PROMOTION EFFECT OF CHONDROITIN SULFATE ON PROLIFERATION OF MYOBLASTS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANGLin ^1,2,3 , ZHOULidong ^1,2 , WUMei ^1,2 , LIULijin ^1,2,3 , XIANGYongping ^1,2,3 , LIUMouyuan ^1,2 ,  LIUAibing ^1,2

1. Experimental Center, General Hospital of Chinese People's Armed Police Forces, Beijing, 100039, P. R. China;
2. Beijing Municipal Key Laboratory of Disaster Rescue Medicine;
3. Jinzhou Medical University;

Corresponding author：

LIUAibing, Email: doctor_liuaibing@aliyun.com

Keywords：

Myoblast; Myotube; Cell fusion; Chondroitin sulfate

DOI：

10.7507/1002-1892.20160262

Video：

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Objective To research the effect of chondroitin sulfate (CS) on the proliferation of myoblasts and the formation of myotube. Methods The myoblasts at passage 5 were used to prepare the cells suspension (1×108 cells/mL), and the experiment was divided into 4 groups based on CS concentration in the medium:group A (0 μg/mL), group B (50 μg/mL), group C (100 μg/mL), and group D (200 μg/mL). The cell morphology and myotube formation were observed by inverted microscope at 4, 5, and 8 days after treatment; MTT was used to detect the cell proliferation at 6 days, and the number of myotube was calculated by HE staining at 8 days. Results Cells showed spindle shape after adherent, with ovoid nuclei and dense cytoplasm under inverted microscope. When the cell adherent rate was 90%, cells arranged in whorls swirled and showed long fusiform adherent growth; and then nuclei fusion resulted in formation of multincleated myotubes. At 8 days, most myoblasts fused to form myotube in group A, but less myotube was observed in groups B and C, and the least myotube in group D. The absorbance (A) values of groups A, B, C, and D were 0.045 2±0.004 4, 0.540 4±0.096 7, 0.660 9±0.143 4, and 1.069 0±0.039 0 respectively, showing significant difference between other groups (P<0.05) except between groups B and C P>0.05). HE staining observation showed that most myoblasts fused to form myotube in group A, but less myotube in groups B and C, and the least myotube in group D. The number of myotube of groups A, B, C, and D were 222.01±30.02, 193.13±42.46, 170.26±11.96, and 136.88±16.78 respectively, showing no significant difference among groups (F=1.658, P=0.252). Conclusion CS can significantly promote the proliferation of myoblast, the promotion is the biggest when CS concentration is 200 μg/mL.

Citation： WANG Lin, ZHOU Lidong, WU Mei, LIU Lijin, XIANG Yongping, LIU Mouyuan, LIU Aibing. PROMOTION EFFECT OF CHONDROITIN SULFATE ON PROLIFERATION OF MYOBLASTS. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(10): 1290-1294. doi: 10.7507/1002-1892.20160262 Copy

