中国修复重建外科杂志

中国修复重建外科杂志

沉默 Piezo1 机械敏感型离子通道对 MC3T3-E1 成骨细胞迁移的影响

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目的 研究 Piezo1 机械敏感型离子蛋白在小鼠 MC3T3-E1 成骨细胞迁移中的作用。 方法 取第 5~10 代小鼠 MC3T3-E1 成骨细胞,分为 Piezo1-小干扰 RNA(small interfering RNA,siRNA)转染组(A 组)、阴性对照组(B 组)和空白对照组(C 组)。A、B 组分别采用 siRNA 转染试剂将 Piezo1-siRNA 或阴性对照 siRNA 转染入小鼠 MC3T3-E1 成骨细胞,C 组仅加入 siRNA 转染试剂,在倒置相差显微镜及荧光显微镜下观察细胞形态并计算转染效率。采用免疫荧光染色和 Western blot 检测各组 Piezo1 蛋白表达水平;采用 Transwell 细胞迁移实验和细胞划痕实验检测 Piezo1-siRNA 转染后 MC3T3-E1 成骨细胞迁移能力的变化。 结果 转染 48 h 后,A 组可见细胞体积较未转染细胞略有增大,细胞突变长增粗,但细胞集落有所减少,悬浮细胞较未转染增多,细胞碎片增多。荧光显微镜下可见 B 组小鼠 MC3T3-E1 成骨细胞中出现绿色荧光,转染效率为 68.56%±4.12%。免疫荧光染色及 Western blot 检测示,A 组细胞内 Piezo1 蛋白表达水平均显著低于 B、C 组,差异有统计学意义(P<0.05);B、C 组间差异无统计学意义(P>0.05)。Transwell 细胞迁移实验及细胞划痕实验检测示,A 组每孔迁移细胞数及培养 1~4 d 的细胞划痕愈合率均明显低于 B、C 组,差异有统计学意义(P<0.05);B、C 组间差异无统计学意义(P>0.05)。 结论 沉默 Piezo1 基因能显著抑制小鼠 MC3T3-E1 成骨细胞的迁移能力。

Objective To discuss the effect of Piezo1 mechanically sensitive protein in migration process of mouse MC3T3-E1 osteoblast cells. Methods The 5th-10th generation mouse MC3T3-E1 osteoblasts were divided into Piezo1-small interfering RNA (siRNA) transfection group (group A), negative control group (group B), and blank control group (group C). Piezo1-siRNA or negative control siRNA was transfected into mouse MC3T3-E1 osteoblasts by siRNA transfection reagent, respectively; group C was only added with siRNA transfection reagent; and the cell morphology was observed under inverted phase contrast microscope and fluorescence microscope, and the transfection efficiency was calculated. The expression of Piezo1 protein was detected by immunofluorescence staining and Western blot. Transwell cell migration assay and cell scratch assay were used to detect the migration of MC3T3-E1 osteoblasts after Piezo1-siRNA transfection. Results After 48 hours of transfection, group A showed a slight increase in cell volume and mutant growth, but cell colonies decreased, suspension cells increased and cell fragments increased when compared with untransfected cells. Under fluorescence microscope, green fluorescence was observed in MC3T3-E1 osteoblasts of group B, and the transfection efficiency was 68.56%±4.12%. Immunofluorescence staining and Western blot results showed that the expression level of Piezo1 protein in group A was significantly lower than that in groups B and C (P<0.05); there was no significant difference between group B and group C (P>0.05). Transwell cell migration assay and cell scratch assay showed that the number of cells per hole and the scratch healing rate of cells cultured for 1-4 days in group A were significantly lower than those in groups B and C (P<0.05); there was no significant difference between group B and group C (P>0.05). Conclusion Piezo1 knocked down by siRNA can inhibit the migration ability of MC3T3-E1 osteoblast cells.

关键词: Piezo1; 小干扰 RNA; MC3T3-E1 成骨细胞; 细胞迁移

Key words: Piezo1; small interfering RNA; MC3T3-E1 osteoblast cells; cell migration

引用本文: 闫亮, 姜金, 马崇文, 李睿, 夏亚一. 沉默 Piezo1 机械敏感型离子通道对 MC3T3-E1 成骨细胞迁移的影响. 中国修复重建外科杂志, 2019, 33(1): 28-34. doi: 10.7507/1002-1892.201806121 复制

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