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目的:探讨牛蒡子苷元(Arctigenin,ARC-G)对软骨细胞的增殖,及对Ⅱ型胶原(Collagen Ⅱ,Col Ⅱ)、Y染色体性别决定相关高泳动蛋白盒基因9(SRY-related high mobility group-box gene 9,SOX9)和基质金属蛋白酶-13(Matrix Metalloproteinase-13,MMP-13)表达的影响。方法:体外分离培养软骨细胞,取第1代细胞进行实验干预。实验分为空白组(ARC-0组)、低浓度牛蒡子苷元组(ARC-L组)和高浓度牛蒡子苷元组(ARC-H组)。各组分别予相应干预措施处理48h后,以SABC免疫细胞化学染色法观察Col Ⅱ的表达情况;以CCK-8法检测软骨细胞的增殖情况;以Real-time quantitative PCR检测Col Ⅱ,SOX9及MMP-13 mRNA的表达情况;以酶联免疫吸附法(ELISA)检测细胞培养上清液中Col Ⅱ的含量。结果:1)ARC-L组和ARC-H组的Col Ⅱ阳性染色强度均强于ARC-0组,其中ARC-H组染色最强;2)与ARC-0组相比,在不同时间点ARC-L组和ARC-H组均可促进软骨细胞的增殖,且ARC-H组促增殖作用更显著,除24h和96h的ARC-L组外其余各时间点差异均有统计学意义(P<0.05);3)与ARC-0组相比,ARC-L组和ARC-H组均可上调ColⅡ和SOX9 mRNA的表达,下调MMP-13 mRNA的表达,且ARC-H组作用更为显著,除ARC-L组Col Ⅱ及MMP-13 mRNA外,差异均有统计学意义(P<0.05);4)与ARC-0组相比,ARC-L组和ARC-H组均可促进Col Ⅱ蛋白的表达,且ARC-H组作用更为显著,除ARC-0组和ARC-L组外,差异均有统计学意义(P<0.05)。结论:牛蒡子苷元可通过上调SOX9和Col Ⅱ mRNA的表达,下调MMP-13 mRNA的表达以促进Col Ⅱ蛋白的合成。
Objective: To investigate the effects of Arctigenin (ARC-G) on the proliferation of chondrocytes and the effects of Collagen Ⅱ and Col Ⅱ, SRY-related high mobility group-box gene 9, SOX9) and matrix metalloproteinase-13 (MMP-13). Methods: Chondrocytes were isolated and cultured in vitro, and the first generation of cells were taken for experimental intervention. The experiment was divided into blank group (ARC-0 group), low concentration arctigenin group (ARC-L group) and high concentration arctigenin group (ARC-H group). The rats in each group were treated with the corresponding interventions for 48h. The expression of Col Ⅱ was observed by SABC immunocytochemical staining. The proliferation of chondrocytes was detected by CCK-8 assay. The expressions of Col Ⅱ, SOX9 and MMP were detected by Real-time quantitative PCR -13 mRNA expression was detected by enzyme-linked immunosorbent assay (ELISA) to detect the cell culture supernatant Col Ⅱ content. Results: 1) Col Ⅱ positive staining intensity of ARC-L group and ARC-H group were stronger than that of ARC-0 group, of which ARC-H group was the strongest staining; 2) Compared with ARC-0 group, ARC-H group and ARC-H group can promote the proliferation of chondrocytes, and ARC-H group was more significant role in promoting proliferation, except ARC-L group 24h and 96h after the rest of the time points were statistically significant difference (P <0.05) .3) Compared with ARC-0 group, both ARC-L group and ARC-H group could up-regulate the expression of ColⅡand SOX9 mRNA and down-regulate the expression of MMP-13 mRNA, and the effect of ARC-H group was more significant (P <0.05) .4) Compared with ARC-0 group, both ARC-L group and ARC-H group could promote the expression of Col Ⅱ protein expression, and ARC-H group more significant effect, with the exception of ARC-0 group and ARC-L group, the difference was statistically significant (P <0.05). CONCLUSION: Arctigenin can promote the synthesis of Col Ⅱ protein by up-regulating the expression of SOX9 and Col Ⅱ mRNA and down-regulating the expression of MMP-13 mRNA.