Hedgehog信号通路阻断剂环巴胺体外对佐剂性关节炎大鼠的关节软骨细胞增殖的影响

Effect of the Hedgehog signaling pathway blockers cyclopamine on the articular cartilage cell proliferation of adjuvant arthritis rat in vitro

目的探讨Hedgehog(Hh)信号通路抑制剂环巴胺体外对佐剂性关节炎大鼠(AA)模型的关节软骨细胞增殖的影响和部分机制。方法弗氏完全佐剂诱导AA大鼠,测量关节炎指数和继发性足肿胀度,HE染色观察两组软骨组织生长情况;取AA大鼠踝关节软骨组织,采用胰蛋白酶-胶原酶法分离、培养、鉴定,环巴胺(0、0.05、0.5、5、20μmol/L)体外给药,MTT法检测AA大鼠踝关节软骨细胞增殖,Annexin V-FITC/PI双染检测AA大鼠踝关节软骨细胞凋亡,Western blotting检测AA大鼠踝关节软骨细胞Shh、Ptch1、Gli1的蛋白表达。结果弗氏完全佐剂诱导后,与正常大鼠相比,AA大鼠关节炎指数和继发性足肿胀度明显升高,HE染色显示,AA大鼠踝关节软骨组织有破坏;甲苯胺蓝和Ⅱ型胶原鉴定体外成功培养AA大鼠踝关节软骨细胞;体外给药环巴胺(0.05、0.5、5、20μmol/L)可升高AA模型关节软骨细胞增殖,流式细胞检测结果显示,环巴胺能降低AA模型软骨细胞凋亡率;与未用环巴胺组相比,环巴胺(0.5、5、20μmol/L)给药对AA软骨细胞中Hh信号通路相关蛋白(Shh、Ptch1、Gli1)表达显著下降。结论弗氏完全佐剂诱导建立AA大鼠模型成立,体外给药环巴胺可抑制AA大鼠软骨细胞的增殖,抑制软骨细胞的凋亡,该作用与抑制AA大鼠软骨细胞Hh信号有关。

Objective To explore the effect of Hedgehog（ Hh） signaling pathway inhibitor cyclopamine on articular cartilage cell proliferation and part of the mechanism in adjuvant arthritis（ AA） rats model in vitro. Methods Freund’s complete adjuvant induced AA rats,measurement of arthritis index and secondary foot swelling degree,rat’s cartilage tissue growth were observed by HE staining; AA rat ankle joint cartilage,pancreatic enzyme-collagen enzymatic isolation,culture, identification, AA rat ankle joint cartilage cell proliferation was determined by MTT method.Cyclopamine（ 0,0. 05,0. 5,5,20 μmol / L） in vitro,cell apoptosis was determined by Annexin V-FITC / PI double dye in AA rats ankle joint cartilage,proteins expressionin in AA rats ankle joint chondrocytes of Shh,Ptch1,Gli1 were determined by Western blotting. Results After freund’s complete adjuvant induced,compared with normal rats,the AA rats arthritis index and secondary foot swelling degree increased significantly,HE staining showed AA rats ankle cartilage destruction; Toluidine blue and type Ⅱ collagen in vitro cultivation of AA rats ankle joint cartilage cells; Cyclopamine（ 0. 05,0. 5,5,20 μmol/L） in vitro could elevated AA model of articular cartilage cell proliferation. Flow cytometry test results showed that cyclopamine reduced apoptosis rate of AA model of chondrocytes; Compared with the model group,cyclopamine（ 0. 5,5,20 μmol / L） declined AA chondrocytes related proteins express（ Shh,Ptch1 and Gli1） of Hhsignaling pathway significantly. Conclusion AA model rat was established by induced Freund ’s complete adjuvant.Cyclopamine may inhibit the proliferation of chondrocytes in AA rats in vitro,inhibit the apoptosis of cartilage cells,which was associated with inhibition of AA rat chondrocytes Hh signal.