不同浓度人源性抗菌肽LL-37/PLGA/β-TCP复合支架对兔骨髓间充质干细胞增殖、分化的影响

Effects of Different Concentrations of Human Antimicrobial Peptide LL-37/PLGA/β-TCP Composite Scaffolds on Proliferation and Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells

目的:研究不同浓度人源性抗菌肽LL-37/聚乳酸聚乙醇酸共聚物(PLGA)/β-磷酸三钙(β-TCP)复合支架对于兔骨髓间充质干细胞(rBMSCs)增殖、分化的影响。方法:制备LL-37/PLGA/β-TCP复合支架材料,按LL-37浓度分为0、1、5、10μmol/L组,另设置空白rBMSCs培养基为N组,每组三个培养皿,细胞密度为5×10^5个/皿。扫描电镜下观察不同浓度LL-37/PLGA/β-TCP复合支架形态。采用CCK-8检查方法检测各组干预rBMSCs 0、24、48、72、96、120 h后rBMSCs细胞增殖活力。干预rBMSCs 24 h后,采用荧光定量聚合酶链反应(qPCR)检测各组成骨相关基因碱性磷酸酶(ALP)、骨形态发生蛋白-1(BMP-1)及骨形态发生蛋白-7(BMP-7)的mRNA相对表达量。干预rBMSCs 24 h后,采用ELISA检测各组ALP、BMP-1及BMP-7的蛋白水平。每个实验独立重复三次。结果:LL-37/PLGA/β-TCP复合支架材料随着LL-37浓度增加而呈现多孔、细胞吸附增多的趋势,促进细胞贴附生长。干预24 h后,10μmol/L组细胞增殖能力明显高于N组(P<0.05);随着干预时间延长,1、5、10μmol/L组均对rBMSCs有促进增殖作用,且呈浓度依赖作用。1、5、10μmol/L组ALP、BMP-1及BMP-7的mRNA相对表达量与蛋白表达水平均高于N组(P<0.05);1、5、10μmol/L组ALP、BMP-1及BMP-7的mRNA相对表达量与蛋白表达水平均呈高表达,且呈浓度依赖。结论:LL-37/PLGA/β-TCP复合支架可以显著促进rBMSCs增殖与分化,是一种潜在的骨组织工程支架。

Objective:To investigate the effects of concentrations of human antimicrobial peptide LL-37/polylactic acid-polyglycolic acid copolymer(PLGA)/β-tricalcium phosphate(β-TCP)composite scaffolds on proliferation and differentiation of rabbit bone marrow mesenchymal stem cells(rBMSCs).Method:LL-37/PLGA/β-TCP composite scaffolds were prepared.The scaffolds were divided into 0,1,5,10μmol/L group according to the concentration of LL-37.In addition,the blank rBMSCs medium was set as group N,three plates in each group and cell density of 5×10^5/plates.The morphology of LL-37/PLGA/β-TCP composite scaffolds with different concentrations were observed under scanning electron microscope.The proliferation activity of rBMSCs was detected by CCK-8 after 0,24,48,72,96 and 120 h of rBMSCs intervention.After 24 h of rBMSCs intervention,the relative mRNA expression of osteogenesis-related genes alkaline phosphatase(ALP),bone morphogenetic protein-1(BMP-1)and bone morphogenetic protein-7(BMP-7)were detected by fluorescence quantitative polymerase chain reaction(qPCR).After 24 h of rBMSCs intervention,the protein levels of ALP,BMP-1 and BMP-7 were detected by ELISA.Each experiment was repeated three times independently.Result:With the increase of LL-37 concentration,LL-37/PLGA/β-TCP composite scaffolds showed a trend of porous and cell adsorption,which promoted cell adhesion and growth.After 24 h of intervention,the proliferation ability of 10μmol/L group was significantly higher than that of N group(P<0.05).With the extension of intervention time,the 1,5 and 10 moL/L group all had the effect of promoting rBMSCs proliferation,and the effect was concentration dependent.The relative mRNA expression and protein expression level of ALP,BMP-1 and BMP-7 in 5,10μmol/L group were higher than those in N group(P<0.05).The relative mRNA expression and protein expression level of ALP,BMP-1 and BMP-7 in 1,5,10μmol/L group were all highly expressed and concentration dependent.Conclusion:The LL-37/PLGA/β-TCP composite scaffold can significantly promote rBMSCs proliferation and differentiation.It is a potential bone tissue engineering scaffold.