• 1 南方醫(yī)科大學(xué)解剖學(xué)教研室 廣東省組織構(gòu)建與檢測(cè)重點(diǎn)實(shí)驗(yàn)室（廣州，510515）;;
  • 2 寧夏醫(yī)科大學(xué)解剖教研室;

目的 探討不同頻率的周期性應(yīng)力加載對(duì)體外培養(yǎng)多層肌管極性與分化的影響，篩選優(yōu)化的肌組織體外應(yīng)力加載培養(yǎng)條件。 方法 體外培養(yǎng)C2C12 成肌細(xì)胞于Sylgard 184 鑄型凹槽誘導(dǎo)分化形成多層肌管組織，采用自制體外細(xì)胞拉伸 儀，對(duì)培養(yǎng)并分化的肌管進(jìn)行間歇性應(yīng)力加載：加載幅度10%，加載頻率分別為0（A 組）、0.25（B 組）、0.50（C 組）、1.00 Hz（D 組），加載時(shí)間3 次/d，每次1 h。連續(xù)加載5、7、10 d 時(shí)，觀察各組肌管形態(tài)； RT-PCR 和實(shí)時(shí)熒光定量PCR（real-time fl uorescent quantitative PCR，QRT-PCR）分析檢測(cè)成肌相關(guān)基因成肌分化抗原（myogenic differentiation antigen，MyoD）、肌細(xì)胞生成素（Myogenin）、結(jié)蛋白（Desmin）、肌球重鏈蛋白（myosin heavy chain，MyHC） mRNA 的表達(dá)
差異。 結(jié)果 倒置相差顯微鏡觀察示，應(yīng)力加載促進(jìn)各組肌管的極性融合及數(shù)量增加，其中B 組加載培養(yǎng)7 d 時(shí)，多層
肌管排列緊密，極性顯著。加載條件能促進(jìn)成肌相關(guān)基因mRNA 的表達(dá)：組內(nèi)隨加載時(shí)間延長，各組MyoD 的mRNA 表
達(dá)逐漸下降，7、10 d 與5 d 比較差異均有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05）；Myogenin、Desmin、MyHC 的mRNA 表達(dá)呈先升高后降低趨勢(shì)，以7 d 表達(dá)量最高；B 組除7 d 與10 d Desmin 的mRNA 表達(dá)比較差異無統(tǒng)計(jì)學(xué)意義（P  gt; 0.05）外，其余各時(shí)間點(diǎn)比較差異均有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05）。同時(shí)間點(diǎn)隨加載頻率增加，MyoD、Myogenin、Desmin、MyHC 的mRNA 表達(dá)呈先升高后降低趨勢(shì)，以B 組表達(dá)量最高；除5 d B、C 組Desmin 和10 d A、B 組MyHC 的mRNA 表達(dá)比較差異無統(tǒng)計(jì)學(xué)意
義（P  gt; 0.05） 外，其余各組與B組各相關(guān)基因mRNA表達(dá)比較，差異均有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05）。 結(jié)論 低頻（0.25 Hz）、適時(shí)（7 d）的周期性應(yīng)力加載有利于生長于Sylgard 184 彈性材料表面的多層肌管極性分化，但隨著應(yīng)力加載時(shí)間延長，肌管的老化加速。

引用本文： 黃維一,劉幸卉,陳榮,馮利強(qiáng),廖華,余磊,曾慧君. 不同頻率周期性應(yīng)力加載對(duì)體外多層肌管極性及分化的影響. 中國修復(fù)重建外科雜志, 2012, 26(6): 735-742. doi: 復(fù)制

1. Wigmore PM, Dunglison GF. The generation of fiber diversity during myogenesis. Int J Dev Biol, 1998, 42(2): 117-125.
2. Bach AD, Beier JP, Stern-Staeter J, et al. Skeletal muscle tissue engineering. J Cell Mol Med, 2004, 8(4): 413-422.
3. Peng H, Huard J. Muscle-derived stem cells for musculoskeletal tissue regeneration and repair. Transpl Immunol, 2004, 12(3-4): 311-319.
4. Stern-Straeter J, Riedel F, Bran G, et al. Advances in skeletal muscle tissue engineering. In Vivo, 2007, 21(3): 435-444.
5. Bian W, Bursac N. Tissue engineering of functional skeletal muscle: challenges and recent advances. IEEE Eng Med Biol Mag, 2008, 27(5): 109-113.
