3种小分子拮抗剂对骨关节炎软骨细胞的作用

Effects of three antagonists on type Ⅱ collagen in osteoarthritis chondrocytes

摘要:

目的 探讨3种小分子拮抗剂（TN14003、T140、AMD3100）阻断SDF-1/CXCR4信号通路对骨关节炎（OA）软骨细胞表达的影响，为寻找一种高效的OA治疗药物奠定实验基础。方法 OA软骨细胞随机分成4组，A组、B组、C组分别加入TN14003、T140及AMD3100，D组为对照组。第2天、第4天时，qRT-PCR检测各组软骨细胞Ⅱ型胶原（ColⅡ）mRNA相对表达量，Western blotting检测各组软骨细胞ColⅡ蛋白相对表达量。第10天时，免疫组织化学染色观察各组软骨细胞ColⅡ表达。结果 培养第2天、第4天时，4组软骨细胞ColⅡ mRNA和蛋白相对表达量比较：①不同时间点软骨细胞ColⅡ mRNA和蛋白相对表达量有差异（P <0.05）；②4组软骨细胞ColⅡ mRNA和蛋白相对表达量有差异（P <0.05）；③4组软骨细胞ColⅡ mRNA和蛋白相对表达量变化趋势有差异（P <0.05）。进一步行两两比较，结果提示第2天、第4天时A组软骨细胞ColⅡ mRNA和蛋白相对表达量均高于其他3组（P <0.05）。第10天时，免疫组织化学染色发现A组软骨细胞ColⅡ染色最深，B组、C组、D组依次变浅。结论 TN14003较T140、AMD3100能明显缓解OA软骨关节退变，有望成为一种高效的抗OA治疗药物。

Abstract:

Objective To explore the effects of three antagonists (TN14003, T140, AMD3100) on type Ⅱ collagen in OA chondrocytes through targeted blocking SDF-1/CXCR4 signaling pathway, and to lay an experimental basis in order to find a novel agent for the treatment of osteoarthritis.Methods OA chondrocytes were divided into four groups randomly, among which added TN14003, T140, and AMD3100 respectively to the Group A, B, and C, while the Group D was selected as a control group. On the 2nd and 4th day after drug intervention, chondrocytes in four groups were harvested for measuring mRNA levels of type Ⅱ collagen with qPCR, and protein levels of type Ⅱcollagen with Western blotting. On the 10th day, the expressions of type Ⅱ collagen in OA chondrocytes in four groups were observed by Immunohistochemical staining.Results The chondrocytes of group of variance was performed to compared the relative expression levels of type Ⅱ collagen mRNA and protein of cells in four groups. The results showed that the relative expression levels of collagen type Ⅱ mRNA and protein were different among distinct time points (P < 0.05); the relative expression levels of type Ⅱ collagen mRNA and protein were different among distinct groups (P < 0.05); the change trends of relative expression levels of type Ⅱ collagen mRNA and protein of cells were different among the groups (P < 0.05). Further pair-wise comparison showed that the relative expression levels of type Ⅱ collagen mRNA and protein in group A were also higher than those in group B, C and D at days 2 and 4 (P < 0.05). The result of immunohistochemical staining for type Ⅱ collagen showed that collagen staining was the deepest in group A, and that was gradually lighter in group B, C, and D.Conclusion Compared with T140 and AMD3100, TN14003 can significantly slow down the degradation on typeⅡ collagen in OA chondrocytes, and is expected to be a highly effective anti-OA drug.