鹿茸多肽纳米复合材料诱导兔骨髓间质细胞向成骨细胞的分化效应

Differentiation of rabbit mesenchymal stem cells into osteoblasts induced by velvet antler polypeptide/nano TCP/gelatin material

目的:探讨鹿茸多肽纳米复合材料诱导兔骨髓间质细胞(MSCs)向成骨细胞分化的效应,阐明复合材料作用机制。方法:将含5mg.g-1鹿茸多肽、20%纳米β-磷酸三钙(β-TCP)和明胶的复合材料(以下简称复合材料)与第3代MSCs共培养48h,用MTT法和流式细胞术检测复合材料对MSCs增殖及细胞周期的影响;AO/EB荧光染色观察MSCs凋亡形态的变化;将MSCs与复合材料共培养,扫描电镜下观察细胞黏附、生长情况;复合材料连续作用于MSCs 21d,全自动生化分析仪检测细胞中碱性磷酸酶(ALP)活性,茜素红染色法观察复合材料对MSCs矿化结节形成的作用。结果:复合材料作用于MSCs 48h,可明显促进MSCs增殖,相对增殖率为126.45%,此时的MSCs主要处于S期,占细胞总数45.84%(对照组为7.96%),凋亡率为2.08%(对照组为13.68%);MSCs在复合材料上黏附、生长良好,呈多角或梭形。复合材料连续作用MSCs 21d,显著增加了矿化结节形成;随着作用时间的延长,细胞的ALP活性也逐渐增强,21d复合材料组与对照组比较明显升高(P<0.001)。结论:复合材料在体外可促进MSCs增殖,并可诱导MSCs向成骨细胞分化,具有良好的成骨效能。

Objective To explore the effects of velvet antler polypeptide/ nano β-tricalcium phosphate(β-TCP)/ gelatin material on the osteogenetic differentiation in rabbit mesenchymal stem cells(MSCs),and clarify the mechanism.Methods Cell cycle and proliferation were examined by flow cytometry and MTT assay in the third generation MSCs treated with 5 mg·g-1 velvet antler polypeptide/20 % nano TCP/gelatin material for 48 h.The growth of MSCs cocultured with the material was observed by scanning electron microscope.The morphology of apoptotic MSCs was observed by AO∕EB fluorescence staining assay.The activity of alkaline phosphatase(ALP) was detected with automatic biochemistry analyzer,and the mineralized nodules in MSCs were counted after 21 d treatment.Results The material could significantly promote the proliferation of MSCs,the relative growth rate was 126.45 % after treated with the material for 48 h.The percentage of MSCs in the S phase was 45.84 %(7.96% in control),and the apoptotic rate was 2.08 %(13.68% in control group).The calcified nodules and the activity of ALP were increased after treatment with material for 21 d.MSCs could adhere to the surface of the material and go well with it.Conclusion The material has the effects on stimulating the proliferation,differentiation and mineralization of rabbit MSCs.