER stress signal way.And the calcium played as the second messenger.

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