1.	王琳, 刘爱兵. 适合人肌母细胞的无血清培养基. 中国组织工程, 2015, 19(51):8271-8275.
2.	Peter K Law, Danlin M Law, Ping Lu, et al. Human myoblast genome therapy. J Geriatric Cardiology, 2006, 3(3):135-149.
3.	Hecht N, Marushima A, Nieminen M, et al. Myoblast-mediated gene therapy improves functional collateralization in chronic cerebral hypoperfusion. Stroke, 2015, 46(1):203-211.
4.	Pagani FD, DerSimonian H, Zawadzka A, et al. Autologous skeletal myoblast transplanted to ischemia-damaged myocardium in humans.Histological analysis of cell survival and diffierentiation. J Am Coll Cardiol, 2003, 41(5):879-888.
5.	Ye L, Haider HKh, Tan R, et al. Transplantation of nanoparticle transfected skeletal myoblasts overexpressing vascular endothelial growth factor-165 for cardiac repair. Circulation, 2007, 116(11 Suppl):113-120.
6.	Ye L, Haider HKh, Jiang S, et al. Improved angiogenic response in pig heart following ischaemic injury using human skeletal myoblast simultaneously expressing VEGF165 and angiopoietin-1. Eur J Heart Fail, 2007, 9(1):15-22.
7.	Ye L, Lee KO, Su LP, et al. Skeletal myoblast transplantation for attenuation of hyperglycaemia, hyperinsulinaemia and glucose intolerance in a mouse model of type 2 diabetes mellitus. Diabetologia, 2009, 52(9):1925-1934.
8.	Cheng Q, Law PK, de Gasparo M, et al. Combination of the dipeptidyl peptidase IV inhibitor LAF237[(S)-1-[(3-hydroxy-1-adamantyl) ammo] acetyl-2-cyanopyrrolidine] with the angiotensin Ⅱ Type 1 receptor antagonist valsartan[N-(1-Oxopentyl)-N-[[2-(1H-tetrazol-5-yl)-[1,1-biphenyl]-4-yl]methyl]-L-valine] enhances pncreatic islet morphology and function in a mouse model of type 2 diabetes. J Pharmacol Exp Ther, 2008, 327(3):683-691.
9.	秦瑞峰, 顾晓明, 黄富国, 等. 流式细胞术测定骨骼肌细胞分化中DNA合成及其细胞周期变化. 第四军医大学学报, 2000, 21(8):921-923.
10.	秦瑞峰, 顾晓明, 张浩. 不同培养条件对骨骼肌细胞增殖及分化的影响. 实用口腔医学杂志, 2000, 16(1):14-16.
11.	刘立斌, 周红雨. 成肌细胞的临床应用现状及前景. 中国修复重建外科杂志, 2007, 21(7):767-770.
12.	刘宁, 刘雅南, 刘涛, 等. 硫酸软骨素的制备研究及发展现状. 食品工业科技, 2014, 35(3):392-395.
13.	陈小宇, 刘成成, 祝爱珍, 等. 硫酸软硫骨素对3T3-L1细胞增殖和分化的影响. 中国医科大学学报, 2013, 42(7):599-603.
14.	Haylock-Jacobs S, Keoμgh MB, Lau L, et al. Chondroitin sulphate proteoglycans:extracellular matrix proteins that regulate immunity of the central nervous system. Autoimmun Rev, 2011, 10(12):766-772.
15.	Lee JY, Lee SH, Kim HJ, et al. The preventive inhibition of chondroitin sulfate against the CCl4-induced oxidative stress of subcellular level. Arch Pharm Res, 2004, 27(3):340-345.
16.	Nishimoto S, Takagi M, Wakitani S, et al. Effect of chondroitin sulfate and hyaluronic acid on gene expression in a three-dimensional culture of chondrocytes. J Biosci Bioeng, 2005, 100(1):123-126.
17.	Dubey L, Sharma M. Cardiogenic shock complicating acute myocardial infarction-a review. Acta Cardiol, 2011, 66(6):691-699.
18.	Peters K, Kaufman M, Dmochowski L, et al. 1340 autologous muscle derived cell therapy for the treatment of female stress urinary incontinence:a multi-center experience. J Urol, 2011, 185(4):e535-536.
19.	Baj A, Bettaccini AA, Casalone R, et al. Culture of skeletal myoblasts from human donors aged over 40 years:dynamics of cell growth and expression of differentiation markers. J Transl Med, 2005, 3(1):21.
20.	Jarocha D, Stangel-Wojcikiewicz K, Basta A, et al. Efficient myoblast expansion for regenerative medicine use. Int J Mol Med, 2014, 34(1):83-91.

