Effect of cyclic stretch on expression of c-fos gene in rat Achilles-derived tendon stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

GAO Shang ¹ ,  TANG Kanglai ¹ , ZHANG Jiqiang ² , YANG Zhijin ¹ , CUI Haifeng ¹ , LI Pao ¹ , TANG Hong ¹ , ZHOU Mei ¹

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

Corresponding author：

TANG Kanglai, Email: tangkanglai@hotmail.com

Keywords：

Tendon stem cells; cyclic stretch; immediate early gene; c-fos; rat

DOI：

10.7507/1002-1892.201609014

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To investigate whether mechanical stretch stimulation affects the expression of the immediate early gene c-fos mRNA in rat Achilles-derived tendon stem cells (TSCs)in vitro. Methods TSCs were isolated from the Achilles tendons of 8 weeks old male Sprague Dawley rats by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated by a uniaxial cyclic stretching loading system under the condition of 1 Hz, respectively with 4% or 8% stretch intensity for 0, 5, 15, 30, 60, and 120 minutes. At each time point, TSCs were collected to detect c-fos mRNA expressions and to find the best time-point T_max by real-time fluorescence quantitative PCR. Then, TSCs were simulated with 2%, 4%, 6%, 8%, or 12% stretch intensity for T_max to observe the relative expressions of c-fos mRNA under different stretch intensities. Next, TSCs were stretched for 0, 5, or 15 minutes respectively and followed by incubation at relax status up to T_max to observe the changes of c-fos mRNA expressions after short period stimulation. Finally, TSCs were stimulated with 4% or 8% stretch intensity respectively for 0, T_max, or 120 minutes to detect the expressions of the tenogenic differentiation related genes [collagen type I, tenomodulin (TNMD)], the osteogenic differentiation related genes [runt related transcription factor 2 (Runx2), distal-less homeobox 5 (Dlx5)], and the adipogenic differentiation related gene [fatty acid binding protein 4 (FABP4)]. Results Under 4% or 8% stretch intensity, the relative expressions of c-fos mRNA significantly increased at 15 minutes (P<0.05), reached the maximum at 30 minutes (P<0.05), and returned to baseline at 60 minutes (P>0.05) when compared with expression at 0 minute. Therefore, T_max was 30 minutes. The stretch intensity of 2% was enough to cause the expression of c-fos mRNA at 30 minutes, and the expression was significantly higher under the stretch intensity of 6%, 8%, and 12% than 2% and 4% (P<0.05). Even for a short period stimulation of 5 minutes, c-fos mRNA expression could still significantly increase at 30 minutes (P<0.05). The relative expressions of differentiation related genes at 30 and 120 minutes showed no significant difference when compared with the expression at 0 minute under 4% stretch intensity (P>0.05); but the relative expression of Runx2 gene significantly increased at 30 minutes, and the relative expressions of collagen type I, TNMD, Dlx5, and Runx2 increased at 120 minutes under 8% stretch intensity (P<0.05). Conclusion Mechanical stretch stimulation can affect the relative expression of the immediate early gene c-fos mRNA of rat Achilles-derived tendon stem cellsin vitro, and there is time- and intensity-dependence. It is suggested that the mechanical stimulation with different time or intensity may affect the differentiation of TSCs at early stage. This study is meaningful for the further study on TSCs intracellular mechanical signal transfer mechanism.

Citation： GAOShang, TANGKanglai, ZHANGJiqiang, YANGZhijin, CUIHaifeng, LIPao, TANGHong, ZHOUMei. Effect of cyclic stretch on expression of c-fos gene in rat Achilles-derived tendon stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(1): 85-90. doi: 10.7507/1002-1892.201609014 Copy

1.	马林, 唐康来, 周兵华, 等. 机械牵伸对体外大鼠肌腱干细胞RhoA/Rho相关蛋白激酶分子表达的影响. 中国修复重建外科杂志, 2014, 28(5): 639-643.
2.	Walz DM, Newman JS, Konin GP, et al. Epicondylitis: pathogenesis, imaging, and treatment. Radiographics, 2010, 30(1): 167-184.
3.	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.
4.	Rui YF, Lui PP, Wong YM, et al. Altered fate of tendon-derived stem cells isolated from a failed tendon-healing animal model of tendinopathy. Stem Cells Dev, 2013, 22(7): 1076-1085.
5.	Zhang J, Wang JH. Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells. PLoS One, 2015, 10(6): e0130454.
6.	Zhang J, Wang JH. The effects of mechanical loading on tendons—anin vivo andin vitro model study. PLoS One, 2013, 8(8): e71740.
7.	Zimitat C, Nixon PF. Glucose induced IEG expression in the thiamin-deficient rat brain. Brain Res, 2001, 892(1): 218-227.
8.	Eliasson P, Andersson T, Hammerman M, et al. Primary gene response to mechanical loading in healing rat Achilles tendons. J Appl Physiol (1985), 2013, 114(11): 1519-1526.
9.	Ying B, Fan H, Wen F, et al. Mechanical strain-induced c-fos expression in pulmonary epithelial cell line A549. Biochem Biophys Res Commun, 2006, 347(1): 369-372.
10.	Sadoshima J, Izumo S. Mechanotransduction in stretch-induced hypertrophy of cardiac myocytes. J Recept Res, 1993, 13(1-4): 777-794.
11.	Dawes NJ, Cox VM, Park KS, et al. The induction of c-fos and c-jun in the stretched latissimus dorsi muscle of the rabbit: responses to duration, degree and re-application of the stretch stimulus. Exp Physiol, 1996, 81(3): 329-339.
12.	Ballermann BJ, Dardik A, Eng E, et al. Shear stress and the endothelium. Kidney Int Suppl, 1998, 67: S100-S108.
13.	Kawata A, Mikuni-Takagaki Y. Mechanotransduction in stretched osteocytes—temporal expression of immediate early and other genes. Biochem Biophys Res Commun, 1998, 246(2): 404-408.
14.	Klein-Nulend J, van der Plas A, Semeins CM, et al. Sensitivity of osteocytes to biomechanical stressin vitro. FASEB J, 1995, 9(5): 441-445.
15.	Roelofsen J, Klein-Nulend J, Burger EH. Mechanical stimulation by intermittent hydrostatic compression promotes bone-specific gene expressionin vitro. J Biomech, 1995, 28(12): 1493-1503.
16.	杨广华, 唐康来, 马林, 等. 体外细胞单轴循环牵伸载荷模型建立的实验研究. 第三军医大学学报, 2014, 36(11): 1142-1146.
17.	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.
18.	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.
19.	Alfredson H, Pietila T, Jonsson P, et al. Heavy-load eccentric calf muscle training for the treatment of chronic Achilles tendinosis. Am J Sports Med, 1998, 26(3): 360-366.
20.	Shynlova OP, Oldenhof AD, Liu M, et al. Regulation of c-fos expression by static stretch in rat myometrial smooth muscle cells. Am J Obstet Gynecol, 2002, 186(6): 1358-1365.
21.	Wang JHC. Mechanobiology of tendon. J Biomech, 2006, 39(9): 1563-1582.
22.	Sadoshima J, Izumo S. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J, 1993, 12(4): 1681-1692.

