慢病毒介导的aggrecanase-2 shRNA转染对类风湿关节炎患者软骨细胞aggrecan的影响

 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200 μl、100 μl、50 μl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-2 mRNA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。

Effects of aggrecanase-2 shRNA transfection on chondrocytes of rheumatoid arthritis patient leaded by lentivirus

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 ɑggrecanase-2 shRNA5, ɑggrecanase-2 shRNA6, ɑggrecanase-2 shRNA7, ɑggrecanase-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.