靶向TNF-α的反义寡核苷酸治疗人工关节磨损微粒诱导的骨溶解的实验研究

Antisense oligonucleotides targeting TNF-α suppress on Co-Cr-Mo particle-induced osteolysis

[目的]研究单次皮下注射靶向肿瘤坏死因子α(tumor necrosis factor,TNF-α)的反义寡核苷酸(antisense oligonucleotides,ASO)治疗人工关节磨损微粒诱导的骨溶解的作用,探讨用于防治人工关节置换后假体松动的可能性。[方法]采用小鼠颅骨建立微粒诱导的骨溶解的动物模型,分为5组:第1组为假手术组,第2组为阳性对照组,第3组为低剂量治疗组,第4组为高剂量治疗组,第5组在第四组处置的基础上再次给予TNF-α。通过甲苯胺蓝染色观察颅骨表面骨吸收陷窝的数量并测量其面积,通过抗酒石酸的酸性磷酸酶(tartrate-resistant acid phospha-tase,TRAP)染色来观察破骨细胞并进行计数,通过定量试剂盒对TRAP的活性进行定量研究。同时采用RT-PCR和ELISA的方法检测TNF-α的表达。[结果]靶向TNF-α的ASO可以显著下调靶基因的表达,对微粒诱导的骨溶解有明显的抑制作用。骨吸收陷窝的数目与面积以及破骨细胞的数量均较阳性对照组有明显下降,定量研究显示TRAP的活性也有明显的下降。这种改变与ASO的用量呈明显的依赖性,高剂量组的治疗结果接近于假手术组。再次给予TNF-α可以逆转这种抑制效果。[结论]靶向炎症因子TNF-α的反义核酸通过抑制TNF-α表达,抑制了由微粒诱导的骨溶解。为防治人工关节置换后假体松动提供了一种很有应用前景的治疗策略。

[ Objective ] To investigate the effect of a single subcutaneous dose of an antisense oligonucleotide （ASO） on particle-induced osteolysis. [ Method ] The routine calvaria osteolysis model was utilized in ICR mice. Bone resorption was measured with the toluidine blue staining. Osteoclasts were detected by tartrate resistant acid phosphatase （TRAP） staining assay and were quantified by a TRAP quantification kit. [ Result] Bone resorption was 0.347 ± 0.09 mm^2 in animals with particle implantation, and decreased to 0. 123 ± 0. 05 mm^2 and 0. 052 ± 0. 02 mm^2 after ASO treatment in low and high doses, respectively. The bone resorption was reestablished in animals given an additional TNF-α. The number of osteoclasts in animal calvaria treated with ASO was reduced obviously compared with those untreated animals and the quantification results indicated that about 90% osteoclastgenesis was suprressed by the ASO. Additionally, the osteoclastgenesis was reestablished by the addition of TNF-α. [ Conclusion]An antisense oligonucleotide targeting an inflammatory factor, TNF-α, has been to suppress the osteolysis induced by particle for the first time. This new finding holds a great promise. It is a therapeutic strategy, for the component loosening.