1. 马林, 唐康来, 周兵华, 等. 机械牵伸对体外大鼠肌腱干细胞RhoA/Rho相关蛋白激酶分子表达的影响. 中国修复重建外科杂志, 2014, 28(5): 639-643.
2. Walz DM, Newman JS, Konin GP, et al. Epicondylitis: pathogenesis, imaging, and treatment. Radiographics, 2010, 30(1): 167-184.
3. 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.
4. Rui YF, Lui PP, Wong YM, et al. Altered fate of tendon-derived stem cells isolated from a failed tendon-healing animal model of tendinopathy. Stem Cells Dev, 2013, 22(7): 1076-1085.
5. Zhang J, Wang JH. Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells. PLoS One, 2015, 10(6): e0130454.
6. Zhang J, Wang JH. The effects of mechanical loading on tendons—anin vivo andin vitro model study. PLoS One, 2013, 8(8): e71740.
7. Zimitat C, Nixon PF. Glucose induced IEG expression in the thiamin-deficient rat brain. Brain Res, 2001, 892(1): 218-227.
8. Eliasson P, Andersson T, Hammerman M, et al. Primary gene response to mechanical loading in healing rat Achilles tendons. J Appl Physiol (1985), 2013, 114(11): 1519-1526.
9. Ying B, Fan H, Wen F, et al. Mechanical strain-induced c-fos expression in pulmonary epithelial cell line A549. Biochem Biophys Res Commun, 2006, 347(1): 369-372.
10. Sadoshima J, Izumo S. Mechanotransduction in stretch-induced hypertrophy of cardiac myocytes. J Recept Res, 1993, 13(1-4): 777-794.
11. Dawes NJ, Cox VM, Park KS, et al. The induction of c-fos and c-jun in the stretched latissimus dorsi muscle of the rabbit: responses to duration, degree and re-application of the stretch stimulus. Exp Physiol, 1996, 81(3): 329-339.
12. Ballermann BJ, Dardik A, Eng E, et al. Shear stress and the endothelium. Kidney Int Suppl, 1998, 67: S100-S108.
13. Kawata A, Mikuni-Takagaki Y. Mechanotransduction in stretched osteocytes—temporal expression of immediate early and other genes. Biochem Biophys Res Commun, 1998, 246(2): 404-408.
14. Klein-Nulend J, van der Plas A, Semeins CM, et al. Sensitivity of osteocytes to biomechanical stressin vitro. FASEB J, 1995, 9(5): 441-445.
15. Roelofsen J, Klein-Nulend J, Burger EH. Mechanical stimulation by intermittent hydrostatic compression promotes bone-specific gene expressionin vitro. J Biomech, 1995, 28(12): 1493-1503.
16. 杨广华, 唐康来, 马林, 等. 体外细胞单轴循环牵伸载荷模型建立的实验研究. 第三军医大学学报, 2014, 36(11): 1142-1146.
17. 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.
18. 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.
19. Alfredson H, Pietila T, Jonsson P, et al. Heavy-load eccentric calf muscle training for the treatment of chronic Achilles tendinosis. Am J Sports Med, 1998, 26(3): 360-366.
20. Shynlova OP, Oldenhof AD, Liu M, et al. Regulation of c-fos expression by static stretch in rat myometrial smooth muscle cells. Am J Obstet Gynecol, 2002, 186(6): 1358-1365.
21. Wang JHC. Mechanobiology of tendon. J Biomech, 2006, 39(9): 1563-1582.
22. Sadoshima J, Izumo S. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J, 1993, 12(4): 1681-1692.

Previous Article
Changes of endogenous Spastin expression after sciatic nerve injury in rats
Next Article
Role and mechanism of stromal cell derived factor 1 on proliferation of vascular endothelial cells

Chinese Journal of Reparative and Reconstructive Surgery

Effect of cyclic stretch on expression of c-fos gene in rat Achilles-derived tendon stem cells

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content