特种肽支架材料对骨髓基质干细胞黏附、增殖及成骨分化的影响

Synthetic RGD-containing peptide K16GRGDSPC affected the adhesion,proliferation and osteogenic differentiation of rabbit bone marrow stromal cells on PLGA-[ASP-PEG]scaffold materials

目的探讨新型含精氨酸-甘氨酸-天冬氨酸(RGD)肽 K16RGDSPC 修饰聚丙交酯-CO-乙交酯(PLGA)-[ASP-PEG]支架材料对骨髓基质干细胞(BMSCs)在其表面黏附、增殖及成骨分化的影响。方法固相合成法合成 K16GRGDSPC 并通过质谱仪和高压液相色谱进行鉴定。用交联剂Sulfo-LC-SPDP 将 K16GRGDSPC 接枝到 PLGA-[ASP-PEG]支架材料上,通过 XPS 图谱检测接枝反应的发生。将改性后的 PLGA-[ASP-PEG]支架材料与兔 BMSCs 在成骨诱导培养基中复合培养,以未改性的支架材料作对照。通过沉淀法、微管吮吸法、MTT 法和考马斯亮蓝法分别检测 BMSCs 的黏附和增殖性能的变化;应用碱性磷酸酶(ALP)试剂盒检测 BMSCs 的 ALP 表达水平并通过 RT-PCR 检测细胞 ALP、骨钙素(OCN)、骨桥蛋白(OPN)和Ⅰ型胶原的表达,通过免疫荧光染色检测核心结合因子 a1(Cbfa1)表达,观察 BMSCs 向成骨细胞方向分化的情况。结果 XPS 图谱证实含 RGD 肽K16GRGDSPC 被成功地接枝到 PLGA-[ASP-PEG]表面。复合 BMSCs 培养结果表明,改性后的 PLGA-[ASP-PEG]表面 BMSCs 的黏附性能和增殖能力明显提高,而且其成骨标志物(ALP、OCN、OPN、Ⅰ型胶原和 Cfbal)的表达均显著高于对照组(P<0.05)。结论含 RGD 肽 K16GRGDSPC 能促进 BMSCs在骨基质材料表面的黏附、增殖并诱导其向成骨方向分化。

Objective To explore the effects of synthetic RGD-eontaining peptide K16GRGDSPC covalent bonding with PLGA-[ ASP-PEG ] scaffold materials on the adhesion, proliferation and osteogenic differentiation of rabbit bone marrow stromal cells （BMSCs）. Methods The peptide was synthesized by solid-phase synthesis method and characterized by mass spectrometry and high pressure liquid chromatography. PLGA-[ ASP-PEG] scaffold materials were modified with the piptide by cross linker Sulfo- LC-SPDP and detected by XPS. The BMSCs obtained from rabbit were cultured on PLGA-[ ASP-PEG] modified with the peptide and those cultured on unmodified PLGA-[ ASP-PEG] were also observed as control group. The adhesion and proliferation behaviors of the cells were analyzed by conventional precipitation method, micropipette aspiration technique, MTT assay and Coomassie Brilliant Blue dyes. The osteogenic differentiation of the cells was showed by the activity of alkaline phosphatase （ALP） assayed by ALP Assyay Kit and the mRNA levels of ALP, osteocalcin（ OCN）, osteopontin（OPN） and collagen Ⅰ assessed by realtime PCR（RT-PCR）. Immunofluorescence stain was also used to detect the expression of core binding factor al （CAbal） which was an osteogenic maker as well. Results The peptide was successfully manufactured and linked to the surface of the PLGA-[ ASP-PEG ] by the cross-linker. The abilities of adhesion and proliferation and the expressions of osteogenic makers of the cells were significantly higher than those of control group （P 〈 0. 05 ）. Conclusion RGD-containing peptide K16GRGDSPC could promote the adhesion, proliferation and osteogenic differentiation of BMSCs on the biomimetic bone-matrix materials.