基底刚度对人宫颈癌HeLa细胞生长影响的体外研究
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摘要
目的探讨基底刚度变化对宫颈癌HeLa细胞生长的影响。方法将人宫颈癌HeLa细胞株培养于基底刚度为0.5、5.0、25.0 kPa水凝胶培养皿和基底刚度约为1.0×10~6kPa的普通塑料/玻璃培养皿中。采用倒置显微镜和环境扫描电子显微镜观察细胞的形态、结构,采用单光子激光共聚焦显微镜观察细胞骨架,采用CCK-8法检测细胞增殖活性并绘制生存曲线。结果随着基底刚度的增加,细胞铺展面积增加,细胞贴壁愈加牢固,细胞形态呈多边形或梭形;不同基底刚度组的最长直径/最短直径、铺展面积比较,差异均有统计学意义(F=15.54、77.21,P﹤0.01);随着基底刚度的增加,细胞伪足逐渐丰富且纤长,细胞骨架应力纤维表达增多;以48 h培养为基础计算细胞倍增时间后显示,0.5、5.0、25.0、1.0×10~6kPa基底刚度上培养的HeLa细胞的倍增时间分别为60.40、39.43、27.79、26.34 h,细胞倍增时间随着基底刚度的增加而缩短,细胞增殖加快。结论较大的基底刚度更利于人宫颈癌HeLa细胞铺展、细胞骨架分布和细胞增殖。
Objective To explore the effect of substrate stiffness on the growth of cervical cancer HeLa cells in vitro. Method Human cervical cancer cell line HeLa was cultured in hydrogel culture dishes with the substrate stiffness of 0.5, 5.0 and 25.0 kPa, as well as in ordinary plastic/glass culture dishes with the substrate stiffness of 1.0×106 kPa under same conditions. Cell morphological structure was observed using inverted microscope and the environmental scanning electron microscope; cell nucleus and cytoskeleton were observed under the single-photon laser-scanning confocal microscope. Cell proliferation activity was detected using the cell counting kit-8 and survival curves were plotted. Result As the substrate stiffness increased, HeLa cells tended to spread, and adhered to culture dishes more firmly, with polygon or spindle morphology; The maximum/minimum diameter and cell spread area among dishes with different stiffness showed statistically significant difference(F=15.54, 77.21, P<0.01); As the substrate stiffness increased, higher stiffness of dishes promoted cells to grow more long pseudopodia and expressed increased cytoskeletal stress fibers; the culture time of 48 h was used to calculate the time for cell doubling, and it was found that, the doubling time of HeLa cells cultured in dishes with substrate stiffness of 0.5, 5.0, 25.0, and 1.0×106 kPa was 60.40, 39.43, 27.79 and 26.34 h, respectively, suggesting that the cell doubling time decreased, while cell proliferation accelerated as the substrate stiffness increased. Conclusion Higher substrate stiffness is favorable for human cervical cancer cells HeLa to spread, and may promote cytoskeletal distribution and cell proliferation.
Objective To explore the effect of substrate stiffness on the growth of cervical cancer HeLa cells in vitro. Method Human cervical cancer cell line HeLa was cultured in hydrogel culture dishes with the substrate stiffness of 0.5, 5.0 and 25.0 kPa, as well as in ordinary plastic/glass culture dishes with the substrate stiffness of 1.0×106 kPa under same conditions. Cell morphological structure was observed using inverted microscope and the environmental scanning electron microscope; cell nucleus and cytoskeleton were observed under the single-photon laser-scanning confocal microscope. Cell proliferation activity was detected using the cell counting kit-8 and survival curves were plotted. Result As the substrate stiffness increased, HeLa cells tended to spread, and adhered to culture dishes more firmly, with polygon or spindle morphology; The maximum/minimum diameter and cell spread area among dishes with different stiffness showed statistically significant difference(F=15.54, 77.21, P<0.01); As the substrate stiffness increased, higher stiffness of dishes promoted cells to grow more long pseudopodia and expressed increased cytoskeletal stress fibers; the culture time of 48 h was used to calculate the time for cell doubling, and it was found that, the doubling time of HeLa cells cultured in dishes with substrate stiffness of 0.5, 5.0, 25.0, and 1.0×106 kPa was 60.40, 39.43, 27.79 and 26.34 h, respectively, suggesting that the cell doubling time decreased, while cell proliferation accelerated as the substrate stiffness increased. Conclusion Higher substrate stiffness is favorable for human cervical cancer cells HeLa to spread, and may promote cytoskeletal distribution and cell proliferation.
