沉默Piezo1机械敏感型离子通道对MC3T3-E1成骨细胞迁移的影响
|
摘要
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 e xpression 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.
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 e xpression 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.
引文
1Sims NA, Martin TJ. Coupling the activities of bone formation and resorption:a multitude of signals within the basic multicellular unit. Bonekey Rep, 2014, 3:481.
2Seeman E. Bone modeling and remodeling. Crit Rev Eukaryot Gene Expr, 2009, 19(3):219-233.
3Lai CF, Cheng SL. Alphavbeta integrins play an essential role in BMP-2 induction of osteoblast differentiation. J Bone Miner Res,2005, 20(2):330-340.
4胥少汀,葛宝丰,徐印坎.实用骨科学. 4版.北京:人民军医出版社, 2012:632-654.
5Tang Y, Wu X, Lei W, et al. TGF-beta1-induced migration of bone mesenchymal stem cells couples bone resorption with formation.Nat Med, 2009, 15(7):757-765.
6Xian L, Wu X, Pang L, et al. Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal stem cells. Nat Med, 2012,18(7):1095-1101.
7Aryal A C S, Miyai K, Izu Y, et al. Nck influences preosteoblastic/osteoblastic migration and bone mass. Proc Natl Acad Sci U S A, 2015, 112(50):15432-15437.
8Coste B, Mathur J, Schmidt M, et al. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science, 2010, 330(6000):55-60.
9Sugimoto A, Miyazaki A, Kawarabayashi K, et al. Piezo type mechanosensitive ion channel component 1 functions as a regulator of the cell fate determination of mesenchymal stem cells.Sci Rep, 2017, 7(1):17696.
10Hansen TH, Madsen MTB, J?rgensen NR, et al. Bone turnover,calcium homeostasis, and vitamin D status in Danish vegans. Eur J Clin Nutr, 2018, 72(7):1046-1054.
11 Campbell EJ, Campbell GM, Hanley DA. The effect of parathyroid hormone and teriparatide on fracture healing. Expert Opin Biol Ther, 2015, 15(1):119-129.
12Eleniste PP, Huang S, Wayakanon K, et al. Osteoblast differentiation and migration are regulated by dynamin GTPase activity. Int J Biochem Cell Biol, 2014, 46:9-18.
13Vishnuprasad CN, Tsuchiya T, Kanegasaki S, et al. Aurantioobtusin stimulates chemotactic migration and differentiation of MC3T3-E1 osteoblast cells. Planta Med, 2014, 80(7):544-549.
14van der Eerden BC, Oei L, Roschger P, et al. TRPV4 deficiency causes sexual dimorphism in bone metabolism and osteoporotic fracture risk. Bone, 2013, 57(2):443-454.
15Zhang Y, Xu J, Ruan YC, et al. Implant-derived magnesium induces local neuronal production of CGRP to improve bonefracture healing in rats. Nat Med, 2016, 22(10):1160-1169.
16Wachter RF, Briggs GP, Pedersen CE Jr. Precipitation of phase I antigen of Coxiella burnetii by sodium sulfite. Acta Virol, 1975,19(6):500.
17Bowman CL, Gottlieb PA, Suchyna TM, et al. Mechanosensitive ion channels and the peptide inhibitor GsMTx-4:history,properties, mechanisms and pharmacology. Toxicon, 2007, 49(2):249-270.
18Bae C, Sachs F, Gottlieb PA. The mechanosensitive ion channel Piezo1 is inhibited by the peptide GsMTx4. Biochemistry, 2011,50(29):6295-6300.
19Wang S, Chennupati R, Kaur H, et al. Endothelial cation channel PIEZO1 controls blood pressure by mediating flow-induced ATP release. J Clin Invest, 2016, 126(12):4527-4536.
20Evans EL, Cuthbertson K, Endesh N, et al. Yoda1 analogue(Dooku1)which antagonizes Yoda1-evoked activation of Piezo1and aortic relaxation. Br J Pharmacol, 2018, 175(10):1744-1759.
21Cahalan SM, Lukacs V, Ranade SS, et al. Piezo1 links mechanical forces to red blood cell volume. Elife, 2015, 4.
22Cheng G, Gao F, Sun X, et al. Paris saponinⅦsuppresses osteosarcoma cell migration and invasion by inhibiting MMP-2/9production via the p38 MAPK signaling pathway. Mol Med Rep,2016, 14(4):3199-3205.
23焦雪峰,黄永灿,黄益洲,等.大鼠BMSCs自发钙化过程中成骨相关基因表达谱的分析.中国修复重建外科杂志, 2014, 28(2):133-141.
