慢病毒介导的Aggrecanase-1 shRNA转染体外培养的类风湿关节炎患者软骨细胞的研究  

The study on lentivirus mediated Aggrecanase-1 shRNA transfection in vitro cultured chondrocytes of rheumatoid arthritis patients

作　　者：杜清峰 王英振[1] 张海宁[1] 刘英魁 杜明 程莉 王永 冯志强 张宝安 宋振 王宁 谭玉春 王亮 杜小卫 

机构地区：[1]青岛大学附属医院关节外科,266003 [2]山东省平原县第一人民医院脊柱关节外科

出　　处：《中华风湿病学杂志》2015年第1期36-41,F0003,共7页Chinese Journal of Rheumatology

基　　金：国家自然科学基金（81171774,81272056）

摘　　要：目的 探讨慢病毒介导的RNA干扰在软骨细胞的可行性,确定是否可以通过抑制软骨细胞中Aggrecanase-1的表达实现对软骨中Aggrecan的保护作用.方法 将设计并构建的shRNAAggrecanase-1慢病毒表达载体转染进RA患者关节软骨细胞.通过荧光定量PCR检测转染后不同时间点不同转染复数下Aggrecanase-1及Aggrecan mRNA水平的变化;通过免疫细胞化学方法测定各组Aggrecan蛋白水平表达量的变化,所有数据利用SPSS 19.0软件,采用配对t检验及方差分析进行统计分析.结果 由慢病毒介导的RNA干扰中,随着Aggrecanase-1 mRNA表达水平显著下降,Aggrecan mRNA 和蛋白表达也相应地增加,特别是shRNA4组变化较为显著,其中Aggrecanase-1 mRNA由开始的1.083±0.133降为0.074±0.004（t=35.71,P＜0.05）;Aggrecan mRNA由开始的1.182±0.160增加到2.922±0.169 （t=19.43,P＜0.01）;同时Aggrecan蛋白多糖由开始的507±265增加到2 270±334（t=11.43,P＜0.05）.结论 由慢病毒介导的RNA干扰可以实现对Aggrecanase-1表达的抑制,且对软骨细胞的表型和生长速度没有负面影响;慢病毒介导的RNA干扰可以通过抑制Aggrecanase-1的表达实现对Aggrecan的保护,是一种缓解软骨退化的有效途径.Objective The major change found in rheumatoid arthritis （RA） is the rapid destruction of cartilage,Aggrecanase-1 plays an important role in Aggrecan degradation in RA patients which is the main components of cartilage matrix.This study aims to identify an effective way to inhibit Aggrecanase-1 expression,to explore if the RNAi mediated by lentivirus in cartilage is feasible,and determine whether the Aggrecan in chondrocyte can be protected by inhibiting the expression of Aggrecanase-1.Methods The lentivirus vectors expressing shRNA targeting Aggrecanase-1 was constructed and transfected into cultured RA patients articular chondrocyte.Gene mRNA levels of Aggrecanase and Aggrecan in different time points after transfection with different MOI were analyzed by RT-PCR.Aggrecan concentration was measured by immuno-histochemistry,the results were processed by SPSS 19.0 software,with the method of paired t test and ANOVA.Results The specific inhibition of Aggrecanase-1 by RNAi mediated by lentivirus had no negative effect on the morphology and growth velocity of chondrocytes.With the mRNA of Aggrecanase-1 decreased significantly,the Aggrecan expression increased accordingly both at mRNA and protein level,especially the change of shRNA4 group was significant,in which the Aggrecanase-1 mRNA decreased from the baseline level 1.083± 0.133 to 0.074±0.004.Aggrecan mRNA increased from the baseline 1.182±0.160 to 2.922t0.169; at the same time,Aggrecan proteoglycan increased from the baseline 507±265 to 2 270±334 （P＜0.05）.Conclusion The results suggest that lentivirus mediated inhibition of Aggrecanase-1 by RNAi can retard Aggrecan degra-dation,without inter-fering with chondrocytic phenotype and viability.RNAi technology can be a useful tool to mitigate cartilage degeneration.

关 键 词：软骨细胞 RNA干扰 蛋白聚糖蛋白酶-1 

分 类 号：R593.22[医药卫生—内科学]