CAJ | 학술논문

目的：研究Hedgehog( Hh)信号通路阻断剂环巴胺对佐剂性关节炎(AIA)大鼠关节软骨细胞增殖凋亡的影响及抗凋亡机制。方法弗氏完全佐剂诱导AIA大鼠模型,胰酶-胶原酶法分离培养关节软骨细胞,环巴胺体外给药处理AIA软骨细胞,MTT法检测细胞增殖,DNA电泳、Hoechst染色、Annexin V-FITC/PI 双染检测细胞凋亡, RT-PCR 检测Shh、Gli1、Ptch1、Bcl-2、Bax 和 Caspase-3 mRNA 的表达。结果环巴胺(0.08、0.4、2、10、50μmol/L)剂量依赖性地提高AIA软骨细胞增殖。 AIA组可见凋亡细胞DNA梯状条带,环巴胺(0.4、2、10μmol/L)给药组的梯状条带明显减少；AIA组存在核碎裂与染色质固缩,环巴胺给药组均匀蓝色荧光的细胞数量增多；流式分析结果表明AIA软骨细胞凋亡率显著升高,环巴胺明显减少细胞凋亡率；与正常组相比, AIA组软骨细胞中Hh信号通路相关基因( Shh、Ptch1、Gli1) mRNA表达显著升高,抗凋亡基因Bcl-2 mRNA表达显著下降,而促凋亡基因Bax、Caspase-3 mRNA表达明显升高。环巴胺体外给药能抑制Hh信号通路过度活化,提高Bcl-2 mR-NA表达,并降低Bax、Caspase-3 mRNA表达。结论环巴胺体外给药能促进AIA软骨细胞的增殖,并抑制AIA软骨细胞的过度凋亡；该抗凋亡作用和调节Bcl-2、Bax和Caspase-3表达密切相关,提示干预软骨细胞Hh信号通路对保护类风湿关节炎关节软骨具有潜在的治疗意义。
목적：연구Hedgehog( Hh)신호통로조단제배파알대좌제성관절염(AIA)대서관절연골세포증식조망적영향급항조망궤제。방법불씨완전좌제유도AIA대서모형,이매-효원매법분리배양관절연골세포,배파알체외급약처리AIA연골세포,MTT법검측세포증식,DNA전영、Hoechst염색、Annexin V-FITC/PI 쌍염검측세포조망, RT-PCR 검측Shh、Gli1、Ptch1、Bcl-2、Bax 화 Caspase-3 mRNA 적표체。결과배파알(0.08、0.4、2、10、50μmol/L)제량의뢰성지제고AIA연골세포증식。 AIA조가견조망세포DNA제상조대,배파알(0.4、2、10μmol/L)급약조적제상조대명현감소；AIA조존재핵쇄렬여염색질고축,배파알급약조균균람색형광적세포수량증다；류식분석결과표명AIA연골세포조망솔현저승고,배파알명현감소세포조망솔；여정상조상비, AIA조연골세포중Hh신호통로상관기인( Shh、Ptch1、Gli1) mRNA표체현저승고,항조망기인Bcl-2 mRNA표체현저하강,이촉조망기인Bax、Caspase-3 mRNA표체명현승고。배파알체외급약능억제Hh신호통로과도활화,제고Bcl-2 mR-NA표체,병강저Bax、Caspase-3 mRNA표체。결론배파알체외급약능촉진AIA연골세포적증식,병억제AIA연골세포적과도조망；해항조망작용화조절Bcl-2、Bax화Caspase-3표체밀절상관,제시간예연골세포Hh신호통로대보호류풍습관절염관절연골구유잠재적치료의의。
Objective To explore the effects of cyclopamine, a hedgehog ( Hh) signal pathway inhibitor, on the proliferation and apoptosis of articular chondrocytes in adjuvant-induced arthritis ( AIA) rats and its anti-apoptotic mechanisms. Methods Freund’ s complete adjuvant was used to induce AIA. Articular chondrocytes were isolated and cultured by trypsin and collagenase digestion method. AIA articular chondrocytes were treated by cyclopamine in vitro. The cell proliferation was detected by MTT assay. The cell apoptosis was evaluated with DNA agarose gel electrophoresis, hoechst staining and flow cytometry analysis. The levels of Shh, Ptch1, Gli1, Bcl-2, Bax and Caspase-3 mRNA were detected by RT-PCR. Results Cyclopamine (0. 08, 0. 4, 2, 10, 50 μmol/L) could in-crease AIA articular chondrocytes proliferation in a dose-dependent. DNA ladder pattern was formed typically in AIA articular chondrocytes, while cyclopamine (0. 4, 2, 10μmol/L) treatment could reduce the formation of DNA ladder . AIA articular chondrocytes displayed nuclei fragmentation with condensed chromatin and cyclopamine could promote the number of uniform blue fluorescent cells. Flow cytometry analysis also indicated cyclopamine could vis-ibly reduce the rate of apoptotic cells in AIA articular chondrocytes. Compared with normal group, the levels of Hh pathway-related genes ( Shh, Ptch-1 and Gli1 ) mRNA apparently increased in AIA articular chondrocytes. Anti-apoptotic gene Bcl-2 mRNA level significantly decreased, while pro-apoptotic genes Bax and Caspase-3 mRNA lev-els were significantly increased. Cyclopamine treatment could inhibit the excessive activation of Hh pathway, up-regulate Bcl-2 mRNA expression, and reduce Bax and Caspase-3 mRNA expressions. Conclusion Cyclopamine could promote the proliferation and inhibit excessive apoptosis of AIA articular chondrocytes, which might be relat-ed to regulation of Bcl-2, Bax, and Caspase-3 expressions. Our results suggest that intervention of Hh signal in ar-ticular chondrocytes has potential therapeutic significance for articular cartilage protection in rheumatoid arthritis.