24 Andolfi L, Bourkoula E, Migliorini E, et al. Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy. PLoS One, 2014, 9(11):e112582.
2Seeman E. Bone modeling and remodeling. Crit Rev Eukaryot Gene Expr, 2009, 19(3):219-233.
3Lai CF, Cheng SL. Alphavbeta integrins play an essential role in BMP-2 induction of osteoblast differentiation. J Bone Miner Res,2005, 20(2):330-340.
4胥少汀,葛宝丰,徐印坎.实用骨科学. 4版.北京:人民军医出版社, 2012:632-654.
5Tang Y, Wu X, Lei W, et al. TGF-beta1-induced migration of bone mesenchymal stem cells couples bone resorption with formation.Nat Med, 2009, 15(7):757-765.
6Xian L, Wu X, Pang L, et al. Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal stem cells. Nat Med, 2012,18(7):1095-1101.
7Aryal A C S, Miyai K, Izu Y, et al. Nck influences preosteoblastic/osteoblastic migration and bone mass. Proc Natl Acad Sci U S A, 2015, 112(50):15432-15437.
8Coste B, Mathur J, Schmidt M, et al. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science, 2010, 330(6000):55-60.
9Sugimoto A, Miyazaki A, Kawarabayashi K, et al. Piezo type mechanosensitive ion channel component 1 functions as a regulator of the cell fate determination of mesenchymal stem cells.Sci Rep, 2017, 7(1):17696.
10Hansen TH, Madsen MTB, J?rgensen NR, et al. Bone turnover,calcium homeostasis, and vitamin D status in Danish vegans. Eur J Clin Nutr, 2018, 72(7):1046-1054.
11 Campbell EJ, Campbell GM, Hanley DA. The effect of parathyroid hormone and teriparatide on fracture healing. Expert Opin Biol Ther, 2015, 15(1):119-129.
12Eleniste PP, Huang S, Wayakanon K, et al. Osteoblast differentiation and migration are regulated by dynamin GTPase activity. Int J Biochem Cell Biol, 2014, 46:9-18.
13Vishnuprasad CN, Tsuchiya T, Kanegasaki S, et al. Aurantioobtusin stimulates chemotactic migration and differentiation of MC3T3-E1 osteoblast cells. Planta Med, 2014, 80(7):544-549.
14van der Eerden BC, Oei L, Roschger P, et al. TRPV4 deficiency causes sexual dimorphism in bone metabolism and osteoporotic fracture risk. Bone, 2013, 57(2):443-454.
15Zhang Y, Xu J, Ruan YC, et al. Implant-derived magnesium induces local neuronal production of CGRP to improve bonefracture healing in rats. Nat Med, 2016, 22(10):1160-1169.
16Wachter RF, Briggs GP, Pedersen CE Jr. Precipitation of phase I antigen of Coxiella burnetii by sodium sulfite. Acta Virol, 1975,19(6):500.
17Bowman CL, Gottlieb PA, Suchyna TM, et al. Mechanosensitive ion channels and the peptide inhibitor GsMTx-4:history,properties, mechanisms and pharmacology. Toxicon, 2007, 49(2):249-270.
18Bae C, Sachs F, Gottlieb PA. The mechanosensitive ion channel Piezo1 is inhibited by the peptide GsMTx4. Biochemistry, 2011,50(29):6295-6300.
19Wang S, Chennupati R, Kaur H, et al. Endothelial cation channel PIEZO1 controls blood pressure by mediating flow-induced ATP release. J Clin Invest, 2016, 126(12):4527-4536.
20Evans EL, Cuthbertson K, Endesh N, et al. Yoda1 analogue(Dooku1)which antagonizes Yoda1-evoked activation of Piezo1and aortic relaxation. Br J Pharmacol, 2018, 175(10):1744-1759.
21Cahalan SM, Lukacs V, Ranade SS, et al. Piezo1 links mechanical forces to red blood cell volume. Elife, 2015, 4.
22Cheng G, Gao F, Sun X, et al. Paris saponinⅦsuppresses osteosarcoma cell migration and invasion by inhibiting MMP-2/9production via the p38 MAPK signaling pathway. Mol Med Rep,2016, 14(4):3199-3205.
23焦雪峰,黄永灿,黄益洲,等.大鼠BMSCs自发钙化过程中成骨相关基因表达谱的分析.中国修复重建外科杂志, 2014, 28(2):133-141.
24 Andolfi L, Bourkoula E, Migliorini E, et al. Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy. PLoS One, 2014, 9(11):e112582.