沉默ski基因对大鼠星形胶质细胞迁移的影响

doi:10.3969/j.issn.1006-9771.2017.08.008

《中国康复理论与实践》 ›› 2017, Vol. 23 ›› Issue (8): 905-911.doi: 10.3969/j.issn.1006-9771.2017.08.008

沉默ski基因对大鼠星形胶质细胞迁移的影响

赵鑫^1,2，王兴文^1,2，寇江力^1,2，李忠浩^1,2，郭永强^1,2，伍亚民³，张海鸿¹

1.兰州大学第二医院骨科，甘肃兰州市 730030；
2.甘肃省骨关节疾病研究重点实验室，甘肃兰州市 730030；
3.第三军医大学大坪医院野战外科研究所三室，创伤、烧伤与复合伤国家重点实验室，重庆市 400042。

收稿日期:2017-04-10 修回日期:2017-05-05 出版日期:2017-08-25 发布日期:2017-08-24
通讯作者: 张海鸿，主任医师，副教授，硕士研究生导师，主要研究方向：脊柱外科。E-mail: zhanghaihong1968@sina.com。
作者简介:赵鑫(1991-)，男，汉族，山东潍坊市人，硕士研究生，主要研究方向：脊柱脊髓损伤。
基金资助:
国家自然科学基金项目(No.30772299)；2.兰州大学第二医院科研基金项目(No.sdkyjj-04)。

Effect of Knocking Down ski on Migration of Astrocytes in Rats　

ZHAO Xin^1,2, WANG Xing-wen^1,2, KOU Jiang-li^1,2, LI Zhong-hao^1,2, GUO Yong-qiang^1,2, WU Ya-min³, ZHANG Hai-hong¹

1. Department of Orthopedics, Second Clinical Medical College of Lanzhou University, Lanzhou, Gansu 730030, China;
2. Key Laboratory of Orthopedics of Gansu Province, Lanzhou, Gansu 730030, China;
3. State Key Laboratory of Trauma, Burns and Combined Injury, the Third Department of Research Institute of Surgery, Daping Hospital, Third Military University, Chongqing 400042, China

Received:2017-04-10 Revised:2017-05-05 Published:2017-08-25 Online:2017-08-24
Contact: ZHANG Hai-hong. E-mail: zhanghaihong1968@sina.com

摘要/Abstract

摘要： 目的研究ski基因在大鼠星形胶质细胞迁移过程中的作用。方法分离大鼠脑皮质星形胶质细胞，体外培养。合成ski基因和阴性对照小干扰片段。设置ski-siRNA转染组、阴性对照组和空白对照组。采用脂质体法将特异性针对ski基因的siRNA和阴性对照siRNA转入大鼠星形胶质细胞。采用Western blotting检测各组ski蛋白的表达水平；采用Transwell细胞迁移实验和细胞划痕实验检测ski-siRNA转染后星形胶质细胞迁移能力的变化。结果与空白对照组和阴性对照组比较，ski-siRNA转染组细胞内ski蛋白的相对表达水平显著降低(F=132.957, P<0.001)。Transwell细胞迁移实验发现，ski-siRNA转染组每孔迁移细胞数少于空白对照组和阴性对照组(F>47.197, P<0.05)。细胞划痕实验显示，转染组细胞1~5 d的划痕愈合率均明显低于对照组(F>69.187, P<0.01)。结论沉默ski基因能显著抑制星形胶质细胞的迁移能力，ski可能参与星形胶质细胞的转移过程，进而调控胶质瘢痕的形成。

关键词: ski, RNA干扰, 星形胶质细胞, Transwell细胞迁移实验, 划痕实验, 大鼠

Abstract: Objective To investigate the effect of ski gene in migration process of astrocytes in rats. Methods Astrocytes were obtained from rats' cerebral cortex and cultured in vitro. siRNA targeting ski gene and negative control sequences were prepared. The ski-siRNA group, siRNA negative control group and untreated group were set in this experiment. The specific siRNA targeting ski gene was transfected into astrocytes with Lipofectamine?RNAiMAX Reagent. Then the ski protein levels were determined with Western blotting. After transfection, the changes in migration of astrocytes were measured with wound scratch assay and Transwell migration assay. Results Western blotting showed that the expression of ski protein was significantly lower in the ski-siRNA group than in the siRNA negative control group and untreated group (F=132.957, P<0.001). Transwell migration assay showed that the number of astrocytes crossing through chambers was less in the ski-siRNA group than in the siRNA negative control group and untreated group (F>47.197, P<0.05). Wound scratch assay showed that the wound healing rate was lower in the ski-siRNA group than in the control group one, two, three, four and five days after transfection (F>69.187, P<0.001). Conclusion Ski knocked down by siRNA could inhibit the migration ability of astrocytes. It is a reminding that ski may take part in the migration process of astrocytes, and moreover, ski may play an important role in the formation of glial scar.

Key words: ski, siRNA, astrocyte, Transwell migration assay, wound scratch assay, rats

中图分类号:

R364.5

赵鑫,王兴文,寇江力,李忠浩,郭永强,伍亚民，张海鸿. 沉默ski基因对大鼠星形胶质细胞迁移的影响[J]. 《中国康复理论与实践》, 2017, 23(8): 905-911.

ZHAO Xin,WANG Xing-wen,KOU Jiang-li, LI Zhong-hao, GUO Yong-qiang,WU Ya-min, ZHANG Hai-hong. Effect of Knocking Down ski on Migration of Astrocytes in Rats　[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2017, 23(8): 905-911.

