细胞外基质蛋白模式对成肌细胞分化的影响
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摘要
成肌细胞分化机理至今尚不明确,为此主要研究细胞外基质蛋白模式对成肌细胞分化的影响。采用微接触印刷技术制备4种不同的纤维粘连蛋白微图案(平行组、随机组、垂直组、对照组)培养成肌细胞,用免疫荧光染色法分析7 d后各组成肌细胞的分化情况,并分析2、6、18 h后各组中的黏附分子、Vinculin以及微丝蛋白F-actin的分布情况,每组实验重复3次,每次取3个样本。肌管形成个数平行排列组与对照组相比,无显著性差异(17.33±0.58 vs 16.00±1.73),与垂直组(7.00±1.00)和无极性组(10.89±0.19)相比则显著高(P<0.05)。在2 h时,微丝在细胞中排列与局部微图案一致;在6 h时,微丝开始沿整体图案长轴方向排列;在12 h时,微丝分布与条带整体图案长轴方向一致;18 h时,所形成的黏着斑会沿着微图案区域分布。细胞外基质蛋白模式会对成肌细胞的分化有影响,不同FN极性排列微图案对成肌细胞的骨架和黏附影响不同,这也可能是后期产生不同分化结果的一个原因。
The differentiation mechanism of skeletal myoblast differentiation is still unclear. The main purpose of this research is to explore the effect of the extracellular matrix(ECM) protein pattern on myogenic differentiation. In this study, four different fibronectin micropatterns(parallel, random, vertical, and control group) were prepared by microcontact printing technology, and the differentiation of C2 C12 cells after 7 day incubation was analyzed by immunofluorescence staining and analyzes the adhesion molecule, Vinculin and F-actin distribution after 2 h, 6 h, 18 h between groups by immunofluorescence staining method, each group experiment repeated 3 times, each time point had 3 parallel samples. The myotubes number formed in the control group had no significant difference compared with that in the parallel alignment group(17.33±0.58 vs 16±1.73), but was significantly higher than the vertical group(7.00±1.00) and the random group significantly(10.89±0.19)(P<0.05). At 2 h, the arrangement of microfilaments in cells was consistent with the local micropatterns. At 6 h, the microfilament began to align along the long axis direction of the whole strips pattern. At 12 h, the distribution of microfilaments was consistent with that of the long axis direction of the whole strips pattern. At 18 h, the adhesion plaques formed was distributed along the micropattern area. In conclusion, the extracellular matrix protein patterns are influential on the differentiation of myoblasts, different fibronectin polarity micropatterns have distinct effects on myoblasts cytoskeleton and adhesion, and it might be one of the reasons that later differentiation of C2 C12 results in different outcomes.
The differentiation mechanism of skeletal myoblast differentiation is still unclear. The main purpose of this research is to explore the effect of the extracellular matrix(ECM) protein pattern on myogenic differentiation. In this study, four different fibronectin micropatterns(parallel, random, vertical, and control group) were prepared by microcontact printing technology, and the differentiation of C2 C12 cells after 7 day incubation was analyzed by immunofluorescence staining and analyzes the adhesion molecule, Vinculin and F-actin distribution after 2 h, 6 h, 18 h between groups by immunofluorescence staining method, each group experiment repeated 3 times, each time point had 3 parallel samples. The myotubes number formed in the control group had no significant difference compared with that in the parallel alignment group(17.33±0.58 vs 16±1.73), but was significantly higher than the vertical group(7.00±1.00) and the random group significantly(10.89±0.19)(P<0.05). At 2 h, the arrangement of microfilaments in cells was consistent with the local micropatterns. At 6 h, the microfilament began to align along the long axis direction of the whole strips pattern. At 12 h, the distribution of microfilaments was consistent with that of the long axis direction of the whole strips pattern. At 18 h, the adhesion plaques formed was distributed along the micropattern area. In conclusion, the extracellular matrix protein patterns are influential on the differentiation of myoblasts, different fibronectin polarity micropatterns have distinct effects on myoblasts cytoskeleton and adhesion, and it might be one of the reasons that later differentiation of C2 C12 results in different outcomes.
引文
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[2] Morimoto Y, Takeuchi S. In vitro construction of skeletal muscle tissues[J]. Clin Calcium, 2017, 27(3):383-389.
