EFFECT OF MECHANICAL TENSION ON EXPRESSIONS OF RhoA/Rho ASSOCIATED PROTEIN KINASES IN RAT TENDON STEM CELLS IN VITRO/_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

MALin ,  TANGKanglai , ZHOUBinghua , YANGGuanghua , CHENWan , MUMiduo , YUANChengsong

Department of Orthopedics, Orthopedic Center of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. Chin;

Corresponding author：

TANGKanglai, Email: tangkanglai@hotmail.com

Keywords：

Tendon stem cells; Tendinopathy; RhoA; Rho associated protein kinases; Rat

DOI：

10.7507/1002-1892.20140141

Video：

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Objective To investigate the effect of different mechanical tensions on the expressions of RhoA/Rho associated protein kinases (ROCK) in rat tendon stem cells (TSCs). Methods TSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged, 2-3 months; weighing, 200-250 g) by enzymatic digestion method and cultured for 2-3 passages, then seeded on micro groovdishes. The 4% (4% stretch group) and 8% (8% stretch group) mechanical stretching was performed for 4 hours every day at 1 Hz. After 1, 2, and 3 days, the protein and mRNA expressions of RhoA and ROCK were measured by Western blot and real-time quantitative PCR. The cell proliferation was measured by cell counting kit 8. The cells were not stretched as control group. Results The TSCs at passage 2 showed a cobble-stone shape and aggregation growth; TSCs seeded on micro groovdishes showed random growth, and the cells grew along the stretching direction after mechanical stretching. The mRNA expressions of RhoA and ROCK in control group, 4%, and 8% stretch groups showed an increasing tendency at 1, 2, and 3 days, showing significant difference between groups (P<0.05). The protein expressions of RhoA and ROCK in 4% and 8% stretch groups were similar to those in control group at 1 day (P>0.05), but the expressions in 4% and 8% stretch groups showed an increasing tendency at 2 and 3 days, which were significantly higher than those in control group (P<0.05). The cell proliferation of 8% stretch group was significantly lower than that of 4% stretch group and control group at each time point (P<0.05), but no significant difference was found between 4% stretch group and control group (P>0.05). Conclusion The expressions of RhoA and ROCK of rat TSCs are positively correlated with stretch intensity. So RhoA/ROCK may be an important molecule in TSCs after mechanical stretching.

Citation： MALin, TANGKanglai, ZHOUBinghua, YANGGuanghua, CHENWan, MUMiduo, YUANChengsong. EFFECT OF MECHANICAL TENSION ON EXPRESSIONS OF RhoA/Rho ASSOCIATED PROTEIN KINASES IN RAT TENDON STEM CELLS IN VITRO/. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(5): 639-643. doi: 10.7507/1002-1892.20140141 Copy

1.	Bi Y, Ehirchiou D, Kilts TM, et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med, 2007, 13(10):1219-1227.
2.	Rui YF, Lui PP, Li G, et al. Isolation and characterization of multipotent rat tendon-derived stem cells. Tissue Eng Part A, 2010, 16(5):1549-1558.
3.	Zhang J, Wang JH. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord, 2010, 11:10.
4.	Zhang J, Wang JH. Mechanobiological response of tendon stem cells:implications of tendon homeostasis and pathogenesis of tendinopathy. J Orthop Res, 2010, 28(5):639-643.
5.	Zhang J, Wang JH. Production of PGE(2) increases in tendons subjected to repetitive mechanical loading and induces differentiation of tendon stem cells into non-tenocytes. J Orthop Res, 2010, 28(2):198-203.
6.	Rui YF, Lui PP, Ni M, et al. Mechanical loading increased BMP-2 expression which promoted osteogenic differentiation of tendon-derived stem cells. J Orthop Res, 2011, 29(3):390-396.
7.	Arnsdorf EJ, Tummala P, Kwon RY, et al. Mechanically induced osteogenic differentiation-the role of RhoA, ROCKⅡ and cytoskeletal dynamics. J Cell Sci, 2009, 122(Pt 4):546-553.
8.	邓银栓, 唐康来, 谢美明, 等. 不同牵伸载荷对体外培养人肌腱细胞纤维型肌动蛋白的影响. 中华医学杂志, 2011, 91(25):1780-1785.
9.	Liu J, Chen L, Tao X, et al. Phosphoinositide 3-kinase/Akt signaling is essential for prostaglandin E2-induced osteogenic differentiation of rat tendon stem cells. Biochem Biophys Res Commun, 2013, 435(4):514-519.
10.	Chang TC, Chen YC, Yang MH, et al. Rho kinases regulate the renewal and neural differentiation of embryonic stem cells in a cell plating density-dependent manner. PLoS One, 2010, 5(2):e9187.
11.	曹洪辉, 唐康来, 陈磊, 等. 新型肌腱细胞循环牵伸载荷系统的研制及运行效果观察. 第三军医大学学报, 2010, 32(6):507-510.
12.	胡超, 唐康来, 陈万, 等. 大鼠跟腱来源肌腱干细胞的分离培养及鉴定. 第三军医大学学报, 2013, 35(11):1097-1101.
13.	Chen L, Dong SW, Liu JP, et al. Synergy of tendon stem cells and platelet-rich plasma in tendon healing. J Orthop Res, 2012, 30(6):991-997.
14.	Chen L, Dong SW, Tao X, et al. Autologous platelet-rich clot releasate stimulates proliferation and inhibits differentiation of adult rat tendon stem cells towards nontenocyte lineages. J Int Med Res, 2012, 40(4):1399-1409.
15.	Zhang J, Wang JH. The effects of mechanical loading on tendons-an in vivo and in vitro model study. PLoS One, 2013, 8(8):e71740.
16.	谢美明, 唐康来, 邓银栓, 等. 不同牵伸强度对体外人肌腱细胞磷脂酶A2/环氧合酶表达的影响. 中华创伤杂志, 2011, 27(9):822-826.
17.	Wang YK, Yu X, Cohen DM, et al. Bone morphogenetic protein-2-induced signaling and osteogenesis is regulated by cell shape, RhoA/ROCK, and cytoskeletal tension. Stem Cells Dev, 2012, 21(7):1176-1186.
18.	Teramura T, Takehara T, Onodera Y, et al. Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells. Biochem Biophys Res Commun, 2012, 417(2):836-841.
19.	Chen JC, Jacobs CR. Mechanically induced osteogenic lineage commitment of stem cells. Stem Cell Res Ther, 2013, 4(5):107.
20.	Shi Y, Fu Y, Tong W, et al. Uniaxial mechanical tension promoted osteogenic differentiation of rat tendon-derived stem cells (rTDSCs) via the Wnt5a-RhoA pathway. J Cell Biochem, 2012, 133(10):3133-3142.
21.	Keung AJ, de Juan-Pardo EM, Schaffer DV, et al. Rho GTPases mediate the mechanosensitive lineage commitment of neural stem cells. Stem Cells, 2011, 29(11):1886-1897.

