环巴胺对佐剂性关节炎大鼠关节软骨细胞增殖和凋亡的影响

目的 观察Hedgehog(Hh)信号通路抑制剂环巴胺对体外培养的佐剂性关节炎(AA)大鼠关节软骨细胞增殖和凋亡的影响.方法 SD大鼠足趾皮内注射弗氏完全佐剂制备AA模型(模型组),对照组足趾皮内注射等量生理盐水;观察继发侧足肿胀,28 d后,HE染色评价踝关节形态学变化;胰酶-胶原酶消化培养AA大鼠软骨细胞,采用甲苯胺蓝染色和Ⅱ型胶原染色鉴定软骨细胞,CCK-8法检测环巴胺处理对AA大鼠软骨细胞增殖的影响,Hoechst 33258检测环巴胺对AA大鼠软骨细胞凋亡影响,Western blot法检测AA大鼠软骨细胞中Hh信号通路相关蛋白(Shh、Ptch1、Gli1)的表达情况.结果 第12天,AA大鼠继发侧关节肿胀明显,HE染色表明AA大鼠踝关节破坏明显;Ⅱ型胶原和甲苯胺蓝染色证实成功培养AA大鼠软骨细胞;不同剂量的环巴胺可促进AA大鼠软骨细胞增殖,抑制其凋亡,降低Shh、Ptch1、Gli1 蛋白表达.结论 环巴胺可促进AA大鼠软骨细胞的增殖,抑制其凋亡,其机制与抑制AA大鼠软骨细胞Hh信号通路有关.

Effect of cyclopamine on the articular cartilage cell proliferation and apoptosis of adjuvant arthritis rat in vitro

Objective To observe the effect of Hedgehog ( Hh) signaling pathway inhibitor cyclopamine on the proliferation of chondrocytes in adjuvant arthritis ( AA) in vitro. Methods AA model was established by injection of Freund's complete adjuvant in the toe skin of SD rats, and the same amount of normal saline was injected into the control group. Secondary swelling was detected, the changes of the ankle joints were evaluated by HE staining after 28 d. Pancreatic enzyme-collagenase cultured cartilage cells of AA model rats, chondrocytes were identified by toluidine blue staining and collagen typeⅡimmunocytochemistry staining, the effect of different dose of cyclopam-ine on cartilage cell proliferation was detected by CCK-8 method, chondrocytes apoptosis in AA rats were detected by Hoechst 33258 staining, Hh signaling pathways related protein (Shh, Ptch1 and Gli1) in chondrocytes expres-sion were detected by Western blot method. Results From 12nd day, the rats showed significant swelling in the secondary arthrosis in AA rats, and HE staining showed that AA rats were damaged significantly. Chondrocytes of AA rats were proved successful by type II collagen and methylaniline blue. The growth of chondrocytes could in-crease significantly, and could inhibit apoptosis and decrease the expression of Shh, Ptch1 and Gli1 protein in high concentrations of cyclopamine. Conclusion Cyclopamine inhibits the proliferation and apoptosis of chondrocytes in AA rats, and its mechanism is related to inhibit Hh signal pathway in AA rats.