摘要
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）、诱导型一氧化氮合酶（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。
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 （Col2al） 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 expres- sion of miRNA-34a in group B and C were significantly up-regulated by 2.70 times and 3.05 times compa- rable to group A, after silencing of miRNA-34a, the expression of miRNA-34a in group B and C signifi- cantly 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1 37. 00% were reduced respectively as comparable to that before silencing. 46. 80% 30. 00% down-regulation were recorded in expression of Col2al in group B and group C as comparable to group A, after silencing, the expression of Col2al were up-regulation in 2 times and 2. 95 times respetive- ly. Immunocytochemistry showed the more Col2al positive ceils 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 chon- drocyte in early osteoarthritis.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第7期1413-1415,共3页
Chinese Journal of Experimental Surgery
基金
国家重点基础研究计划（973）资助项目（2012CB619105）
