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干霖,李玉才,赵超前,潘珍,余将明,叶晓健.负载SVVYGLR多肽的骨支架对血管生成和成骨分化的促进作用[J].脊柱外科杂志,2024,22(5):313-320.
负载SVVYGLR多肽的骨支架对血管生成和成骨分化的促进作用     点此下载全文 (Fulltext)
干霖1  李玉才2  赵超前3  潘珍2  余将明2*  叶晓健2*
1. 上海交通大学医学院附属同仁医院康复科, 上海 2003362;
2. 上海交通大学医学院附属同仁医院骨科, 上海 2003363;
3. 中国科学院上海硅酸盐研究所高性能陶瓷与超微结构国家重点实验室, 上海 200050
基金项目:上海市科学技术委员会科技计划项目(20ZR1469800)
DOI:10.3969/j.issn.1672-2957.2024.05.005
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摘要:
      目的 探讨负载SVVYGLR多肽的骨支架对血管生成和成骨分化的促进作用。方法 将β-TCP支架(BK组)和负载SVVYGLR多肽的β-TCP支架(SV组)与人脐静脉内皮细胞(HUVEC)共培养,观察SVVYGLR多肽对HUVEC增殖与活力的影响。用BK组和SV组支架浸提液培养大鼠骨髓间充质干细胞(BMSC),观察SVVYGLR多肽对BMSC促成骨分化的影响。采用实时荧光定量聚合酶链反应(RT-qPCR)分析HUVEC血管基因(VEGF、CD31)的mRNA表达水平以及BMSC中骨桥蛋白(OPN)、骨钙蛋白(OCN)、Runt相关转录因子2(Runx2)的mRNA表达水平。在SD大鼠皮下植入BK组和SV组支架6周,进行苏木精-伊红(HE)、Masson染色和OCN、CD31免疫组化分析,观察动物体内血管生成和成骨分化情况。结果 共聚焦激光扫描显微镜(CLSM)和扫描电子显微镜(SEM)观察结果显示,SV组HUVEC细胞增殖能力更强。碱性磷酸酶(ALP)染色及茜素红S(ARS)染色结果显示,SV组促成骨分化能力优于BK组,差异有统计学意义(P<0.05)。RT-qPCR分析结果显示,SV组成骨基因OPN、OCN和Runx2及血管生成基因VEGF和CD31的mRNA表达量高于BK组,差异均有统计学意义(P<0.05)。体内实验观察到SV组比BK组有更多的胶原纤维和更高的OCN、CD31蛋白表达。结论 负载SVVYGLR多肽的骨支架可以促进细胞血管生成和成骨分化。
关键词:人脐静脉内皮细胞  间质干细胞  骨桥蛋白  组织支架  骨再生  新生血管,生理学
Effect of bone scaffolds loaded with SVVYGLR peptide on promoting angiogenesis and osteogenic differentiation    Fulltext
Gan Lin1  Li Yucai2  Zhao Chaoqian3  Pan Zhen2  Yu Jiangming2*  Ye Xiaojian2*
1. Department of Rehabilitation Medicine, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200336, China;
2. Department of Orthopaedics, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200336, China;
3. State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China
Fund Project:
Abstract:
      Objective To investigate the promoting effect of bone scaffolds loaded with SVVYGLR peptides on angiogenesis and osteogenic differentiation. Methods β-TCP scaffolds(BK group) and β-TCP scaffolds loaded with SVVYGLR peptides(SV group) were co-cultured with human umbilical vein endothelial cells(HUVEC),and the effect of SVVYGLR peptides on HUVEC proliferation and viability was observed. Rat bone mesenchymal stem cells(BMSC) were cultivated with BK and SV groups’ scaffold extracts,and the effect of SVVYGLR peptide on BMSC induced bone differentiation were observed. Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was used to analyze the mRNA expression levels of vascular genes(VEGF,CD31) in HUVEC,as well as the mRNA expression levels of osteopontin(OPN),osteocalcin(OCN),and runt related transcription factor 2(Runx2) in BMSC. Subcutaneous implantation of BK and SV groups’ scaffolds in SD rats for 6 weeks,then hematoxylin-eosin(HE),Masson staining,and OCN and CD31 immunohistochemical analysis were performed to observe angiogenesis and osteogenic differentiation in the animals. Results The observation results of confocal laser scanning microscopy(CLSM) and scanning electron microscopy(SEM) showed that the SV group had stronger proliferation ability of HUVEC cells. The results of alkaline phosphatase(ALP) staining and alizarin reds(ARS) staining showed that the SV group had better ability of promoting bone differentiation than the BK group,and the difference was statistically significant(P<0.05). The RT-qPCR analysis results showed that the mRNA expression levels of bone genes OPN,OCN,and Runx2,as well as angiogenesis genes VEGF and CD31 in the SV group were higher than those in the BK group,and the differences were statistically significant(P<0.05). In vivo experiments observed that the SV group had more collagen fibers and higher expression of OCN and CD31 proteins than the BK group. Conclusion Bone scaffolds loaded with SVVYGLR peptides can promote cell angiogenesis and osteogenic differentiation.
Keywords:Human umbilical vein endothelial cells  Mesenchymal stem cells  Osteopontin  Tissue scaffolds  Bone regeneration  Neovascularization,physiologic
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