Cyclic mechanical stretch influences cell adhesion and spreading of immortalized human keratinocytes

doi:10.3969/j.issn.2095-4344.2015.01.021

Abstract

Abstract:

BACKGROUND: The mechanical environment of skin tissue and spreading state of epithelial cells are closely related with the wound healing and scar formation process.
OBJECTIVE: To analyze the effect of extracellular mechanical stimulation on cell spreading, to test the cell proliferation in order to analyze the effect of spreading form on cell proliferation and other physiological activities.
METHODS: Cyclic sine wave mechanical stretching was exerted on immortalized human keratinocyte by using FX-4000 flexible substrate loading system, on the condition of 0.2 Hz and at frequency of 10% amplitudes. The spreading form was compared at 0, 24 and 48 hours, the cell proliferation was analyzed with flow cytometry, and the distribution of vinculin was analyzed with immunofluorescence staining.
RESULTS AND CONCLUSION: human keratinocyte would keep the spreading state and could induce more cell proliferation by 24 hours mechanical stretching stimulation. Conversely, after stimulated for 48 hours, the morphology of the human keratinocyte was significantly changed, and the number of human keratinocyte in the division stage was larger than that in the static control group; under tensile stress, the distribution of vinculin was transformed from the surrounding nucleus membrane area to the cell edge. The results indicate that proper mechanical stimulate can increase cell proliferation with keeping cell spreading and adhesion state; the stimulating time of continuous cyclic stretching is the major factor to determine cell spreading morphology and adhesion regions of immortalized human keratinocyte.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: Biomechanics, Stress, Mechanical, Cell Adhesion, Cell Proliferation

CLC Number:

R318

Liu Kun, An Mei-wen, Wang Li, Huang Jing-jing. Cyclic mechanical stretch influences cell adhesion and spreading of immortalized human keratinocytes[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(1): 119-123.

Figures/Tables 1

2.1 周期性拉伸应力对HaCaT细胞铺展形态的影响正常培养的对数生长期HaCaT细胞呈典型上皮细胞铺路石样特征，细胞三角形或不规则形状，排列紧密，轮廓清楚折光性好(图1A)。经过周期性正弦波拉伸刺激后24 h，绝大多数细胞铺展形态没有明显的变化，有个别细胞在拉伸刺激作用下，形态发生的变化，呈现出梭形或者其他不规则形状(图1B)。拉伸48 h后的细胞与对照组对比，形态近梭形、长条形等的细胞明显增多，有的细胞延伸出很长的突触，HaCaT细胞的铺展方式随着拉伸应力作用时间的延长在发生改变(图1C)。 2.2 周期性力学刺激对于HaCaT细胞内纽蛋白表达的影响通过免疫荧光标记技术，观察纽蛋白在静态对照组和拉伸应力作用下的分布，见图2。图2A是静态对照HaCaT细胞的扭蛋白分布，可以看出纽蛋白主要分布在核以外的胞质区域，而且纽蛋白在细胞的铺展边缘并没有明显分布。拉伸应力作用24 h后，纽蛋白的分布区域与静态组相似。拉伸应力作用48 h后，可以看出随着细胞铺展形态的改变和细胞突出的延伸，纽蛋白在细胞的异常突出部分有明显分布(图C，箭头所示)。纽蛋白主要存在于细胞与基底发生粘着的部位，也是应力纤维形成的的区域，其分布的改变表明细胞的整体黏附方式发生改变。可以看到，由于不是单周拉伸作用，48 h处理组细胞的延伸方向并不相同，细胞隆起，与基底的黏附面积减少并形成明显的的极性，纽蛋白向细胞边缘和细胞突出延伸部分的重分布表示细胞在改变自身黏附以适应力学环境的改变。这种因为力学因素引起的细胞形态改变和黏附相关因子的重分布也许会影响细胞增殖、分化等生理活动。 2.3 周期性正弦波拉伸对HaCaT细胞增殖的影响为了进一步研究力学刺激作用于细胞铺展和黏附，进而对细胞正常生理活动造成的影响，测定了力学加载下分裂期细胞的含量。流式细胞仪检测得到24，48 h拉伸组组和静态对照组G0/G1，S，G2/M各期细胞的百分率见图3。从图中可看出G2/M期细胞所对应的横坐标值大约是G0/G1期细胞对应的横坐标值的2倍，可以看出，24 h拉伸组比24 h静态对照组S期DNA含量明显增多；而48 h拉伸组比48 h静态对照组S期DNA含量略有降低。静态对照组HaCaT细胞24，48 h增殖指数分别为44.63%，56.58%；拉伸组HaCaT细胞24，48 h增殖指数分别为60.89%，51.69%。从以上结果看出，24 h周期性拉伸应力作用下的HaCaT细胞较静态对组增殖明显增加，经过48 h的拉伸刺激之后，处于分裂期的细胞较48 h静态对照组和24 h拉伸应力处理组均有减少。因此认为力学刺激在幅度10%，频率0.2 Hz的周期性拉伸应力作用下，合理的加载时间能够促进细胞增殖，过长的加载时间会引起细胞增殖降低或者导致细胞凋亡增多。与上述铺展和黏附的观测结果相比较来看，细胞在外界力学刺激作用下能够维持其正常的黏附和铺展状态的时间内，会使细胞增殖更为明显。可见HaCaT能够响应一定的外界力学刺激，对自身铺展和增殖做出适应性改变，但是时间过长的力学刺激会引起细胞生理活动的根本性改变。适度的力学刺激和合理的应力作用时间能够促进皮肤细胞更迅速的增殖。"