[3] Clark P, Dunn G, Knibbs A, et al. Alignment of myoblasts on ultrafine gratings inhibits fusion in vitro [J]. The International Journal of Biochemistry & Cell Biology, 2002, 34(7): 816-825.
[4] Tourovskaia A, Barber T, Wickes BT, et al. Micropatterns of chemisorbed cell adhesion-repellent films using oxygen plasma etching and elastomeric masks [J]. Langmuir, 2003, 19(11): 4754-4764.
[5] Zatti S, Zoso A, Serena E, et al. Micropatterning topology on soft substrates affects myoblast proliferation and differentiation [J]. Langmuir, 2012, 28(5): 2718-2726.
[6] Sun Y, Duffy R, Lee A, et al. Optimizing the structure and contractility of engineered skeletal muscle thin films [J]. Acta Biomaterialia, 2013, 9(8): 7885-7894.
[7] Lynch MP, Stein JL, Stein GS, et al. The influence of type I collagen on the development and maintenance of the osteoblast phenotype in primary and passaged rat calvarial osteoblasts: modification of expression of genes supporting cell growth, adhesion, and extracellular matrix mineralization[J]. Experimental Cell Research, 1995, 216(1):35-45.
[8] Hedin U, Bottger BA, Forsberg E, et al. Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells[J]. Journal of Cell Biology, 1988, 107(1):307-319.
[9] Snyder J, Rin SA, Hamid Q, et al. Mesenchymal stem cell printing and process regulated cell properties[J]. Biofabrication, 2015, 7(4):044106.
[10] Théry M, Piel M. Adhesive micropatterns for cells: a microcontact printing protocol.[J]. Cold Spring Harbor Protocols, 2009, 2009(7):637-641.
[11] Verhulsel M, Vignes M, Descroix S, et al. A review of microfabrication and hydrogel engineering for micro-organs on chips[J]. Biomaterials, 2014, 35(6):1816-1832.
[12] Tourovskaia A, Barber T, Wickes BT, et al. Micropatterns of chemisorbed cell adhesion-repellent films using oxygen plasma etching and elastomeric masks [J]. Langmuir, 2003, 19(11): 4754-4764.
[13] Li B, Lin M, Tang Y, et al. A novel functional assessment of the differentiation of micropatterned muscle cells [J]. Journal of Biomechanics, 2008, 41(16): 3349-3353.
[14] Ye NN, Qin JH, Shi WW, et al. Characterizing doxorubicin-induced apoptosis in HepG2 cells using an integrated microfluidic device [J]. Electrophoresis, 2007, 28(7): 1146-1153.
[15] Wang PY, Yu HT, Tsai WB. Modulation of alignment and differentiation of skeletal myoblasts by submicron ridges/grooves surface structure [J]. Biotechnology and Bioengineering, 2010, 106(2): 285-294.
[16] Shen JY, Chan-Park MB, Feng ZQ, et al. UV-embossed microchannel in biocompatible polymeric film: application to control of cell shape and orientation of muscle cells[J]. Journal of Biomedical Materials Research Part B Applied Biomaterials, 2010, 77B(2):423-430.
[17] Song M, Uhrich KE. Optimal micropattern dimensions enhance neurite outgrowth rates, lengths, and orientations[J]. Annals of Biomedical Engineering, 2007, 35(10):1812-1820.
[18] Fujie T, Shi X, Ostrovidov S, et al. Spatial coordination of cell orientation directed by nanoribbon sheets[J]. Biomaterials, 2015, 53:86-94.
[19] Takayama Y, Wagatsuma A, Hoshino T, et al. Simple micropatterning method for enhancing fusion efficiency and responsiveness to electrical stimulation of C2C12 myotubes[J]. Biotechnology Progress, 2015, 31(1):220-225.
[20] Liu X, Liu Y, Zhao F, et al. Regulation of cell arrangement using a novel composite micropattern.[J]. Journal of Biomedical Materials Research Part A, 2017, 105(11):3093-3101.
[21] Moraes C, Kim BC, Zhu X, et al. Defined topologically-complex protein matrices to manipulate cell shape via three-dimensional fiber-like patterns.[J]. Lab on a Chip, 2014, 14(13):2191-2201.