CAJ | 학술논문

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-1 Ⅰ型受体(IL-IR Ⅰ)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1×107TU/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,Westem blot法测定软骨细胞中IL-1R Ⅰ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-1R I以及MMP-1蛋白表达显著降低.结论慢病毒介导的大鼠IL-IR Ⅰ基因干扰能够显著降低体外培养的软骨细胞中IL-IR Ⅰ的表达,并进一步降低下游蛋白MMP-1的表达.
목적 구건대서백세포개소-1 (IL-1)Ⅰ형수체기인RNA간우(RNAi)만병독표체재체,병관찰기대체외대서연골세포적작용.방법 사선학정IL-1 Ⅰ형수체(IL-IR Ⅰ)기인적RNAi유효파서렬,사용293T세포포장산생만병독.수집293T세포상청액병농축,득도적도위1×107TU/ml적병독.사용휴대유효간우편단적만병독분별이전염배수(MOI)치20화50감염체외배양적연골세포,Westem blot법측정연골세포중IL-1R Ⅰ이급기질금속단백매-1(MMP-1)적표체.결과 괄합감염연골세포적MOI위50,감염솔＞70％.경과RNAi후,체외배양적연골세포IL-1R I이급MMP-1단백표체현저강저.결론 만병독개도적대서IL-IR Ⅰ기인간우능구현저강저체외배양적연골세포중IL-IR Ⅰ적표체,병진일보강저하유단백MMP-1적표체.
Objective To construct lentiviral vectors targeting rat interleukin-1 receptor type Ⅰ(IL-1RⅠ) gene by RNA interference ( RNAi ) and to study its effects on chondrocytes in vitro.Methods Effective sequence of siRNA targeting IL-1 RⅠ gene was designed and determined and 239T cells were were transfected with pGCL-GFP vector containing the inserted DNA sequence.The lentivirus containing the effective sequence of siRNA targeting IL-1 RⅠ 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-1-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.Results The best MOI for lentivirus infecting chondrocytes was 50.Lentiviruses demonstrated a high ( above 70％ ) infection efficiency of chondrocytes.The expression of IL-1 RⅠ and MMP-1 was both repressed significantly in rat chondrocytes by RNAi.Conclusion The recombinant lentivirus shows significantly inhibitory effects on IL-1RⅠ and MMP-1 (the downstream protein) in rat chondrocytes.