RGD多肽修饰多孔钽可激活MG63细胞整合素/黏着斑激酶信号通路  

Porous tantalum coated with RGD polypeptide can activate the integrin/focal adhesion kinase signaling pathway of MG63 cells

作　　者：张辉[1] 王少华 王茜 王辉[4] 甘洪全 崔逸爽 李琪佳 王志强 Zhang Hui;Wang Shaohua;Wang Qian;Wang Hui;Gan Hongquan;Cui Yishuang;Li Qijia;Wang Zhiqiang(First Department of Joint Surgery,Second Hospital of Tangshan,Tangshan 063000,Hebei Province,China;Tianjin Emergency Center,Tianjin 300000,China;Medical Research Center,North China University of Science and Technology,Tangshan 063210,Hebei Province,China;First Department of Hand Surgery,Second Hospital of Tangshan,Tangshan 063000,Hebei Province,China;Department of Orthopedics,Affiliated Hospital of North China of Science and Technology,Tangshan 063000,Hebei Province,China)

机构地区：[1]唐山市第二医院关节一科,河北省唐山市063000 [2]天津市急救中心,天津市300000 [3]华北理工大学医学实验研究中心,河北省唐山市063210 [4]唐山市第二医院手一科,河北省唐山市063000 [5]华北理工大学附属医院骨科,河北省唐山市063000

出　　处：《中国组织工程研究》2021年第16期2535-2540,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家科技部科技支撑课题资助项目(2012BAE06B03),项目负责人:王志强;河北省科技支撑资助项目(16277776D),项目负责人:李琪佳;河北省医学科学研究重点课题计划资助项目(20160225),项目负责人:王志强;华北理工大学博士科研启动基金资助项目(28606299),项目负责人:王茜;河北省卫计委资助课题(20180733),项目负责人:王茜。

摘　　要：背景:在材料表面复合RGD多肽可诱导成骨细胞整合素基因的表达,促进成骨细胞黏附在生物材料的表面并分化成熟,促进新骨形成。目的:分析RGD多肽修饰国产多孔钽后对MG63细胞整合素/黏着斑激酶信号通路的影响。方法:制备RGD多肽修饰的多孔钽材料。将MG63细胞分别接种于多孔钽、RGD多肽修饰的多孔钽材料表面,以单纯培养的MG63细胞为空白组,培养1,3,5,7 d时,采用CCK-8法检测细胞增殖;培养1,3,5 d时,倒置显微镜下观察细胞生长状态;培养3,5 d时,扫描电镜观察细胞黏附;培养5 d时,采用Western-blot与RT-PCR法检测Ⅰ型胶原、整合素β1与黏着斑激酶表达。结果与结论:①RGD修饰组培养3,5,7 d的细胞增殖快于多孔钽组、空白组(P<0.05),多孔钽组各时间点细胞增殖与空白组比较差异无显著性意义(P>0.05);②倒置显微镜显示,培养1 d时,多孔钽组、RGD修饰组细胞贴附在材料边缘,细胞数量随着培养时间的延长逐渐增加,其中RGD修饰组细胞数量及密集程度优于多孔钽组;③扫描电镜显示,培养3 d时,多孔钽组、RGD修饰组材料表面均可见细胞黏附生长,细胞在材料孔壁及孔隙内黏附生长,伸出伪足嵌入孔洞内;5 d时,细胞分泌大量细胞外基质,细胞通过基质相互连接并逐渐覆盖材料表面,其中RGD修饰组细胞生长状态、基质分泌及细胞覆盖面积优于多孔钽组;④Western-blot检测显示,RGD修饰组Ⅰ型胶原、整合素β1蛋白表达高于多孔钽组、空白组(P<0.05),多孔钽组Ⅰ型胶原、整合素β1、黏着斑激酶蛋白表达高于空白组(P<0.05);⑤RT-PCR检测显示,RGD修饰组Ⅰ型胶原、整合素β1与黏着斑激酶mRNA表达高于多孔钽组、空白组(P<0.05),并且多孔钽组高于空白组(P<0.05);⑥结果表明,RGD多肽修饰多孔钽后可上调细胞膜上Ⅰ型胶原、整合素β1表达激活整合素/黏着斑激酶信号通路,促进细胞黏附、生长。BACKGROUND:The compounding of RGD polypeptide on the surface of the material can induce the expression of osteoblast integrin gene,promote the adhesion of osteoblasts to the surface of biomaterials and differentiate into mature cells,and promote the formation of new bone.OBJECTIVE:To analyze the effect of domestic porous tantalum modified by RGD polypeptide on integrin/focal adhesion kinase signaling pathway in MG63 cells.METHODS:Porous tantalum material modified by RGD polypeptide was prepared.MG63 cells were inoculated on the surface of porous tantalum and porous tantalum materials modified with RGD polypeptide.MG63 cells cultured alone were used as the blank group.When cultured for 1,3,5,and 7 days,the cell proliferation was detected by the CCK-8 method.At 1,3,and 5 days,the cell growth status was observed under an inverted microscope.At 3,5 days of culture,cell adhesion was observed with scanning electron microscope.At 5 days of culture,RT-PCR and western blot assay were used to detect type I collagen and integrinβ1 and focal adhesion kinase expression.RESULTS AND CONCLUSION:(1)The cell proliferation of the RGD modified group cultured at 3,5,and 7 days was faster than that of the porous tantalum group and the blank group(P<0.05).There was no significant difference in cell proliferation between the porous tantalum group and the blank group at each time point(P>0.05).(2)Observation by an inverted phase contrast microscope showed that the cells of the porous tantalum group and the RGD modified group were attached to the edge of the material when cultured for 1 day,and the number of cells gradually increased with the extension of the culture time.The number and density of cells in the RGD modified group were better than that of the porous tantalum group.(3)Observation by scanning electron microscope showed that cells adhered to the surface of the porous tantalum group and RGD modified group after 3 days of culture.The cells adhered to the material pore walls and pores,and protruded pseudopods into the pores.When

关 键 词：骨 材料 蛋白质 信号通路 RGD多肽 多孔钽 MG63细胞 Ⅰ型胶原 整合素 黏着斑激酶 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]