中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (34): 5430-5436.doi: 10.3969/j.issn.2095-4344.2017.34.004

• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials • 上一篇    下一篇

血小板裂解液联合国产多孔钽对MG63细胞增殖以及ITGβ1/Vinculin/F-actin信号通路表达的影响

孙福斋1，2，王少华1、2，邓华民1，王  茜3，李琪佳3，甘洪全1，王志强1 
  

  1. 1华北理工大学附属医院骨科，河北省唐山市  063000；2华北理工大学基础医学院、河北省慢性疾病重点实验室、唐山市慢性病临床基础研究重点实验室，河北省唐山市  063000；3华北理工大学医学中心实验室，河北省唐山市  063000
  • 收稿日期:2017-10-19 出版日期:2017-12-08 发布日期:2018-01-04
  • 通讯作者: 王志强，华北理工大学附属医院骨科，河北省唐山市 063000
  • 作者简介:孙福斋，男，1990年生，河北省廊坊市人，华北理工大学硕士研究生在读，主要从事骨组织工程方面的研究。
  • 基金资助:
    国家科技部科技支撑课题(2012BAE06B03)；河北省科技支撑资助项目(16277776D)；河北省医学科学研究重点课题计划资助项目(20160225)；河北省卫生计生委临床医学优秀人才研究项目(361036)；华北理工大学博士科研启动基金资助项目(286062299)；华北理工大学研究生创新项目(2017S40)

Platelet lysate combined with domestic porous tantalum promotes MG63 proliferation and activates integrin beta1/Vinculin/F-actin signaling pathway

Sun Fu-zhai1, 2, Wang Shao-hua1, 2, Deng Hua-min1, Wang Qian3, Li Qi-jia3, Gan Hong-quan1, Wang Zhi-qiang1
  

  1. 1Department of Orthopaedics, Affiliated Hospital of North China University of Science and Technology, Tangshan 063000, Hebei Province, China; 2Hebei Key Laboratory for Chronic Disease, Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan 063000, Hebei Province, China; 3Medical Central Laboratory, North China University of Science and Technology, Tangshan 063000, Hebei Province, China
  • Received:2017-10-19 Online:2017-12-08 Published:2018-01-04
  • Contact: Wang Zhi-qiang, Department of Orthopaedics, Affiliated Hospital of North China University of Science and Technology, Tangshan 063000, Hebei Province, China
  • About author:Sun Fu-zhai, Studying for master’s degree, Department of Orthopaedics, Affiliated Hospital of North China University of Science and Technology, Tangshan 063000, Hebei Province, China; Hebei Key Laboratory for Chronic Disease, Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan 063000, Hebei Province, China
  • Supported by:
     the National Key Technology Research and Development Program of the Ministry of Science and Technology of China, No. 2012BAE06B03; the Science and Technology Research Project of Hebei Province, No. 16277776D; the Key Medical Research Project of Hebei Province, No. 20160225; the Clinical Talent Research Project of Hebei Provincial Health and Family Planning Commission, No. 361036; the Doctoral Scientific Research Foundation of North China University of Science and Technology, No. 286062299; the Postgraduate Innovation Research Project of North China University of Science and Technology, No. 2017S40

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摘要：

文章快速阅读：

 

文题释义：
血小板裂解液：是血小板浓缩物进一步裂解获得的液体成分，含有多种活性因子，如血小板衍生长因子、转化生长因子β、胰岛素样生长因子、血管内皮生长因子等。血小板裂解液去除了血小板残余的细胞结构，降低免疫原性的同时，保留的多种液态生长因子被证实能促进骨的再生修复。同时，血小板裂解后的上清液能促进成骨细胞的增殖。血小板裂解液所具备的促成骨属性决定了其可成为诱导修复骨缺损的辅助材料。
 
