异常机械拉伸应力下 shRNA-Piezo1 诱导的髓核细胞过度凋亡的机制研究。
Study on the mechanism of excessive apoptosis of nucleus pulposus cells induced by shRNA-Piezo1 under abnormal mechanical stretch stress.
机构信息
Department of Joint Orthopaedic Surgery, Jinhua Municipal Central Hospital, Zhejiang University, Jinhua, China.
出版信息
J Cell Biochem. 2019 Mar;120(3):3989-3997. doi: 10.1002/jcb.27683. Epub 2018 Sep 27.
OBJECTIVE
The aim of the study was to explore the mechanism of excessive apoptosis of nucleus pulposus cells induced by short hairpin RNA (shRNA) Piezo type mechanosensitive ion channel component 1 (Piezo1) under abnormal mechanical stretch stress.
METHODS
In vitro mechanical stretch stress model of nucleus pulposus cells in vitro was established, in which the expression of Piezo1 was interfered by transfection of shRNA-Piezo1 interfering vector. Both messenger RNA and protein level of Piezo1 were measured by reverse-transcription polymerase chain reaction and Western blot analysis, respectively. Cytoplasmic Ca was detected by Fluo3-AM kit, and changes of mitochondrial membrane potential in cells were detected using Cell Meter Assay kit. Finally, the apoptosis was evaluated with annexin V-fluorescein isothiocyanate kit.
RESULTS
The highest transfection efficiency of lentivirus titer was 1 × 10 TU/mL and the nucleus pulposus cells were transfected with plural multiplicity of infection = 50. Homo-3201 sequence exhibited the most effective silencing effect and was used in subsequent experiments as the default sequence of shRNA-Piezo1. The calcium content in the cytoplasm of the tension stress group increased significantly compared with that in the blank control group ( q = 3.773; P < 0.05). The level of cytosolic calcium in shRNA-interference group was significantly lower than that in stretch stress group ( q = 5.159; P < 0.05). Stretch stress treatment resulted in an elevated ratio of mitochondrial membrane potential turnover as opposed to blank control group ( q = 4.332; P < 0.05), while shRNA-interference group showed smaller ratio of mitochondrial membrane potential turnover than that in stretch stress group ( q = 4.974; P < 0.05). Similar results were also observed in apoptosis rate analysis ( q = 3.175; P < 0.05).
CONCLUSION
ShRNA-Piezo1 can protect cells by reducing the level of intracellular Ca and the change of mitochondrial membrane potential.
目的
研究短发夹 RNA（shRNA）Piezo 型机械敏感离子通道成分 1（Piezo1）在异常机械拉伸应力下诱导过多的髓核细胞凋亡的机制。
方法
建立体外髓核细胞机械拉伸应激模型，通过转染 shRNA-Piezo1 干扰载体干扰 Piezo1 的表达。分别采用逆转录聚合酶链反应和 Western blot 分析检测 Piezo1 的信使 RNA 和蛋白水平。用 Fluo3-AM 试剂盒检测胞浆 Ca 变化，用 Cell Meter Assay 试剂盒检测细胞线粒体膜电位变化。最后，用 Annexin V-荧光素异硫氰酸酯试剂盒评估细胞凋亡。
结果
慢病毒滴度的最高转染效率为 1×10 TU/mL，转染复数感染量=50。Homo-3201 序列表现出最有效的沉默效果，在后续实验中作为 shRNA-Piezo1 的默认序列。与空白对照组相比，张力组细胞胞浆内 Ca 含量明显增加（ q=3.773；P<0.05）。shRNA 干扰组胞浆内 Ca 水平明显低于拉伸应激组（ q=5.159；P<0.05）。与空白对照组相比，拉伸应激处理导致线粒体膜电位转换的比例升高（ q=4.332；P<0.05），而 shRNA 干扰组与拉伸应激组相比，线粒体膜电位转换的比例较小（ q=4.974；P<0.05）。在凋亡率分析中也观察到了类似的结果（ q=3.175；P<0.05）。
结论
shRNA-Piezo1 通过降低细胞内 Ca 水平和线粒体膜电位变化来保护细胞。
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