摘要
目的 探讨慢病毒介导的agggrecanase-2 shRNA对类风湿关节炎患者软骨细胞aggrecan的影响.方法 术中切取类风湿关节炎患者关节软骨,通过胰酶＋Ⅱ型胶原酶两步消化法消化软骨块并培养,取第2～3代软骨细胞.以慢病毒为载体将aggrecanase-2 shRNA5～8转染进入软骨细胞,观察转染后细胞的生长变化;以荧光定量PCR检测转染后第2、5、10天aggrecanase-2 mRNA水平的变化及aggrecan mRNA水平的变化,以循环阈值（cycle threshold,Ct）表示;用免疫组织化学方法检测aggrecan蛋白水平的变化,以积分光密度（integral optical density,IOD）表示,筛选最佳抑制序列.结果 以慢病毒为载体介导aggrecanase-2 shRNA转染软骨细胞后对细胞生长速度及形态无明显影响.加入病毒液200μl、100μl、50μl后感染复数分别为100、50、25,转染效率分别为90％、60％、30％.转染后aggrecanase-2mRNA的表达水平明显下降,特别是mRNA5,由开始的0.876 3±0.115 6下降至0.069 9±0.015 1 （P＜ 0.05）;aggrecan mRNA水平显著上调,由开始的0.992 1±0.201 3增加至3.049 2±0.278 2（P＜ 0.05）;aggrecan蛋白表达水平显著提高,由开始的496.160 5±225.673 7增加至4 525.433 0±1 131.813 0（P＜0.05）;最佳抑制序列为aggrecanase-2 shRNA5.结论 以慢病毒为载体介导aggrecanase-2shRNA转染骨关节炎患者软骨细胞可有效干扰aggrecanase-2 mRNA表达,相应地增加aggrecan表达,是一种保护aggrecan的有效途径.
Objective To investigate the effects of aggrecanase-2 knockdown in chondrocytes from rheumatoid arthritis patient by shRNA infection.Methods Cartilage harvested from rheumatoid arthritis patients who underwent total knee arthroplasty was digested by pancreatin and type Ⅱ collagen enzyme to obtain chondrocytes.Then chondrocytes were cultured and passaged to second or third generation.After aggrecanase-2 shRNA5,aggrecanase-2 shRNA6,aggrecanase-2 shRNA7,aggrecanase-2 shRNA8 infection,growth and morphological changes of the chondrocytes were examined.To select the best target sequence,mRNA expression of aggrecanase-2 and aggrecan was detected by RT-qPCR assay on day 2,5,10,represented with Ct （Cycle threshold） value.Expression of aggrecan protein was detected by immunocytochemistry,represented with IOD （integral optical density） value.Results aggrecanase-2 knockdown had no obvious effects on the morphology and growth of the chondrocytes.MOI （multiplicity of infection） was 100,50,25,and infection efficiency was 90％,60％,30％ with the corresponding viral load of 200 μl,100 μl,50 μl after transfection.mRNA expression of aggrecanase-2 was suppressed significantly,especially the group of shRNA5 which suppressed aggrecanase-2 expression from 0.876 3±0.115 6 to 0.069 9±0.015 1 （P ＜ 0.05）.mRNA and protein expression of aggrecan were significantly upregulated after infection.mRNA expression of aggrecan increased from 0.992 1±0.201 3 to 3.049 2±0.278 2 （P ＜ 0.05） and protein expression of aggrecan increased from 496.160 5± 225.673 7 to 4 525.433 0±1 131.813 0 （P ＜ 0.05）.Conclusion aggrecanase-2 suppression in chondrocytes by lentivirius infection is an effective method to protect the expression of aggrecan.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2014年第9期936-944,共9页
Chinese Journal of Orthopaedics
基金
国家自然科学基金（81171774,81272056）