背景：课题组前期研究发现国产多孔钽有利于MG63细胞的早期黏附、增殖，可用作骨组织工程支架材料。血小板裂解液是富血小板血浆进一步优化后的产物，更适合用于诱导修复骨缺损。
目的：在前期研究基础上，深入探索血小板裂解液联合国产多孔钽支架材料对MG63细胞增殖以及ITGβ1/ Vinculin/F-actin信号通路表达的影响。
方法：培养MG63细胞并接种在国产多孔钽支架上，加入3%、5%、7%、10%血小板裂解液，应用CCK-8法筛选出最佳的体积分数7%进行以下实验。实验分组：空白对照组MG63细胞培养；血小板裂解液组：7%血小板裂解液与MG63细胞共同培养；多孔钽组：多孔钽支架材料与MG63细胞共同培养；血小板裂解液-多孔钽组：7%血小板裂解液、多孔钽与MG63细胞共同培养。扫描电镜观察国产多孔钽及血小板裂解   液-多孔钽-MG63细胞复合物的表面形态；CCK-8法检测MG63细胞的增殖状态；qPCR、免疫细胞化学染色、Western-blot检测MG63细胞ITGβ1、Vinculin、F-actin的mRNA和蛋白表达。
结果与结论：①扫描电镜显示，MG63细胞均良好地黏附在多孔钽支架表面；②与空白对照组比较，血小板裂解液组与多孔钽组均可促进MG63细胞的增殖(P < 0.05)；4组中，血小板裂解液-多孔钽组MG63细胞的增殖最显著(P < 0.05)；③qPCR、免疫细胞化学染色、Western-blot结果显示，4组中血小板裂解液-多孔钽组MG63细胞的ITGβ1、Vinculin、F-actin的mRNA及蛋白表达最多(P < 0.05)。说明血小板裂解液和国产多孔钽支架材料能协同促进MG63 细胞的增殖，并能上调ITGβ1/Vinculin/F-actin信号通路mRNA及蛋白的表达，该信号通路的激活可能有助于MG63细胞的增殖、黏附及分化。

关键词: 生物材料, 骨生物材料, 血小板裂解液, 多孔钽, MG63细胞, 整合素, 黏着斑蛋白, 束状肌动蛋白

Abstract:

BACKGROUND: The preliminary study found that domestic porous tantalum is conducive to the early adhesion and proliferation of MG63 cells, which can be used as a scaffold material for bone tissue engineering. As an optimized product of platelet-rich plasma, platelet lysate is more suitable for bone induction in the bone repair.
OBJECTIVE: To further investigate the effect of platelet lysate and domestic porous tantalum scaffold constructs on the proliferation of MG63 cells and expression of integrin β1 (ITGβ1)/Vinculin/F-actin signaling pathway based on our previous findings.
METHODS: MG3 cells were cultured and inoculated onto domestic porous tantalum scaffolds with the addition of 3%, 5%, 7% and 10% platelet lysates. Then, 7% as the best volume fraction of platelet lysate was screened by cell counting kit-8 method. There were four experimental groups including blank group (normally cultured MG63 cells), platelet lysate group (MG63 cells were cultured in 7% platelet lysate), porous tantalum scaffold group (MG63 cells were cultured on the domestic porous tantalum scaffold), and combined group (MG63 cells were cultured with 7% platelet lysate and porous tantalum scaffold. Scanning electron microscope was used to observe the surface morphology of domestic porous tantalum and platelet lysate-porous tantalum scaffold-MG63 cell complex. Cell counting kit-8 method was used to detect the proliferation of MG63 cells. Real-time fluorescence quantitative PCR (qPCR), immunocytochemical staining and western blot were used to detect the expression of ITGβ1, Vinculin, F-actin in MG63 cells at mRNA and protein levels.
RESULTS AND CONCLUSION: Under the scanning electron microscope, MG63 cells adhered well to the scaffold surface. Compared with the blank group, the proliferation of MG63 cells could be significantly promoted by either platelet lysate or porous tantalum scaffold (P < 0.05). Moreover, the proliferation of MG63 cells was significantly improved in the combined group compared with the other three groups (P < 0.05). Findings from qPCR, immunocytochemical staining and western blot showed the highest expression of ITGβ1, Vinculin, F-actin mRNA and protein in the combined group (P < 0.05). These results indicate that platelet lysate and the domestic porous tantalum scaffold can synergistically promote the proliferation of MG63 cells, and up-regulate the expression of ITGβ1, Vinculin and F-actin mRNA and protein. Activation of the ITGβ1/Vinculin/F-actin signaling pathway may contribute to the proliferation, adhesion and differentiation of MG63 cells.

Key words: Tantalum, Integrins, Biocompatible Materials, Tissue Engineering

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