1. 王琳, 刘爱兵. 适合人肌母细胞的无血清培养基. 中国组织工程, 2015, 19(51):8271-8275.
2. Peter K Law, Danlin M Law, Ping Lu, et al. Human myoblast genome therapy. J Geriatric Cardiology, 2006, 3(3):135-149.
3. Hecht N, Marushima A, Nieminen M, et al. Myoblast-mediated gene therapy improves functional collateralization in chronic cerebral hypoperfusion. Stroke, 2015, 46(1):203-211.
4. Pagani FD, DerSimonian H, Zawadzka A, et al. Autologous skeletal myoblast transplanted to ischemia-damaged myocardium in humans.Histological analysis of cell survival and diffierentiation. J Am Coll Cardiol, 2003, 41(5):879-888.
5. Ye L, Haider HKh, Tan R, et al. Transplantation of nanoparticle transfected skeletal myoblasts overexpressing vascular endothelial growth factor-165 for cardiac repair. Circulation, 2007, 116(11 Suppl):113-120.
6. Ye L, Haider HKh, Jiang S, et al. Improved angiogenic response in pig heart following ischaemic injury using human skeletal myoblast simultaneously expressing VEGF165 and angiopoietin-1. Eur J Heart Fail, 2007, 9(1):15-22.
7. Ye L, Lee KO, Su LP, et al. Skeletal myoblast transplantation for attenuation of hyperglycaemia, hyperinsulinaemia and glucose intolerance in a mouse model of type 2 diabetes mellitus. Diabetologia, 2009, 52(9):1925-1934.
8. Cheng Q, Law PK, de Gasparo M, et al. Combination of the dipeptidyl peptidase IV inhibitor LAF237[(S)-1-[(3-hydroxy-1-adamantyl) ammo] acetyl-2-cyanopyrrolidine] with the angiotensin Ⅱ Type 1 receptor antagonist valsartan[N-(1-Oxopentyl)-N-[[2-(1H-tetrazol-5-yl)-[1,1-biphenyl]-4-yl]methyl]-L-valine] enhances pncreatic islet morphology and function in a mouse model of type 2 diabetes. J Pharmacol Exp Ther, 2008, 327(3):683-691.
9. 秦瑞峰, 顾晓明, 黄富国, 等. 流式细胞术测定骨骼肌细胞分化中DNA合成及其细胞周期变化. 第四军医大学学报, 2000, 21(8):921-923.
10. 秦瑞峰, 顾晓明, 张浩. 不同培养条件对骨骼肌细胞增殖及分化的影响. 实用口腔医学杂志, 2000, 16(1):14-16.
11. 刘立斌, 周红雨. 成肌细胞的临床应用现状及前景. 中国修复重建外科杂志, 2007, 21(7):767-770.
12. 刘宁, 刘雅南, 刘涛, 等. 硫酸软骨素的制备研究及发展现状. 食品工业科技, 2014, 35(3):392-395.
13. 陈小宇, 刘成成, 祝爱珍, 等. 硫酸软硫骨素对3T3-L1细胞增殖和分化的影响. 中国医科大学学报, 2013, 42(7):599-603.
14. Haylock-Jacobs S, Keoμgh MB, Lau L, et al. Chondroitin sulphate proteoglycans:extracellular matrix proteins that regulate immunity of the central nervous system. Autoimmun Rev, 2011, 10(12):766-772.
15. Lee JY, Lee SH, Kim HJ, et al. The preventive inhibition of chondroitin sulfate against the CCl4-induced oxidative stress of subcellular level. Arch Pharm Res, 2004, 27(3):340-345.
16. Nishimoto S, Takagi M, Wakitani S, et al. Effect of chondroitin sulfate and hyaluronic acid on gene expression in a three-dimensional culture of chondrocytes. J Biosci Bioeng, 2005, 100(1):123-126.
17. Dubey L, Sharma M. Cardiogenic shock complicating acute myocardial infarction-a review. Acta Cardiol, 2011, 66(6):691-699.
18. Peters K, Kaufman M, Dmochowski L, et al. 1340 autologous muscle derived cell therapy for the treatment of female stress urinary incontinence:a multi-center experience. J Urol, 2011, 185(4):e535-536.
19. Baj A, Bettaccini AA, Casalone R, et al. Culture of skeletal myoblasts from human donors aged over 40 years:dynamics of cell growth and expression of differentiation markers. J Transl Med, 2005, 3(1):21.
20. Jarocha D, Stangel-Wojcikiewicz K, Basta A, et al. Efficient myoblast expansion for regenerative medicine use. Int J Mol Med, 2014, 34(1):83-91.

Chinese Journal of Reparative and Reconstructive Surgery

PROMOTION EFFECT OF CHONDROITIN SULFATE ON PROLIFERATION OF MYOBLASTS

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