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目的:观察MC3T3-E1细胞在Fe_3O_4涂层多巴胺修饰聚对苯二甲酸乙二醇酯(PET)人工韧带材料表面的粘附、增殖及成骨分化情况。方法:按PET材料表面涂层情况分为4组:PET组,即PET表面未处理组;多巴胺(DA)组,即PET表面进行多巴胺涂层组;100 Fe_3O_4组和200 Fe_3O_4组,分别为PET经多巴胺处理后表面涂有100μg/ml和200μg/ml的Fe_3O_4溶液组。在上述4组PET材料上分别培养MC3T3-E1细胞,进行各项检测。扫描电镜(SEM)和X射线能谱分析(EDS)材料涂层情况。细胞在材料上培养3天和7天后行荧光染色,观察细胞粘附情况。培养1、3、7天后,行CCK-8检测MC3T3-E1细胞的增殖。诱导培养基培养7天检测碱性磷酸酶活性;培养21天行茜素红染色,检测细胞矿化情况;培养14天行Real Time PCR检测骨钙蛋白和胶原I基因的表达。结果:SEM和EDS检测验证涂层成功。荧光染色显示涂层组有效增加了细胞的粘附。细胞增殖实验示涂层组对细胞增殖有促进作用,各涂层组与未涂层组差异具有统计学意义(P<0.05)。涂层材料组碱性磷酸酶OD值明显高于未涂层组(P<0.05)。茜素红染色示各组都有较多矿化结节形成,定量分析显示100 Fe_3O_4组和200 Fe_3O_4组与PET组比较差异有统计学意义(P<0.05)。PCR检测显示涂层组骨钙蛋白和I型胶原基因的表达都较未涂层组增加,骨钙蛋白:Fe_3O_4组与PET组比较差异有统计学意义(P<0.01);I型胶原:DA组、Fe_3O_4组与PET组比较差异具有统计学意义(P<0.05)。结论:经多巴胺修饰四氧化三铁涂层处理的PET人工韧带材料,能明显促进MC3T3-E1细胞的粘附、增殖和分化。
OBJECTIVE: To observe the adhesion, proliferation and osteogenic differentiation of MC3T3-E1 cells on the surface of Fe3O4-coated dopamine-modified polyethylene terephthalate (PET) artificial ligament. Methods: According to the situation of PET material surface coating, it was divided into four groups: PET group, that is, PET untreated group; dopamine (DA) group, ie, PET coated with dopamine coating group; 100 Fe 3 O 4 group and 200 Fe 3 O 4 group, After dopamine treatment, the surface was coated with 100μg / ml and 200μg / ml Fe_3O_4 solution group. MC3T3-E1 cells were cultured on the above four groups of PET materials for various tests. Scanning electron microscopy (SEM) and X-ray energy dispersive spectroscopy (EDS) material coating. The cells were cultured on the material for 3 days and 7 days after the fluorescence staining to observe cell adhesion. After 1,3,7 days of culture, the proliferation of MC3T3-E1 cells was examined by CCK-8. Alkaline phosphatase activity was detected by induction medium for 7 days. Alizarin red staining was performed on day 21 for cell mineralization. Real-time PCR was used to detect osteocalcin and collagen I gene expression on day 14 after culture. Results: The SEM and EDS tests verified the coating was successful. Fluorescent staining showed that the coating group effectively increased cell adhesion. Cell proliferation experiments showed that the coating group promoted cell proliferation, the difference between the coating group and the uncoated group was statistically significant (P <0.05). The OD value of alkaline phosphatase in the coating material group was significantly higher than that in the uncoated group (P <0.05). Alizarin red staining showed that each group had more mineralized nodules. Quantitative analysis showed that there was significant difference between 100 Fe 3 O 4 group and 200 Fe 3 O 4 group and PET group (P <0.05). The results of PCR showed that the expression of osteocalcin and type I collagen gene in the coating group was higher than that in the uncoated group, and the difference was statistically significant (P <0.01) between the osteocalcin: Fe 3 O 4 group and the PET group. Type I collagen: DA Group, Fe 3 O 4 group and PET group, the difference was statistically significant (P <0.05). Conclusion: The PET artificial ligament material treated with dopamine-modified ferroferric oxide coating can significantly promote the adhesion, proliferation and differentiation of MC3T3-E1 cells.