Lysine-Cyclic RGD肽对骨髓间充质干细胞黏附、增殖和分化的影响

Effect of bifunctional Lysine-Cyclic RGD peptide on adhesion,proliferation and differentiation of mesenchymal stem cells

目的研究物理电荷与生物配基两种因素相结合的新型表面修饰剂Lysine-Cyclic RGD(LcRGD)肽对骨髓间充质干细胞(mesenchymal stem cells,MSCs)的黏附、增殖及分化的影响。方法以光学接触角仪衡量LcRGD肽修饰表面的亲水性;以离心黏附实验、震荡黏附实验评价LcRGD肽对MSCs的黏附性能;以CCK-8实验评价LcRGD表面改性DBM上MSCs的增殖活力;以Western blot法检测LcRGD表面改性脱钙骨基质(decalcified bone matrix,DBM)上MSCs的Runx2和OCN蛋白的表达。结果 LcRGD肽修饰表面接触角为(29.33±4.06)°,远低于普通cRGD肽修饰表面的接触角(63.72±8.65)°,提示LcRGD肽具有更好的亲水性(P<0.01);离心黏附实验和震荡黏附实验显示相对于普通cRGD肽,LcRGD肽从细胞黏附早期开始便具有更好的细胞黏附性能;CCK-8实验提示MSCs在LcRGD修饰的DBM支架材料上的增殖活力高于普通cRGD肽修饰的DBM支架材料(P<0.05);Western blot结果提示相对于普通cRGD肽修饰的DBM,MSCs在LcRGD修饰的DBM上表达更高的成骨标志蛋白水平,提示LcRGD肽具有更好的骨诱导性。结论相对于普通的cRGD肽,物理电荷与生物配基相结合的LcRGD肽具有更好的细胞黏附、促增殖和骨诱导性能。

Objective To investigate the effect of Lysine-Cyclic RGD（ LcRGD） peptide,which is new surface modifier based on biological ligand and physical positive charge,on the adhesion,proliferation and differentiation of mesenchymal stem cells（ MSCs）. Methods The hydrophilicity of LcRGD peptidemodified cover glass was measured via contact angle that was detected with an optical tensiometer. The adhesion ability of MSCs to LcRGD peptide was measured by centrifugal cell adhesion assay and vortex cell adhesion assay. The proliferation and differentiation ability of the MSCs was detected by CCK-8 assay and Western blotting. Results LcRGD peptide-modified cover glass possessed contact angle（ 29. 33 ± 4. 06） °lower than（ 63. 72 ± 8. 65） ° of cRGD peptide-modified cover glass,indicating better hydrophilicity（ P〈0. 01）. The results of centrifugal cell adhesion assay and vortex cell adhesion assay confirmed the LcRGD peptide had better cell adhesion（ P〈0. 01） than cRGD peptide from the initial stage of cell adhesion. The results of CCK-8 assay and Western blotting showed that the MSCs seeded on LcRGD peptide-modified decalcified bone matrix（ DBM） showed higher proliferation activity as compared with cRGD peptide-modified DBM（ P〈0. 05）,and Runx2 and OCN protein levels were significantly increased. Conclusion LcRGD peptide holds satisfactory cell adhesion,biocompatibility and osteoinduction,suggesting it is a preferable modifier for tissue engineered bone scaffold.