氧化石墨烯/聚己内酯复合静电纳米纺丝材料对大鼠棕色脂肪干细胞成心肌分化的影响

Effect of graphene oxide/polycaprolactone composite electrospun nanofibrous materials on the differentiation of rat brown adipose stem cells into cardiomyocytes

目的制备不同浓度的氧化石墨烯(GO)/聚己内酯(PCL)复合基质纳米材料,观察其在体外对大鼠棕色脂肪干细胞(BASC)成心肌分化的影响。方法利用静电纺丝技术制备GO/PCL复合纳米纤维材料。通过将BASC接种于纯PCL和GO/PCL复合纳米纤维上培养后进行CCK-8法测试、对纳米材料的扫描电镜(SEM)检测及电导率测定,表征GO/PCL复合纳米材料的生物相容性及性状特征。采用细胞学、免疫荧光染色等方法,检测心肌细胞相关蛋白的表达情况,系统评价GO/PCL复合材料对BASC活力、增殖及成心肌分化效率的影响。结果该GO/PCL复合基质纳米材料在GO浓度为0.1 mg/ml时无明显细胞毒性。蛋白荧光统计结果表明,与PCL组相比,GO/PCL组细胞的心肌细胞特异蛋白α-辅肌动蛋白(α-actinin)及闰盘相关蛋白缝隙连接蛋白-43(connexin-43,CX-43)表达水平显著提高(P<0.05)。细胞骨架染色结果表明,与PCL组相比,GO/PCL组的细胞表现出类似天然心肌细胞束的长梭样伸展,且生长方向具有一定的极性。结论本研究成功制备了GO/PCL复合纳米材料。该GO/PCL复合纳米材料可促进BASC向心肌细胞分化,并促进闰盘相关蛋白的表达。

Objective To prepare graphene oxide(GO)/polycaprolactone(PCL)composite matrix nanomaterials with different concentrations,and investigate effects of the nanomaterials on the myocardial differentiation of rat brown adipose stem cells(BASC)in vitro.Methods The GO/PCL composite nanofiber materials were prepared by electrospinning technology.The biocompatibility of the nanomaterials was tested by the CCK-8 assay after cultivation of the BASC on the pure PCL and GO/PCL composite nanofibers,and the characterization of the nanofiber materials was performed by the scanning electron microscopy(SEM)and electrical conductivity measurement.The expression of cardiomyocyte-related proteins was examined by the cytological method and immunofluorescence staining.The GO/PCL composite nanofiber materials were systematically evaluated for their effect on the viability,proliferation and myocardial differentiation of BASC.Results The GO/PCL composite nanofiber materials showed no obvious cytotoxicity at the 0.1 mg/ml GO concentration.The statistical results for the protein fluorescence intensities showed that the expression of the cardiomyocyte specific protein,α-actinin,and the intercalated disc-related protein,connexin-43(CX-43),was significantly increased in the GO/PCL group than in the PCL group(P<0.05).The cytoskeletal staining results showed that,compared with the PCL group,the cells in the GO/PCL group showed a long spindle-like stretch similar to the natural myocardial cell bundle,and the growth direction had a certain polarity.Conclusion This study successfully prepared GO/PCL composite nanomaterials,which could promote the differentiation of BASCs into cardiomyocytes and the expression of intercalated disc-related proteins.