引文
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[2]Engle AJ,Griffin MA,Sen S,et al.Myotubes differentiate optimally on substrates with tissue-like stiffness:pathological implications for soft or stiff microenvironments[J].Cell Biol,2004,166(6):877-887.
[3]Hartman CD,Isenberg BC,Chua SG,et al.Extracellular matrix type modulates cell migration on mechanical gradients[J].Exp Cell Res,2017,359(2):361-366.
[4]Yeung T,Georges PC,Flanagan LA,et al.Effects of substrate stiffness on cell morphology,cytoskeletal structure and adhesion[J].Cell Motil Cytoskeleton,2005,60(1):24-34.
[5]Pan Q,Li DG,Lu HM,et al.A new immortalized rat cell line,hepatic stellate cell-PQ,exhibiting characteristics of hepatic stellate cell[J].Hepatobiliary Pancreat Dis Int,2005,4(2):281-284.
[6]Liu F,Tschumperlin DJ.Micro-mechanical characterization of lung tissue using atomic force microscopy[J].J Vis Exp,2011,(54):2911.
[7]Leventa I,Georgesa P,Janmey P.Soft biological materials and their impact on cell function[J].Soft Matter,2007,3(3):299-306.
[8]Palmeri ML,Feltovich H,Homyk AD,et al.Evaluating the feasibility of acoustic radiation force impulse shear wave elasticity imaging of the uterine cervix with an intracavity array:a simulation study[J].IEEE Trans Ultrason Ferroelectr Freq Control,2013,60(10):2053-2064.
[9]Zhao X,Zhong Y,Ye T.Discrimination between cervical cancer cells and normal cervical cells based on longitudinal elasticity using atomic force microscopy[J].Nanoscale Res Lett,2015,10(1):482.
[10]Lu P,Weaver VM,Werb Z.The extracellular matrix:a dynamic niche in cancer progression[J].J Cell Biol,2012,196(4):395-406.
[11]Trappmann B,Gautrot JE,Connelly JT,et al.Extracellularmatrix tethering regulates stem-cell fate[J].Nat Mater,2012,11(7):642-649.
[12]Tilghman RW,Cowan CR,Mih JD,et al.Matrix rigidity regulates cancer cell growth and cellular phenotype[J].PLo S One,2010,5(9):e12905.
[13]Wang Y,Wang G,Luo X,et al.Substrate stiffness regulates the proliferation,migration,and differentiation of epidermal cells[J].Burns,2012,38(3):414-420.
[14]Teixeira AI,Ilkhanizadeh S,Wigenius JA,et al.The promotion of neuronal maturation on soft substrates[J].Biomaterials,2009,30(27):4567-4572.
[15]O’connor RS,Hao X,Shen K,et al.Substrate rigidity regulates human T cell activation and proliferation[J].J Immunol,2012,189(3):1330-1339.
[16]Hui L,Zhang J,Ding X,et al.Matrix stiffness regulates the proliferation,stemness and chemoresistance of laryngeal squamous cancer cells[J].Int J Oncol,2017,50(4):1439-1447.
[17]Feng J,Tang Y,Xu Y,et al.Substrate stiffness influences the outcome of antitumor drug screening in vitro[J].Clin Hemorheol Microcirc,2013,55(1):121-131.