慢病毒介导的大鼠白细胞介素-1I型受体基因RNA干扰对体外软骨细胞的作用

Suppressive effect of lentivirus-mediated RNA interference on interleukin-1 receptor type I gene in rat chondrocytes in vitro

目的构建大鼠白细胞介素-1（IL-1）I型受体基因RNA干扰（RNAi）慢病毒表达载体，并观察其对体外大鼠软骨细胞的作用。方法筛选确定IL-1I型受体（IL—1RI）基因的RNAi有效靶序列，使用293T细胞包装产生慢病毒。收集293T细胞上清液并浓缩，得到滴度为1×10^7TU／ml的病毒。使用携带有效干扰片段的慢病毒分别以转染倍数（MOI）值20和50感染体外培养的软骨细胞，Westernblot法测定软骨细胞中IL—1RI以及基质金属蛋白酶-1（MMP-1）的表达。结果适合感染软骨细胞的MOI为50，感染率〉70％。经过RNAi后，体外培养的软骨细胞IL-1RI以及MMP-1蛋白表达显著降低。结论慢病毒介导的大鼠IL-1RI基因干扰能够显著降低体外培养的软骨细胞中IL-1RI的表达，并进一步降低下游蛋白MMP-1的表达。

Objective To construct lentiviral vectors targeting rat interleukin-1 receptor type I（IL- 1 RI） gene by RNA interference （RNAi） and to study its effects on chondrocytes in vitro. Methods Effec- tire sequence of siRNA targeting IL-1RI gene was designed and determined and 239T cells were were trans- fected with pGCL-GFP vector containing the inserted DNA sequence. The lentivirus containing the effective sequence of siRNA targeting IL-1RI gene with concentration of 1 × 107 TU/ml was obtained after supernatant of 293T cells were collected and concentrated. The recombinant lentivirus was used to infect IL-l-stimulated rat chondrocytes previously with multiple of infection （MOI） 20 and 50 seperately in vitro and the expression of IL-1RI and matrix metalloproteinase （MMP） -1 was detected by using Western blotting 3 days later. Re- suits The best MOI for lentivirus infecting chondrocytes was 50. Lentiviruses demonstrated a high （above 70% ） infection efficiency of chondrocytes. The expression of IL-1RI and MMP-1 was both repressed signifi- cantly in rat chondrocytes by RNAi. Conclusion The recombinant lentivirus shows significantly inhibitory effects on IL-1RIand MMP-1 （the downstream protein） in rat chondrocytes.