[敲低机械敏感蛋白Piezo1对MC3T3-E1成骨细胞迁移的影响]
[Effect of knocking down Piezo1 mechanically sensitive protein on migration of MC3T3-E1 osteoblast cells].
作者信息
Yan Liang, Jiang Jin, Ma Chongwen, Li Rui, Xia Yayi
机构信息
Department of Orthopedics, Gansu Key Laboratory of Orthopaedics, Lanzhou University Second Hospital, Lanzhou Gansu, 730000, P.R.China.
Department of Orthopedics, Gansu Key Laboratory of Orthopaedics, Lanzhou University Second Hospital, Lanzhou Gansu, 730000,
出版信息
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2019 Jan 15;33(1):28-34. doi: 10.7507/1002-1892.201806121.
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 ( <0.05); there was no significant difference between group B and group C ( >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 ( <0.05); there was no significant difference between group B and group C ( >0.05).
CONCLUSION
Piezo1 knocked down by siRNA can inhibit the migration ability of MC3T3-E1 osteoblast cells.
目的
探讨Piezo1机械敏感蛋白在小鼠MC3T3-E1成骨细胞迁移过程中的作用。
方法
将第5-10代小鼠MC3T3-E1成骨细胞分为Piezo1小干扰RNA（siRNA）转染组（A组）、阴性对照组（B组）和空白对照组（C组）。分别用siRNA转染试剂将Piezo1-siRNA或阴性对照siRNA转染至小鼠MC3T3-E1成骨细胞；C组仅加入siRNA转染试剂；在倒置相差显微镜和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蛋白质印迹法检测Piezo1蛋白的表达。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成骨细胞的迁移情况。
结果
转染48小时后，A组细胞体积略有增大，生长变异性增加，但与未转染细胞相比，细胞集落减少，悬浮细胞增多，细胞碎片增多。荧光显微镜下，B组MC3T3-E1成骨细胞可见绿色荧光，转染效率为68.56%±4.12%。免疫荧光染色和蛋白质印迹结果显示，A组Piezo1蛋白表达水平明显低于B组和C组（P<0.05）；B组和C组之间差异无统计学意义（P>0.05）。Transwell细胞迁移实验和细胞划痕实验显示，A组培养1-4天的每孔细胞数和细胞划痕愈合率均明显低于B组和C组（P<0.05）；B组和C组之间差异无统计学意义（P>0.05）。
结论
siRNA敲低Piezo1可抑制MC3T3-E1成骨细胞的迁移能力。
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