白藜芦醇-固体脂质纳米粒促BMSCs成骨分化实验研究

Experimental study of resveratrol-solid lipid nanoparticles in promotion of osteogenic differentiation of bone marrow mesenchymal stem cells

目的探讨固体脂质纳米粒(solid lipid nanoparticles,SLNs)增强白藜芦醇(resveratrol,Res)促BMSCs体外成骨分化的效果,为骨稳态失衡疾病的治疗提供一种方法。方法通过高温乳化-低温固化法制备Res-SLNs,然后将分离培养的第2~3代SD大鼠BMSCs与不同浓度(0、0.1、1、5、10、20μmol/L)Res和ResSLNs共培养,通过细胞计数试剂盒8(cell counting kit 8,CCK-8)和活/死细胞染色检测Res和Res-SLNs对BMSCs细胞活性的影响;通过成骨诱导分化后的ALP染色和茜素红S(alizarin red S,ARS)染色检测Res和ResSLNs对BMSCs成骨分化的影响,并确定用于基因检测的Res-SLNs最适浓度;通过抗骨钙素(osteocalcin,OCN)免疫荧光染色和实时荧光定量PCR(real-time fluorescent quantitative PCR,RT-qPCR)检测Res和Res-SLNs对BMSCs成骨相关基因(ALP和OCN)的影响。结果活/死细胞染色示,Res和Res-SLNs各浓度组内的死细胞数量无明显差异;CCK-8检测示,浓度为20μmol/L时,Res组BMSCs活性显著降低(P<0.05);而浓度对ResSLNs活性无影响(P>0.05)。成骨分化诱导后两组ALP和ARS染色强度均呈浓度依赖性,Res组和Res-SLNs组ALP阳性染色面积百分比和矿化结节面积百分比分别在10μmol/L和1μmol/L浓度时达最大值(P<0.05),然后逐渐减小;确定Res-SLNs最适浓度为1μmol/L。成骨诱导分化培养14 d OCN免疫荧光染色和RT-qPCR检测示Res-SLNs组OCN表达量及ALP、OCN mRNA相对表达量均显著高于Res组(P<0.05)。结论SLNs包封处理可以提高Res促进成骨的药物效果,在更低浓度达到BMSCs成骨分化的最佳效果,有望用于骨稳态失衡疾病的治疗。

Objective To investigate the effect of solid lipid nanoparticles(SLNs)on enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in vitro by resveratrol(Res),and provide a method for the treatment of bone homeostasis disorders.Methods Res-SLNs were prepared by high-temperature emulsification and low-temperature solidification method,and then the 2nd-3rd generation BMSCs from Sprague Dawley rat were cocultured with different concentrations(0,0.1,1,5,10,20μmol/L)of Res and Res-SLNs.The effects of Res and Res-SLNs on the cell viability of BMSCs were detected by cell counting kit 8(CCK-8)and live/dead cell staining;the effects of Res and Res-SLNs on the osteogenic differentiation of BMSCs were detected by alkaline phosphatase(ALP)staining and alizarin red S(ARS)staining after osteogenic differentiation induction,and the optimal concentration of Res-SLNs for gene detection was determined.Anti-osteocalcin(OCN)immunofluorescence staining and real-time fluorescent quantitative PCR(RT-qPCR)were used to detect the effect of Res and Res-SLNs on osteoblast-related genes(ALP and OCN)of BMSCs.Results Live/dead cell staining showed that there was no significant difference in the number of dead cells between Res and Res-SLNs groups;CCK-8 detection showed that the activity of BMSCs in Res group was significantly reduced at the concentration of 20μmol/L(P<0.05),while Res-SLNs activity was not affected by Res concentration(P>0.05).After osteogenic differentiation,the staining intensity of ALP and ARS in both groups was dose-dependent.The percentage of ALP positive staining area and the percentage of mineralized nodule area in Res group and Res-SLNs group reached the maximum at the concentrations of 10μmol/L and 1μmol/L,respectively(P<0.05),and then decreased gradually;the most effective concentration of Res-SLNs was 1μmol/L.The expression of OCN and the relative expression of ALP and OCN mRNA in Res-SLNs group were significantly higher than those in Res group(P<0.05).Conclusion Encapsulation of SLNs can improve the effect of Res on promoting osteogenesis,and achieve the best effect of osteogenic differentiation of BMSCs at a lower concentration,which is expected to be used in the treatment of bone homeostasis imbalance diseases.