6. Wang PY, Yu HT, Tsai WB. Modulation of alignment and differentiation of skeletal myoblasts by submicron ridges/grooves surface structure. Biotechnol Bioeng, 2010, 106(2): 285-294.
7. Lee WY, Cheng WY, Yeh YC, et al. Magnetically directed self-assembly of electrospun superparamagnetic fibrous bundles to form three-dimensional tissues with a highly ordered architecture. Tissue Eng Part C Methods, 2011, 17(6): 651-661.
8. Flaibani M, Boldrin L, Cimetta E, et al. Muscle differentiation and myotubes alignment is influenced by micropatterned surfaces and exogenous electrical stimulation. Tissue Eng Part A, 2009, 15(9): 2447-2457.
9. Powell CA, Smiley BL, Mills J, et al. Mechanical stimulation improves tissue-engineered human skeletal muscle. Am J Physiol Cell Physiol, 2002, 283(5): C1557-1565.
10. Kroehne V, Heschel I, Schugner F, et al. Use of a novel collagen matrix with oriented pore structure for muscle cell differentiation in cell culture and in grafts. J Cell Mol Med, 2008, 12(5A): 1640-1648.
11. Grossi A, Yadav K, Lawson MA. Mechanical stimulation increases proliferation, differentiation and protein expression in culture: stimulation effects are substrate dependent. J Biomech, 2007, 40(15): 3354-3362.
12. Formigli L, Meacci E, Sassoli C, et al. Sphingosine 1-phosphate induces cytoskeletal reorganization in C2C12 myoblasts: physiological relevance for stress fibres in the modulation of ion current through stretch-activated channels. J Cell Sci, 2005, 118(Pt 6): 1161-1171.
13. Kook SH, Lee HJ, Chung WT, et al. Cyclic mechanical stretch stimulates the proliferation of C2C12 myoblasts and inhibits their differentiation via prolonged activation of p38 MAPK. Mol Cells, 2008, 25(4): 479-486.
14. Grossi A, Karlsson AH, Lawson MA. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation. Cell Biol Int, 2008, 32(6): 615-622.
15. Soltow QA, Lira VA, Betters JL, et al. Nitric oxide regulates stretch-induced proliferation in C2C12 myoblasts. J Muscle Res Cell Motil, 2010, 31(3): 215-225.
16. Otis JS, Burkholder TJ, Pavlath GK. Stretch-induced myoblast proliferation is dependent on the COX2 pathway. Exp Cell Res, 2005, 310(2): 417-425.
17. Boonen KJ, Langelaan ML, Polak RB, et al. Effects of a combined mechanical stimulation protocol: Value for skeletal muscle tissue engineering. J Biomech, 2010, 43(8): 1514-1521.
18. Pennisi CP, Olesen CG, de Zee M, et al. Uniaxial cyclic strain drives assembly and differentiation of skeletal myocytes. Tissue Eng Part A, 2011, 17(19-20): 2543-2550.
19. 王齊, 廖華, 秦建強(qiáng), 等. C2C12細(xì)胞誘導(dǎo)構(gòu)建三維骨骼肌組織. 解剖學(xué)報(bào), 2010, 41(4): 606-610.
20. Vandenburgh HH, Hatfaludy S, Karlisch P, et al. Mechanically induced alterations in cultured skeletal muscle growth. J Biomech, 1991, 24 Suppl 1: 91-99.
21. Schultz E, McCormick KM. Skeletal muscle satellite cells. Rev Physiol Biochem Pharmacol, 1994, 123: 213-257.
22. Zhan M, Jin B, Chen SE, et al. TACE release of TNF-alpha mediates mechanotransduction-induced activation of p38 MAPK and myogenesis. J Cell Sci, 2007, 120(Pt 4): 692-701.
  1. 1. Wigmore PM, Dunglison GF. The generation of fiber diversity during myogenesis. Int J Dev Biol, 1998, 42(2): 117-125.
  2. 2. Bach AD, Beier JP, Stern-Staeter J, et al. Skeletal muscle tissue engineering. J Cell Mol Med, 2004, 8(4): 413-422.
  3. 3. Peng H, Huard J. Muscle-derived stem cells for musculoskeletal tissue regeneration and repair. Transpl Immunol, 2004, 12(3-4): 311-319.
