摘要
背景:生长分化因子5作为骨形态发生蛋白的一员在软骨及骨组织修复领域表现出良好的应用潜力,增强生长分化因子5与骨组织的亲和力是提高蛋白使用效率的关键,因而开发具有骨靶向性的生长分化因子5蛋白具有重要意义。目的:利用双膦酸盐修饰生长分化因子5并探讨改性后蛋白对小鼠成骨前体细胞生长分化的影响。方法:采用化学交联法将生长分化因子5与帕米膦酸钠偶联,得到偶联帕米膦酸钠的生长分化因子5,采用傅里叶变换红外光谱、圆二色谱对其基团及结构进行表征,利用ELISA试剂盒测定生长分化因子5与磷酸钙的结合量以及生长分化因子5的体外释放量,用于表征其体外骨靶向性。将生长分化因子5(对照组)、偶联帕米膦酸钠的生长分化因子5(实验组)分别与成骨前体细胞MC3T3-E1共培养,以单独培养的细胞为空白对照,评价复合物对细胞增殖及分化等的影响。结果与结论:①红外光谱及圆二色谱结果表明,实验成功制备了双膦酸盐/生长分化因子5复合物且蛋白二级结构无显著变化;体外磷酸钙吸附结果表明,偶联帕米膦酸钠后,生长分化因子5与磷酸钙的吸附率增加了约1倍;在半胱氨酸存在条件下,偶联帕米膦酸钠的生长分化因子5的蛋白可释放出来;②CCK-8实验结果显示,实验组培养4,7 d的吸光度值高于对照组、空白对照组(P<0.0001);培养7 d后,实验组碱性磷酸酶表达明显高于对照组、空白对照组(P<0.0001);培养13 d后,实验组钙结节含量明显高于对照组、空白对照组(P<0.0001);qRT-PCR结果检测结果显示,培养7 d后,实验组碱性磷酸酶、骨钙素及Runx2的mRNA表达高于对照组、空白对照组(P<0.01,P<0.001,P<0.0001);③结果表明,双膦酸盐修饰有利于增强生长分化因子5与磷酸钙的结合能力,同时有利于增强其生物活性。
BACKGROUND:As a member of bone morphogenetic proteins,growth differentiation factor-5 shows promising potential in the application of cartilage and bone repair.The affinity of growth differentiation factor-5 onto bone tissue determines protein use efficiency,so it is of great significance to prepare growth differentiation factor-5 with bone targeting capability.OBJECTIVE:To modify growth differentiation factor-5 using bisphosphonates and investigate the effects of modified protein on the growth of preosteoblasts in mice.METHODS:Pamidronate disodium/growth differentiation factor-5 complex was prepared using chemical crosslinking to couple growth differentiation factor-5 with pamidronate disodium.The functional groups and structures of the complex were characterized using Fourier transform infrared spectroscopy and circular dichromatography.To determine the bone targeting in vitro,the binding of the modified growth differentiation factor-5 with calcium phosphate and in vitro release amount of growth differentiation factor-5 were measured with an ELISA kit.Growth differentiation factor-5(control group)and the pamidronate disodium/growth differentiation factor-5 complex(experimental group)were co-cultured with preosteoblasts MC3T3-E1.Individually cultured cells were blank controls.The effect of the complex on cell proliferation and differentiation was evaluated.RESULTS AND CONCLUSION:(1)The infrared spectroscopy and circular dichromatography results indicated that the bisphosphonate/growth differentiation factor-5 complex was successfully prepared without significant changes in the protein secondary structure.In vitro protein adsorption results showed that growth differentiation factor-5 adsorption on calcium phosphate was increased by about one time after coupling with a bisphosphonate.In the presence of cysteine,growth differentiation factor-5 could be released from the bisphosphonate/growth differentiation factor-5 complex.(2)CCK-8 assay results showed that the absorbance value of the experimental group cultured for 4 and 7 days was higher than that of the control group and blank control group(P<0.0001).After 7 days of culture,the expression of alkaline phosphatase in the experimental group was significantly higher than that in the control group and blank control group(P<0.0001).After 13 days of culture,the content of calcium nodules in the experimental group was significantly higher than that in the control group and the blank control group(P<0.0001).The results of qRT-PCR showed that the mRNA expression of alkaline phosphatase,osteocalcin and Runx2 in the experimental group was higher than that in the control group and the blank control group after 7 days of culture(P<0.01,P<0.001,P<0.0001).(3)These findings exhibit that bisphosphonate modification can enhance the binding capacity of growth differentiation factor-5 to calcium phosphate as well as improve its biological activity.
作者
李立斯
张成栋
李小龙
叶姿妤
蒲超
杨在君
匙峰
肖东琴
Li Lisi;Zhang Chengdong;Li Xiaolong;Ye Ziyu;Pu Chao;Yang Zaijun;Shi Feng;Xiao Dongqin(Organization and Repair Materials Engineering Technology Collaborative Innovation Center,West China Normal University,Nanchong 637000,Sichuan Province,China;Second Clinical College of North Sichuan Medical College·Institute of Tissue Engineering and Stem Cells of Nanchong Central Hospital,Nanchong 637000,Sichuan Province,China;College of Life Sciences,West China Normal University,Nanchong 637000,Sichuan Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2024年第3期373-379,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(82002289),项目负责人:肖东琴
四川省自然科学基金(2022NSFSC0685),项目负责人:肖东琴
四川省自然科学基金(23NSFSC3371),项目负责人:张成栋
南充市市校合作科研项目(22SXJCQN0002),项目负责人:肖东琴
南充市市校合作项目(22SXQT0385),项目负责人:蒲超。
关键词
生长分化因子5
双膦酸盐
释放
MC3T3-E1细胞
增殖
分化
growth differentiation factor-5
bisphosphonate
release
MC3T3-E1 cell
proliferation
differentiation
