精氨酸-甘氨酸-天冬氨酸多肽表面修饰多孔钽对软骨细胞黏附、增殖和分泌功能的促进作用

Promotion effect of RGD peptides-grafted porous tantalum on adhesion,proliferation and secretion of chondrocytes in vitro

目的:研究精氨酸-甘氨酸-天冬氨酸(RGD)多肽表面修饰多孔钽(Ta)对软骨细胞黏附、增殖和分泌功能等生物学行为的影响,探讨RGD/软骨细胞/多孔钽复合物作为软骨组织工程支架材料修复软骨缺损、促进软骨再生的可行性。方法:通过物理吸附的方法将不同浓度RGD肽及Ⅱ型胶原(ColⅡ)吸附于多孔钽表面,分为Ta-RGD组、Ta-ColⅡ组、Ta-RGD/ColⅡ0.2g·L-1组、Ta-RGD/ColⅡ1.0g·L-1组、Ta-RGD/ColⅡ5.0g·L-1组、Ta-RGD/ColⅡ10.0g·L-1组及纯Ta组。分离培养新西兰幼兔关节软骨细胞,取第2代细胞种植于修饰前后多孔钽使其成为RGD/软骨细胞/多孔钽复合物;扫描电镜观察RGD/软骨细胞/多孔钽复合物形貌特征以及软骨细胞生长状态,沉淀法检测多孔钽修饰前后软骨细胞黏附率,MTT法检测多孔钽修饰前后软骨细胞的增殖水平,羟脯氨酸法测定软骨细胞分泌功能。结果:多孔钽经RGD修饰后各组软骨细胞黏附率均高于修饰前(P<0.05),其中黏附效果最好的是4h的Ta-RGD/ColⅡ5.0g·L-1组,同时各组间黏附率比较差异有统计学意义(P<0.05)。扫描电镜下各组软骨细胞在修饰后的多孔钽表面生长状态良好,细胞在多孔钽表面及孔隙内生长,分泌细胞外基质覆盖于多孔钽表面;MTT检测,多孔钽经RGD修饰后各组软骨细胞增殖水平均高于修饰前(P<0.05),其中增殖效果最好的是13d的Ta-RGD/ColⅡ1.0g·L-1组;羟脯氨酸检测,Ta-RGD/ColⅡ1.0g·L-1组羟脯氨酸水平高于其他各组(P<0.05)。结论:RGD多肽可有效加强软骨细胞在多孔钽表面及孔隙内的黏附及增殖,对软骨细胞的分泌有促进作用。

Objective To investigate the protective effects of Schisandra Chinensis lignans（SCL）on the oxidative stress injury of PC12 cells induced by hydrogen peroxide and influence in NF-κB/iNOS/NO signaling pathway,and to explore the mechanisms.Methods The PC12 cells were divided into control group,model group（H2O2200μmol·L^-1）,high dose of SCL group（SCL 30mg·L^-1＋ H2O2200μmol·L^-1,SCL1group）and low dose of SCL group（SCL 10mg·L^-1＋ H2O2200μmol·L^-1,SCL2 group）.The PC12 cells were untreated（control group）or treated with H2O2（200μmol·L^-1）for 6h（model group）.The PC12 cells in SCL1 and SCL2 group were firstly pretreated with different concentrations of SCL for 2h,and then continuously with SCL and exposed to200μmol·L^-1 H2O2 for 6hat the same time.The survival rate of PC12 cells was evaluated by MTT assay.The activities of lactate dehydrogenase（LDH）,superoxide dismutase（SOD）and the levels of malondialdehyde（MDA）and nitric oxide（NO）were detected by microplate reader.The fluorescence intensity of reactive oxygen species（ROS）was assessed with flow cytometry.Immunohistochemistry and Western blotting methods were used to detect the protein expressions of inducible nitric oxide synthase（iNOS）and nuclear factor kappa B P65（NF-κB P65）.Results Compared with control group,the survival rates of PC12 cells in model group were significantly decreased（P〈0.01）;the LDH activities in the supernatant were increased（P〈0.01）,the SOD activities were decreased（P〈0.01）,the MDA and NO levels were increased（P〈0.01）,the cell ROS level was increased（P〈0.01）,the number of positive cells of NF-κB were increased（P〈0.01）,and the expression levels of iNOS and NF-κB P65 protein were increased（P〈0.01）.Compared with model group,the survival rates of PC12 cells in SCL1 and SCL2groups were increased（P〈0.05 or P〈0.01）,the LDH activities in the supernatant were decreased（P〈0.05 or P〈0.01）,the SOD activities were increased（P〈0.01）,the MDA and NO levels were decreased（P〈0.05 or P〈0.01）,the cell ROS levels was decreased（P〈0.01）,the number of positive cells of NF-κB was decreased（P〈0.05 or P〈0.01）,and the expression levels of iNOS and NF-κB P65 protein were decreased（P〈0.05）.Conclusion SCL could protect the oxidative stress injury of PC12 cells,its mechanism may be related to inhibiting the NF-κB/iNOS/NO signaling pathway,reducing the free radical generation and alleviating lipid peroxidation damage.