  4. 4. Stern-Straeter J, Riedel F, Bran G, et al. Advances in skeletal muscle tissue engineering. In Vivo, 2007, 21(3): 435-444.
  5. 5. Bian W, Bursac N. Tissue engineering of functional skeletal muscle: challenges and recent advances. IEEE Eng Med Biol Mag, 2008, 27(5): 109-113.
  6. 6. Wang PY, Yu HT, Tsai WB. Modulation of alignment and differentiation of skeletal myoblasts by submicron ridges/grooves surface structure. Biotechnol Bioeng, 2010, 106(2): 285-294.
  7. 7. Lee WY, Cheng WY, Yeh YC, et al. Magnetically directed self-assembly of electrospun superparamagnetic fibrous bundles to form three-dimensional tissues with a highly ordered architecture. Tissue Eng Part C Methods, 2011, 17(6): 651-661.
  8. 8. Flaibani M, Boldrin L, Cimetta E, et al. Muscle differentiation and myotubes alignment is influenced by micropatterned surfaces and exogenous electrical stimulation. Tissue Eng Part A, 2009, 15(9): 2447-2457.
  9. 9. Powell CA, Smiley BL, Mills J, et al. Mechanical stimulation improves tissue-engineered human skeletal muscle. Am J Physiol Cell Physiol, 2002, 283(5): C1557-1565.
  10. 10. Kroehne V, Heschel I, Schugner F, et al. Use of a novel collagen matrix with oriented pore structure for muscle cell differentiation in cell culture and in grafts. J Cell Mol Med, 2008, 12(5A): 1640-1648.
  11. 11. Grossi A, Yadav K, Lawson MA. Mechanical stimulation increases proliferation, differentiation and protein expression in culture: stimulation effects are substrate dependent. J Biomech, 2007, 40(15): 3354-3362.
  12. 12. Formigli L, Meacci E, Sassoli C, et al. Sphingosine 1-phosphate induces cytoskeletal reorganization in C2C12 myoblasts: physiological relevance for stress fibres in the modulation of ion current through stretch-activated channels. J Cell Sci, 2005, 118(Pt 6): 1161-1171.
  13. 13. Kook SH, Lee HJ, Chung WT, et al. Cyclic mechanical stretch stimulates the proliferation of C2C12 myoblasts and inhibits their differentiation via prolonged activation of p38 MAPK. Mol Cells, 2008, 25(4): 479-486.
  14. 14. Grossi A, Karlsson AH, Lawson MA. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation. Cell Biol Int, 2008, 32(6): 615-622.
  15. 15. Soltow QA, Lira VA, Betters JL, et al. Nitric oxide regulates stretch-induced proliferation in C2C12 myoblasts. J Muscle Res Cell Motil, 2010, 31(3): 215-225.
  16. 16. Otis JS, Burkholder TJ, Pavlath GK. Stretch-induced myoblast proliferation is dependent on the COX2 pathway. Exp Cell Res, 2005, 310(2): 417-425.
  17. 17. Boonen KJ, Langelaan ML, Polak RB, et al. Effects of a combined mechanical stimulation protocol: Value for skeletal muscle tissue engineering. J Biomech, 2010, 43(8): 1514-1521.
  18. 18. Pennisi CP, Olesen CG, de Zee M, et al. Uniaxial cyclic strain drives assembly and differentiation of skeletal myocytes. Tissue Eng Part A, 2011, 17(19-20): 2543-2550.
  19. 19. 王齊, 廖華, 秦建強(qiáng), 等. C2C12細(xì)胞誘導(dǎo)構(gòu)建三維骨骼肌組織. 解剖學(xué)報(bào), 2010, 41(4): 606-610.
  20. 20. Vandenburgh HH, Hatfaludy S, Karlisch P, et al. Mechanically induced alterations in cultured skeletal muscle growth. J Biomech, 1991, 24 Suppl 1: 91-99.
  21. 21. Schultz E, McCormick KM. Skeletal muscle satellite cells. Rev Physiol Biochem Pharmacol, 1994, 123: 213-257.
  22. 22. Zhan M, Jin B, Chen SE, et al. TACE release of TNF-alpha mediates mechanotransduction-induced activation of p38 MAPK and myogenesis. J Cell Sci, 2007, 120(Pt 4): 692-701.