CAJ | 학술논문

目的观察早期骨关节炎(OA)软骨细胞中微小RNA(miRNA)-34a以及凋亡细胞的变化,并应用miRNA-34a的抑制剂锁核苷酸(LNA-miRNA-34a)观察miRNA-34a沉默对软骨细胞miRNA-34a的表达和凋亡的影响.方法建立6周兔早期OA模型,并分别获得正常(A组)、3周(B组)和6周(C组)软骨细胞,应用实时定量聚合酶链反应(Real-time PCR)和免疫细胞化学等技术分析软骨细胞中miRNA-34a以及Ⅱ型胶原(Col2a1)、诱导型—氧化氮合酶(iNOS)的表达及其规律,同时采用原位末端转移酶标记染色法(TUNEL)观察细胞凋亡.转染LNA-miRNA-34a后再用同样方法获得上述各项结果并进行比较分析.结果 B组和C组软骨细胞中miRNA-34a的表达水平分别是A组的2.70倍和3.05倍;在转染LNA-miRNA-34a后,B组和C组miRNA-34a的表达降低了2.07倍和3.03倍.转染前,B组和C组iNOS的表达与A组比较分别增加了13.50倍和16.70倍,转染后B组和C组中iNOS表达与转染前比较分别降低了41.00％和37.00％;转染前B组和C组Col2a1的表达水平和A组比较,分别降低了46.80％和30.00％;转染后,Col2a1的表达和转染前比较,B组增加了2.00倍,而C组增加2.95倍;免疫组织化学染色结果显示,转染后B组和C组Col2a1阳性细胞增多,阳性染色增强,甲苯胺蓝和TUNEL染色显示凋亡细胞数显著减少.结论早期OA软骨细胞miRNA-34a表达增加,LNA-miRNA-34a沉默miRNA-34a基因可以显著减少早期OA软骨细胞的凋亡.
목적 관찰조기골관절염(OA)연골세포중미소RNA(miRNA)-34a이급조망세포적변화,병응용miRNA-34a적억제제쇄핵감산(LNA-miRNA-34a)관찰miRNA-34a침묵대연골세포miRNA-34a적표체화조망적영향.방법 건립6주토조기OA모형,병분별획득정상(A조)、3주(B조)화6주(C조)연골세포,응용실시정량취합매련반응(Real-time PCR)화면역세포화학등기술분석연골세포중miRNA-34a이급Ⅱ형효원(Col2a1)、유도형—양화담합매(iNOS)적표체급기규률,동시채용원위말단전이매표기염색법(TUNEL)관찰세포조망.전염LNA-miRNA-34a후재용동양방법획득상술각항결과병진행비교분석.결과 B조화C조연골세포중miRNA-34a적표체수평분별시A조적2.70배화3.05배;재전염LNA-miRNA-34a후,B조화C조miRNA-34a적표체강저료2.07배화3.03배.전염전,B조화C조iNOS적표체여A조비교분별증가료13.50배화16.70배,전염후B조화C조중iNOS표체여전염전비교분별강저료41.00％화37.00％;전염전B조화C조Col2a1적표체수평화A조비교,분별강저료46.80％화30.00％;전염후,Col2a1적표체화전염전비교,B조증가료2.00배,이C조증가2.95배;면역조직화학염색결과현시,전염후B조화C조Col2a1양성세포증다,양성염색증강,갑분알람화TUNEL염색현시조망세포수현저감소.결론 조기OA연골세포miRNA-34a표체증가,LNA-miRNA-34a침묵miRNA-34a기인가이현저감소조기OA연골세포적조망.
Objective The purpose of this study are to investigate the expression of microRNA (miRNA)-34a in chondrocyte relatived to apoptosis in early osteoarthritis,and to investigate the effect of silencing miRNA-34a on chondrocyte apoptosis in a rabbit OA model.Methods 6 weeks rabbit OA model were established,and the chondrocyte were collected in normal(A group),and in 3 weeks and 6 weeks of OA,respectively.The expression of miRNA-34a,inducible nitric oxide synthase (iNOS) and type Ⅱ collagen (Col2a1) were analysis by the real time quantitative polymerase chain reaction (Real-time PCR),immunocytochemistry,the apoptosis were analysis by toluidine blue staining,and TdT-mediated dUTP nick end labeling (TUNEL) before and after silencing miRNA-34a by LNA-miRNA-34a.Results The expression of miRNA-34a in group B and C were significantly up-regulated by 2.70 times and 3.05 times comparable to group A,after silencing of miRNA-34a,the expression of miRNA-34a in group B and C significantly down-regulation in 2.07 times and 3.03 times respectively.The up-regulation of iNOS in 13.50 times and 16.70 times in group B and C comparable to A group before silencing were observed,after silencing,41.00％和 37.00％ were reduced respectively as comparable to that before silencing.46.80％和30.00％ down-regulation were recorded in expression of Col2a1 in group B and group C as comparable to group A,after silencing,the expression of Col2a1 were up-regulation in 2 times and 2.95 times respetively.Immunocytochemistry showed the more Col2a1 positive cells after silencing,and the apoptosis cells were significantly reduced.Conclusion The expression of miRNA-34a in early OA were up-regulation,it could be inhibited by LNA-miRNA-34a,miRNA-34a silencing could significantly reduce apoptosis of chondrocyte in early osteoarthritis.