1. Bi Y, Ehirchiou D, Kilts TM, et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med, 2007, 13(10):1219-1227.
2. Rui YF, Lui PP, Li G, et al. Isolation and characterization of multipotent rat tendon-derived stem cells. Tissue Eng Part A, 2010, 16(5):1549-1558.
3. Zhang J, Wang JH. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord, 2010, 11:10.
4. Zhang J, Wang JH. Mechanobiological response of tendon stem cells:implications of tendon homeostasis and pathogenesis of tendinopathy. J Orthop Res, 2010, 28(5):639-643.
5. Zhang J, Wang JH. Production of PGE(2) increases in tendons subjected to repetitive mechanical loading and induces differentiation of tendon stem cells into non-tenocytes. J Orthop Res, 2010, 28(2):198-203.
6. Rui YF, Lui PP, Ni M, et al. Mechanical loading increased BMP-2 expression which promoted osteogenic differentiation of tendon-derived stem cells. J Orthop Res, 2011, 29(3):390-396.
7. Arnsdorf EJ, Tummala P, Kwon RY, et al. Mechanically induced osteogenic differentiation-the role of RhoA, ROCKⅡ and cytoskeletal dynamics. J Cell Sci, 2009, 122(Pt 4):546-553.
8. 邓银栓, 唐康来, 谢美明, 等. 不同牵伸载荷对体外培养人肌腱细胞纤维型肌动蛋白的影响. 中华医学杂志, 2011, 91(25):1780-1785.
9. Liu J, Chen L, Tao X, et al. Phosphoinositide 3-kinase/Akt signaling is essential for prostaglandin E2-induced osteogenic differentiation of rat tendon stem cells. Biochem Biophys Res Commun, 2013, 435(4):514-519.
10. Chang TC, Chen YC, Yang MH, et al. Rho kinases regulate the renewal and neural differentiation of embryonic stem cells in a cell plating density-dependent manner. PLoS One, 2010, 5(2):e9187.
11. 曹洪辉, 唐康来, 陈磊, 等. 新型肌腱细胞循环牵伸载荷系统的研制及运行效果观察. 第三军医大学学报, 2010, 32(6):507-510.
12. 胡超, 唐康来, 陈万, 等. 大鼠跟腱来源肌腱干细胞的分离培养及鉴定. 第三军医大学学报, 2013, 35(11):1097-1101.
13. Chen L, Dong SW, Liu JP, et al. Synergy of tendon stem cells and platelet-rich plasma in tendon healing. J Orthop Res, 2012, 30(6):991-997.
14. Chen L, Dong SW, Tao X, et al. Autologous platelet-rich clot releasate stimulates proliferation and inhibits differentiation of adult rat tendon stem cells towards nontenocyte lineages. J Int Med Res, 2012, 40(4):1399-1409.
15. Zhang J, Wang JH. The effects of mechanical loading on tendons-an in vivo and in vitro model study. PLoS One, 2013, 8(8):e71740.
16. 谢美明, 唐康来, 邓银栓, 等. 不同牵伸强度对体外人肌腱细胞磷脂酶A2/环氧合酶表达的影响. 中华创伤杂志, 2011, 27(9):822-826.
17. Wang YK, Yu X, Cohen DM, et al. Bone morphogenetic protein-2-induced signaling and osteogenesis is regulated by cell shape, RhoA/ROCK, and cytoskeletal tension. Stem Cells Dev, 2012, 21(7):1176-1186.
18. Teramura T, Takehara T, Onodera Y, et al. Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells. Biochem Biophys Res Commun, 2012, 417(2):836-841.
19. Chen JC, Jacobs CR. Mechanically induced osteogenic lineage commitment of stem cells. Stem Cell Res Ther, 2013, 4(5):107.
20. Shi Y, Fu Y, Tong W, et al. Uniaxial mechanical tension promoted osteogenic differentiation of rat tendon-derived stem cells (rTDSCs) via the Wnt5a-RhoA pathway. J Cell Biochem, 2012, 133(10):3133-3142.
21. Keung AJ, de Juan-Pardo EM, Schaffer DV, et al. Rho GTPases mediate the mechanosensitive lineage commitment of neural stem cells. Stem Cells, 2011, 29(11):1886-1897.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF MECHANICAL TENSION ON EXPRESSIONS OF RhoA/Rho ASSOCIATED PROTEIN KINASES IN RAT TENDON STEM CELLS IN VITRO/

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