miR-145体内转染对小鼠骨关节炎模型的影响△

Effect of transfection of miRNA-145 in vivo on the articular cartilage of osteoarthritis model in mice

摘要:

［目的］探讨 miR-145 体内转染对骨性关节炎模型小鼠关节软骨的影响。［方法］8 周龄雄性 C57 小鼠 40 只，随机分为 4 组，每组 10 只。分别为正常对照组 （normal control, NC）、骨性关节炎模型组 （osteoarthritis model, OAM）、正常动物转染组 （transferred normal animal, TNA） 及 OA 模型转染组 （transferred OAM, TOAM）。对 OAM 和 TOAM 组动物切断全部内侧半月板-胫骨韧带建立 OA 模型。7、10 d 时，对 TOAM 组和 TNA 组关节腔内注射载有 miR-145 的腺病毒液 20 μl。再过 4 d 后过度麻醉处死动物。行大体和组织学观察，并采用 qPCR 和 Western blot 检测相应标志物。［结果］ 形态方面，与 NC 组相比， OAM 组关节软骨结构明显紊乱，纤维细胞增生，基质着色主要呈现蓝绿色。TNA 组的软骨结构完整，但陷窝中出现空泡，基质着色呈蓝绿色。相比之下，TOAM 组软骨结构完整，关节软骨基质着色呈红色，与 NC 组接近。免疫组化检测显示，与 NC 组相比，OAM 组和 TNA 组的 FRS2、LC3 和 p62 染色的 OD 值均显著增加（P<0.05）；而 TOAM 组的 FRS2、LC3 和 p62 染色的 OD 值显著低于 OAM 组和 TNA 组（P>0.05）。定量检测方面，与 NC 组相比，OAM 组的 miRNA-145 的 mRNA 表达水平显著下降 （P<0.05），而体内转染后，TNA 组和 TOAM 组的 miRNA-145 的 mRNA 表达水平显著高于 NC 组 （P<0.05）。OAM 组的 LC3II/I 比值降低且 p62 表达水平显著升高 （P<0.05）。TOAM 组的 p62 的表达水平显著低于 OAM 组和 TNA 组 （P<0.05）。［结论］miR-145 体内转染可减轻关节失稳状的软骨退变，其机理可能与改变软骨自噬功能有关。

Abstract:

[Objective] To investigate the effect of miR-145 transfection in vivo on articular cartilage of osteoarthritis (OA) model in mice. [Methods] Forty male C57 mice aged 8 weeks were randomly divided into 4 groups with 10 mice in each group, including normal con- trol group (NC) , osteoarthritis model group (OAM) , transfected normal animal (TNA) , and transfected OA model (TOAM) . The animals in the OAM and TOAM groups received total resection of medial meniscus-tibial ligament to create OA model. At 7 d and 10 d, 20 μl of ade- nosis vector carrying miR-145 was injected into the joint cavity of the mice in the TOAM group and TNA group. The animals were sacri- ficed after another 4 days by excessive anesthesia, and then gross and histological observation were conducted, while qPCR and western blot assays were used to detect corresponding markers. [Results] In terms of morphology, the structure of articular cartilage in OAM group was obviously disordered, with fibrocyte proliferation and matrix stained in mainly blue-green compared with the NC group, while which preserved intact in structure, but with vacuoles in the lacunae, and the matrix in blue-green in the TNA group, whereas intact cartilage structure was noted in the TOAM group with red color of articular cartilage matrix similar to the NC group. In term of immunohistochemis- try, the OD values of FRS2, LC3 and p62 staining were significantly increased in OAM group compared with those in the NC group and TNA group (P＜0.05) , whereas which in the TOAM group were significantly lower than those in OAM group and TNA group (P＜0.05) . In terms of the quantitative detections, mRNA expression level of miR-145 in OAM group was significantly decreased (P＜0.05) , while which significantly ramped up in the TNA group and TOAM group compared with NC group (P＜0.05) . In OAM group the LC3II/I ratio significant- ly decreased (P＜0.05) and p62 expression level were significantly increased compared with those in NC group (P＜0.05) , while which in the TOAM group was significantly lower than that in OAM group and TNA group (P＜0.05) . [Conclusion] In vivo transfection of miR-145 does alleviate cartilage impairment in this joint instable state for marking OA model, and its mechanism may be related to the change of autopha- gy function of cartilage.