| Piezo1机械敏感型离子通道介导流体剪切力对 MC3T3-E1 成骨细胞迁移的影响 | |
| Alternative Title | Effect of Piezo1 Mechanically Sensitive Ion Channel Mediated Fluid Shear Force on Migration in |
| 闫亮 | |
| Subtype | 硕士 |
| Thesis Advisor | 夏亚一 |
| 2019-01-15 | |
| Degree Grantor | 兰州大学 |
| Place of Conferral | 兰州 |
| Degree Name | 硕士 |
| Degree Discipline | 外科学 |
| Keyword | 流体剪切力 MC3T3-E1成骨细胞 piezo1蛋白 机械敏感型离子通道 小干扰 RNA 细胞迁移 |
| Abstract | 目的：研究加载不同时间流体剪切力（Fluid Shear Stress，FSS）对MC3T3-E1成骨细胞中piezo1机械敏感型离子蛋白表达的影响研究以及Piezo1 机械敏感型离子蛋白对小鼠 MC3T3-E1 成骨细胞迁移的影响。 方法：利用自行设计的平行平板FSS加载装置，对MC3T3-E1成骨细胞施加12 dyn/cm2 FSS 0、15、30、45、60、90 min，采用Western blot和免疫荧光染色实验检测piezo1机械敏感型离子蛋白的表达水平。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：免疫荧光清晰显示piezo1蛋白明显表达于MC3T3-E1成骨细胞细胞质及细胞核，细胞质尤为明显。Western blot 检测和免疫荧光强度显示，对体外培养的MC3T3-E1细胞加载12 dyn/cm2 FSS，随着加载时间的延长，piezo1蛋白表达逐渐上调，在45 min左右达到高峰，之后再次下降。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：加载不同时间的12 dyn/cm2 FSS能够上调MC3T3-E1成骨细胞piezo1机械敏感型离子蛋白，且加载45 min最合适。沉默 Piezo1 基因能显著抑制小鼠 MC3T3-E1 成骨细胞的迁移能力。 |
| Other Abstract | Objective: To discuss the variations of the expression of piezo1 mechanically sensitive ion protein in MC3T3-E1 osteoblasts with the increasing loading time of fluid shear stress (FSS) and the effect of Piezo1 protein in migration process of mouse MC3T3-E1 osteoblast cells. Methods: With the use of a self-designed parallel plate flow system, FSS (12 dyn/cm2) was acted on MC3T3- E1 osteoblasts for 0, 15, 30, 45, 60, 90 min. The expression level of piezo1 protein was detected by immunofluorescence and Western blot. Results Piezo1 was obviously expressed in the cytoplasm and nucleus of osteoblasts, especially in the cytoplasm. FSS of 12 dyn /cm2 was acted on MC3T3-E1 cells cultured in vitro. With the increase of loading time, the expression of piezo1 protein was upregulated. At 45 minutes after loading, the expression of piezo1 protein reached the maximum. 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: Loading 12 dyn/cm2 FSS can upgrade the expression of piezo1 mechanosensitive ion protein in MC3T3-E1 osteoblasts, and the optimal loading time of FSS is 45 minutes. 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: Loading 12 dyn/cm2 FSS can upgrade the expression of piezo1 mechanosensitive ion protein in MC3T3-E1 osteoblasts, and the optimal loading time of FSS is 45 minutes. Piezo1 knocked down by siRNA can inhibit the migration ability of MC3T3-E1 osteoblast cells. |
| Pages | 55 |
| URL | 查看原文 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | https://ir.lzu.edu.cn/handle/262010/338695 |
| Collection | 第二临床医学院 |
| Affiliation | 第二临床医学院 |
| First Author Affilication | Second Clinical School |
| Recommended Citation GB/T 7714 | 闫亮. Piezo1机械敏感型离子通道介导流体剪切力对 MC3T3-E1 成骨细胞迁移的影响[D]. 兰州. 兰州大学,2019. |
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