参考文献

[1] Hol EM, Pekny M. Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system [J]. Curr Opin Cell Biol, 2015, 32: 121-130.
[2] Oliveira JF, Sardinha VM, Guerra-Gomes S, et al. Do stars govern our actions? Astrocyte involvement in rodent behavior [J]. Trends Neurosci, 2015, 38(9): 535-549.
[3] Martín R, Bajo-Gra?eras R, Moratalla R, et al. Circuit-specific signaling in astrocyte-neuron networks in basal ganglia pathways [J]. Science, 2015, 349(6249): 730-734.
[4] Chen PS, Peng GS, Li G, et al. Valproate protects dopaminergic neurons in midbrain neuron/glia cultures by stimulating the release of neurotrophic factors from astrocytes [J]. Mol Psychiatry, 2006, 11(12): 1116-1125.
[5] Sofroniew MV. Reactive astrocytes in neural repair and protection [J]. Neuroscientist, 2005, 11(5): 400-407.
[6] Pekny M, Wilhelmsson U, Pekna M. The dual role of astrocyte activation and reactive gliosis [J]. Neurosci Lett, 2014, 565: 30-38.
[7] Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation [J]. Trends Neurosci, 2009, 32(12): 638-647.
[8] Barnabé-Heider F, G?ritz C, Sabelstr?m H, et al. Origin of new glial cells in intact and injured adult spinal cord [J]. Cell Stem Cell, 2010, 7(4): 470-482.
[9] Zhan JS, Gao K, Chai RC, et al. Astrocytes in migration [J]. Neurochem Res, 2017, 42(1): 272-282.
[10] 周开升,朱彦东,张海鸿,等. Ski在神经系统中的作用及机制的研究进展[J]. 中国康复理论与实践, 2016, 22(7): 797-800.
[11] Bonnon C, Atanasoski S. c-Ski in health and disease [J]. Cell Tissue Res, 2012, 347(1): 51-64.
[12] 周开升,朱彦东,赵鑫,等. Ski在大鼠脊髓损伤后不同时间的表达变化[J]. 中国康复理论与实践, 2016, 22(9): 1015-1019.
[13] Zhou K, Nan W, Feng D, et al. Spatiotemporal expression of Ski after rat spinal cord injury[J]. Neuroreport, 2017, 28(3): 149-157.
[14] Stavnezer E, Barkas AE, Brennan LA, et al. Transforming Sloan-Kettering viruses generated from the cloned v-ski oncogene by in vitro and in vivo recombinations [J]. J Virol, 1986, 57(3): 1073-1083.
[15] Liu X, Sun Y, Weinberg RA, et al. Ski/Sno and TGF-beta signaling [J]. Cytokine Growth Factor Rev, 2001, 12(1): 1-8.
[16] Song L, Chen X, Gao S, et al. Ski modulate the characteristics of pancreatic cancer stem cells via regulating sonic hedgehog signaling pathway [J]. Tumour Biol, 2016, 37(12): 16115-16125.
[17] Wang P, Chen Z, Meng ZQ, et al. Dual role of Ski in pancreatic cancer cells: tumor-promoting versus metastasis-suppressive function [J]. Carcinogenesis, 2009, 30(9): 1497-1506.
[18] Li P, Liu P, Xiong RP, et al. Ski, a modulator of wound healing and scar formation in the rat skin and rabbit ear [J]. J Pathol, 2011, 223(5): 659-671.
[19] Sofroniew MV. Astrogliosis [J]. Cold Spring Harb Perspect Biol, 2014, 7(2): a020420.
[20] Jain P, Wadhwa PK, Jadhav HR. Reactive astrogliosis: role in Alzheimer's disease [J]. CNS Neurol Disord Drug Targets, 2015, 14(7): 872-879.
[21] Middeldorp J, Hol EM. GFAP in health and disease [J]. Prog Neurobiol, 2011, 93(3): 421-443.
[22] Orlandin JR, Ambrósio CE, Lara VM. Glial scar-modulation as therapeutic tool in spinal cord injury in animal models [J]. Acta Cir Bras, 2017, 32(2): 168-174.
[23] Gesteira TF, Coulson-Thomas YM, Coulson-Thomas VJ. Anti-inflammatory properties of the glial scar [J]. Neural Regen Res, 2016, 11(11): 1742-1743.
[24] Raposo C, Schwartz M. Glial scar and immune cell involvement in tissue remodeling and repair following acute CNS injuries [J]. Glia, 2014, 62(11): 1895-1904.
[25] Helwig K, Seeger F, H?lschermann H, et al. Elevated serum glial fibrillary acidic protein (GFAP) is associated with poor functional outcome after cardiopulmonary resuscitation [J]. Neurocrit Care, 2017, doi: 10.1007/s12028-016-0371-6. [Epub ahead of print].
[26] Lepekhin EA, Eliasson C, Berthold CH, et al. Intermediate filaments regulate astrocyte motility [J]. J Neurochem, 2001, 79(3): 617-625.
[27] Pekny M, Johansson CB, Eliasson C, et al. Abnormal reaction to central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin [J]. J Cell Biol, 1999, 145(3): 503-514.
[28] Cheng G, Gao F, Sun X, et al. Paris saponin VII suppresses osteosarcoma cell migration and invasion by inhibiting MMP2/9 production via the p38 MAPK signaling pathway [J]. Mol Med Rep, 2016, 14(4): 3199-3205.
[29] Andolfi L, Bourkoula E, Migliorini E, et al. Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy [J]. PLoS One, 2014, 9(11): e112582.

沉默ski基因对大鼠星形胶质细胞迁移的影响

Effect of Knocking Down ski on Migration of Astrocytes in Rats

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