References

[1] Takei T, Rivas-Gotz C, Delling CA,et al.Effect of strain on human keratinocytes in vitro.J Cell Physiol. 1997;173(1): 64-72.
[2] Görmar FE, Bernd A, Bereiter-Hahn J,et al.A new model of epidermal differentiation: induction by mechanical stimulation.Arch Dermatol Res. 1990;282(1):22-32.
[3] Boukamp P, Petrussevska RT, Breitkreutz D,et al.Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988;106(3):761-771.
[4] Amano M, Chihara K, Kimura K,et al.Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase.Science. 1997;275(5304):1308-1311.
[5] Palazzo AF, Eng CH, Schlaepfer DD,et al.Localized stabilization of microtubules by integrin- and FAK-facilitated Rho signaling.Science. 2004;303(5659):836-839.
[6] Ingber D.Integrins as mechanochemical transducers.Curr Opin Cell Biol. 1991;3(5):841-848.
[7] Kippenberger S, Loitsch S, Müller J,et al.Melanocytes respond to mechanical stretch by activation of mitogen-activated protein kinases (MAPK).Pigment Cell Res. 2000;13(4):278-280.
[8] Fornaro M, Steger CA, Bennett AM,et al.Differential role of beta(1C) and beta(1A) integrin cytoplasmic variants in modulating focal adhesion kinase, protein kinase B/AKT, and Ras/Mitogen-activated protein kinase pathways.Mol Biol Cell. 2000;11(7):2235-2249.
[9] Bush JA, Ferguson MW, Mason T, et al.Skin tension or skin compression? Small circular wounds are likely to shrink, not gape. J Plast Reconstr Aesthet Surg.2008;61(5):529-534..
[10] Reichelt J.Mechanotransduction of keratinocytes in culture and in the epidermis.Eur J Cell Biol. 2007;86(11-12):807-816.
[11] Khwaja A, Rodriguez-Viciana P, Wennström S,et al.Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway.EMBO J. 1997;16(10):2783-2793.
[12] Renò F, Traina V, Cannas M.Mechanical stretching modulates growth direction and MMP-9 release in human keratinocyte monolayer.Cell Adh Migr. 2009;3(3):239-242.

[1]	Xu Feng, Kang Hui, Wei Tanjun, Xi Jintao. Biomechanical analysis of different fixation methods of pedicle screws for thoracolumbar fracture [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(9): 1313-1317.
[2]	Chen Xinmin, Li Wenbiao, Xiong Kaikai, Xiong Xiaoyan, Zheng Liqin, Li Musheng, Zheng Yongze, Lin Ziling. Type A3.3 femoral intertrochanteric fracture with augmented proximal femoral nail anti-rotation in the elderly: finite element analysis of the optimal amount of bone cement [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(9): 1404-1409.
[3]	Zhou Jihui, Li Xinzhi, Zhou You, Huang Wei, Chen Wenyao. Multiple problems in the selection of implants for patellar fracture [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(9): 1440-1445.
[4]	Li Cai, Zhao Ting, Tan Ge, Zheng Yulin, Zhang Ruonan, Wu Yan, Tang Junming. Platelet-derived growth factor-BB promotes proliferation, differentiation and migration of skeletal muscle myoblast [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(7): 1050-1055.
[5]	Liu Cong, Liu Su. Molecular mechanism of miR-17-5p regulation of hypoxia inducible factor-1α mediated adipocyte differentiation and angiogenesis [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(7): 1069-1074.
[6]	Xu Yulin, Shen Shi, Zhuo Naiqiang, Yang Huilin, Yang Chao, Li Yang, Zhao Heng, Zhao Lu. Biomechanical comparison of three different plate fixation methods for acetabular posterior column fractures in standing and sitting positions [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(6): 826-830.
[7]	Cai Qunbin, Zou Xia, Hu Jiantao, Chen Xinmin, Zheng Liqin, Huang Peizhen, Lin Ziling, Jiang Ziwei. Relationship between tip-apex distance and stability of intertrochanteric femoral fractures with proximal femoral anti-rotation nail: a finite element analysis [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(6): 831-836.
[8]	Song Chengjie, Chang Hengrui, Shi Mingxin, Meng Xianzhong. Research progress in biomechanical stability of lateral lumbar interbody fusion [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(6): 923-928.
[9]	Ma Zetao, Zeng Hui, Wang Deli, Weng Jian, Feng Song. MicroRNA-138-5p regulates chondrocyte proliferation and autophagy [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(5): 674-678.
[10]	Xie Chongxin, Zhang Lei. Comparison of knee degeneration after anterior cruciate ligament reconstruction with or without remnant preservation [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(5): 735-740.
[11]	Wang Yujiao, Liu Dan, Sun Song, Sun Yong. Biphasic calcium phosphate loaded with advanced platelet rich fibrin can promote the activity of rabbit bone marrow mesenchymal stem cells [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(4): 504-509.
[12]	Zhou Jihui, Yao Meng, Wang Yansong, Li Xinzhi, Zhou You, Huang Wei, Chen Wenyao. Influence of novel nanoscaffolds on biological behaviors of neural stem cells and the related gene expression [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(4): 532-536.
[13]	Nie Shaobo, Li Jiantao, Sun Jien, Zhao Zhe, Zhao Yanpeng, Zhang Licheng, Tang Peifu. Mechanical stability of medial support nail in treatment of severe osteoporotic intertrochanteric fracture [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(3): 329-333.
[14]	Tan Jiachang, Yuan Zhenchao, Wu Zhenjie, Liu Bin, Zhao Jinmin. Biomechanical analysis of elastic nail combined with end caps and wire fixation for long oblique femoral shaft fractures [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(3): 334-338.
[15]	Chen Lu, Zhang Jianguang, Deng Changgong, Yan Caiping, Zhang Wei, Zhang Yuan. Finite element analysis of locking screw assisted acetabular cup fixation [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(3): 356-361.