CAJ | 학술논문

目的探讨siRNA干扰大鼠含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶2(ADAMTS2)基因后对大鼠肝星状细胞(HSC)生物学特性的影响及其机制,评估ADAMTS2基因作为一种新的靶向基因在抗肝纤维化治疗中的应用前景.方法设计并化学合成3对针对大鼠ADAMTS2 mRNA 2237、2597及690位点的siRNAs.用筛选出的抑制效率最高的siRNA体外转染HSC-T6,应用实时PCR、Westem blot及MTT等方法研究沉默ADAMTS2基因对大鼠HSC活化、增殖等生物学特性及对基因ADAMTS2、α-SMA、COL1α1、COL(I)、TGF-β1、MMP-2和TIMP-3表达的影响.结果在相同注射剂量(50 nmol/L)及时间(48 h)下,2237位点的siRNA-ADAMTS2具有最高的抑制效率；能显著抑制ADAMTS2基因mRNA和蛋白表达(抑制率80％以上)；显著抑制COL(I)、α-SMA和TGF-β1在HSC中的表达.HSC的增殖能力显著降低.结论化学合成的siRNA-ADAMTS2干扰载体能有效抑制ADAMTS2基因在大鼠HSC-T6细胞系中的表达,抑制HSC的活化和增殖,同时显著降低COL(I)、α-SMA和TGF-β1的表达,具有潜在的阻止甚至逆转肝纤维化的作用.ADAMTS2可能是抗肝纤维化治疗的有效靶位,抑制ADAMTS2在肝脏的表达可能是一种有效的抗肝纤维化治疗策略.
목적 탐토siRNA간우대서함Ⅰ형혈소판결합단백기서적해취단백양금속단백매2(ADAMTS2)기인후대대서간성상세포(HSC)생물학특성적영향급기궤제,평고ADAMTS2기인작위일충신적파향기인재항간섬유화치료중적응용전경.방법 설계병화학합성3대침대대서ADAMTS2 mRNA 2237、2597급690위점적siRNAs.용사선출적억제효솔최고적siRNA체외전염HSC-T6,응용실시PCR、Westem blot급MTT등방법연구침묵ADAMTS2기인대대서HSC활화、증식등생물학특성급대기인ADAMTS2、α-SMA、COL1α1、COL(I)、TGF-β1、MMP-2화TIMP-3표체적영향.결과 재상동주사제량(50 nmol/L)급시간(48 h)하,2237위점적siRNA-ADAMTS2구유최고적억제효솔；능현저억제ADAMTS2기인mRNA화단백표체(억제솔80％이상)；현저억제COL(I)、α-SMA화TGF-β1재HSC중적표체.HSC적증식능력현저강저.결론 화학합성적siRNA-ADAMTS2간우재체능유효억제ADAMTS2기인재대서HSC-T6세포계중적표체,억제HSC적활화화증식,동시현저강저COL(I)、α-SMA화TGF-β1적표체,구유잠재적조지심지역전간섬유화적작용.ADAMTS2가능시항간섬유화치료적유효파위,억제ADAMTS2재간장적표체가능시일충유효적항간섬유화치료책략.
Objective To explore the preventive and therapeutic role of silencing type Ⅰ rat platelet-binding protein motifs depolymerization protein-like metalloproteinase 2(ADAMTS2)by siRNA on experimental liver fibrosis in vitro.By studying the mechanism of siRNA silencing of ADAMTS2,we also aim to evaluate the feasibility of ADAMTS2 as a target for anti-liver fibrosis therapy.Methods Three pairs of siRNAs targeting ADAMTS2 mRNA 2237,2597 and 690 targets were designed and synthesized by utilizing RNA design software.The most effective siRNA was chosen to transfect HSC-T6 cell line to test the tendency of hepatic stellate cell(HSC)activation and ex pression of ADAMTS2,COL1α1,COL(I),α-SMA,TGF-β1,MMP-2 and TIMP-3.These were quantified using real time-PCR,Western blotting,and MTT assays.Results Of the same dosage and time of injection,siRNA 2237 inhibited ADAMTS2 gene expression significantly more than other siRNAs.siRNA-ADAMTS2 2237 markedly inhibited ADAMTS2 gene and protein expression of HSCT6 with more than 80％ efficiency.Conversely,siRNA-ADAMTS2 2237 markedly reduced the gene and protein expressions of COL(I),α-SMA and TGF-β1 on HSC-T6 and inhibited the proliferation of HSC.Conclusions siRNA-ADAMTS2 2237 could effectively knockdown the gene and protein expression of ADAMTS2 in HSC-T6 cell lines.Silencing ADAMTS2 by siRNA significantly inhibited the activation,proliferation of HSC and the gene and protein expressions of COL(I),α SMA,and TGF-β1,and it may have a potential anti-fibrotic effect.ADAMTS2 might be an efficient target for anti-fibrotic therapy.