金属蛋白酶组织抑制因子-1 RNA干扰靶位的筛选

Target screening for RNA interference of the tissue inhibitor of metalloproteinase-1 gent

目的筛选金属蛋白酶组织抑制因子-1(tissue inhibitor of metalloproteinase-1,TIMP-1)的RNA干扰靶位,从而有效抑制肝星状细胞的活化,阻断纤维化进程。方法针对TIMP-1基因,设计3个RNA干扰候选靶位,化学合成双链RNA(dsRNA),分别转染经TGF-β1刺激活化的大鼠肝星状细胞。48h后通过荧光定量PCR测定TIMP-1、前胶原-α1(procol-α1)mRNA相对表达量,计算抑制率;并取上清液检测肝纤维化指标,进行比较分析。结果针对TIMP-1基因的3个靶位,dsRNA都能不同程度抑制TIMP-1基因表达(TIMP-1 mRNA相对表达抑制率在3个靶位分别为26.61%、17.42%和68.55%),其下游Procol-α1基因表达也受到抑制(抑制率在3个靶位分别为6.01%、6.57%和62.84%);肝纤维化指标Ⅲ型胶原(Co1-Ⅲ)、透明质酸(HA)和层粘蛋白(LN)的表达也降低,而且针对第3靶位时降低最明显。结论针对TIMP-1基因第3靶位的dsRNA,即"TIMP-1-3组"对TIMP-1及其下游基因表达的抑制作用最强。成功筛选出TIMP-1最有效的RNA干扰靶位,为进一步探索肝纤维化基因治疗的新途径打了基础。

Objective To scan and select the RNA interference（RNAi）most effeetive target in the connective tissue growth factor gene for the more effective inhibition of the activation of hepatic stellate cell（HSC） and for the blocking of the liver fibrosis process. Methods Three candidate sites targeting the TIMP-1 gene were designed, and these double-stranded RNA RNA （dsRNA）were chemically synthesized. Then, the chemically synthesized were transinfected into the rat hepatic stellate cells （HSC） activated by transforming growth factor β（TGF-β） in advance, respectively. RT-real-time PCR was employed to determine the relative mRNA expression of TIMP-1 and procollagen-α1（procol-α1）, and the liver fibrosis markers, such as collagen-lll （ Col- Ⅲ）, hyaluronic acid （ HA）, and laminin （LN） in the cultural supematant were measured. Results dsRNA targeting the three sites of TIMP-1 gene could inhibite the TIMP-1 gene expression to varying degrees （TIMP-1 mRNA relative expression inhi- bition rate in the three targets were 26. 61%, 17.42% and 68.55%, respectively）, and the downstream Procol-cd gene expres- sion was also inhibited （ inhibition rate at the three targets were 6. 01%, 6. 57% and 62. 84%, respectively）. The liver fibrosis markers ,collagen In （Col-Ⅲ） ,hyaluronic acid（HA） and laminin（LN） were reduced,moreover,dsRNA targeting the third site of TIMP-1 gene （named as TIMP-1-3 group） reduced these markers most obviously. Conclusion Among the three dsRNA, the de- signed dsRNA targeting the third site of the TIMP-1 gene inhibited the TIMP-1 and its downstream gene expression most obviously. The most effective dsRNA had been screened out. This achievement provided an experimental basis for RNA interference in gene therapy for liver fihrosis.