纤维蛋白-1c促进老年膝骨关节炎软骨细胞增殖代谢的机制

Effect of different concentrations of Fibulin-1c on the proliferation and metabolism of elderly chondrocytes

目的观察纤维蛋白-1c(Fibulin-1c)对老年膝骨关节炎(OA)软骨细胞增殖代谢的影响。方法2018年8月至2019年8月,收集山西白求恩医院因外伤截肢年轻人和人工膝关节置换术时老年膝OA患者10例的血清、膝关节软骨标本,检测标本中Fibulin-1c的含量变化。实验分4组,实验组1为培养液含rFibulin-1c(10μg/L);实验组2为培养液含rFibulin-1c(100μg/L);实验组3为培养液含rFibulin-1c(200μg/L);对照组为单纯培养液。常规培养人软骨细胞,实验组培养液中加入不同浓度(10、100、200μg/L)重组人Fibulin-1c蛋白,培养48 h后,5-乙炔基-2’脱氧尿嘧啶核苷(EdU)检测软骨细胞增殖,蛋白质印迹法(Western blot)和实时定量反转录聚合酶链反应(RT-qPCR)检测软骨细胞Col2、基质金属蛋白酶(MMP)-13、Smad3和pSmad3的表达。组间比较采用t检验和单因素方差分析。结果年轻人血清Fibulin-1c蛋白相对表达量为(1.009±0.109),老年人为(0.695±0.071),两者比较差异有统计学意义(t=2.406,P<0.05);年轻人软骨中蛋白相对表达量(1.021±0.097)高于老年人(0.733±0.079,t=2.297,P<0.05),差异有统计学意义。实验组EdU阳性细胞百分比[(25.330±1.453)%、(34.170±2.822)%]高于对照组[(13.170±1.447)%,F=24.300,P<0.01],差异有统计学意义;与对照组(0.786±0.042)比较,实验组软骨细胞Col2含量增高(1.470±0.105、1.880±0.120,F=30.110,P<0.01),差异有统计学意义;Col2的基因表达水平与蛋白表达水平一致。结论Fibulin-1c可以促进老年膝关节软骨细胞增殖代谢能力。

Objective To observe the effect of different concentrations of Fibulin-1c on proliferation of elderly chondrocytes.Methods During August 2018 to August 2019,knee cartilage specimens of young people[n=10,(25.34±7.91)years old]who had amputation due to trauma and elderly patients with knee OA[n=10,(67.36±8.43)years old]who underwent artificial knee replacement were collected,and the changes in the content of Fibulin-1c in the specimens were detected.Human chondrocytes were cultured routinely,and recombinant human Fibulin-1c protein of different concentrations(10,100,200μg/L)was added into the culture medium.After 48 h of culture,the proliferation of chondrocytes was examined by 5-Ethynyl-2’-deoxyuridine(EdU),and the expression levels of matrix metalloproteinase(MMP)-13 Smad3 and pSmad3 in chondrocytes were detected by Western blotting and real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).The statistical methods were t test and one-way ANOVA.Results The relative expression level of serum Fibulin-1c was(1.009±0.109)in the young people and(0.695±0.071)in the elderly patients,and the difference was statistically significant(t=2.406,P<0.05).The relative protein expression in cartilage of young people(1.021±0.097)was significantly higher than that in the elderly patients(0.733±0.079,t=2.297,P<0.05).The percentage of EdU positive cells in the experimental group[(25.330±1.453)%,(34.170±2.822)%]was significantly higher than that in the control group[(13.170±1.447)%,F=24.300,P<0.01].As compared with the control group(0.786±0.042),the Col2 expression of chondrocytes in the experimental group significantly increased(1.470±0.105,1.880±0.120,F=30.110,P<0.01).The gene expression level of Col2 was consistent with the protein expression level.Conclusion Fibulin-1c can promote the proliferation of elderly chondrocytes.