硫代反义寡核苷酸对肝纤维化大鼠TIMP-1基因及蛋白表达的抑制作用

Inhibiting effect of antisense oligonucleotides phosphorothioate on gene and protein expression of TIMPs in rat liver fibrosis

目的 观察硫代反义寡核苷酸 (asON)对实验性免疫性肝纤维化大鼠肝组织中TIMP 1基因和蛋白表达的抑制作用。方法 根据TIMP 1二级结构基因组的调控序列、结构蛋白、编码区序列等分析 ,设计 4组不同的asON。利用尾静脉注射将其导入肝纤维化大鼠模型体内 ;通过逆转录 聚合酶链反应 (RT PCR)、Ⅰ型胶原和Ⅲ型胶原的免疫组化、原位杂交法、胶原纤维特殊染色及电镜等观察asON对大鼠肝纤维化的影响。结果 针对TIMP 1设计的asON经硫代修饰后在活体内能确切表达并能在mRNA水平上封闭实验性肝纤维化大鼠肝组织中TIMP 1的基因和蛋白表达 ,其结果可促进肝脏中Ⅰ、Ⅲ型胶原的降解 (P <0 .0 1)。肝纤维化病理学分级和电镜观察结果显示asON对肝纤维化的逆转具有一定效果。结论 针对TIMP 1设计的硫代asON在动物体内具有良好的抗肝纤维化效应 ,从而为研制新一代抗肝纤维化基因治疗药物奠定了基础。

Objective To observe the inhibition of asON phosphorothioate to the TIMP-1 gene and protein expression in the liver tissue of immune- induced hepatic fibrosis rats. Methods According to the analysis of modulator, structure protein, encoding sequence of TIMP-1 genome, we designed four different groups of asONs. These asONs were injected into the hepatic fibrosis rat models through coccygeal vein. The results were observed by RT-PCR, immunohistochemistry and in situ hybridization with collagen Ⅰ、Ⅲ, special staining of collagen fiber, electron microscope. Results The asON phosphorothioate of TIMP-1 could be expressed in vivo, and could block the TIMP-1 gene and protein expression in the liver of immune- induced hepatic fibrosis rats on the level of mRNA, which could promote the degradation of collagen Ⅰ、Ⅲ(P<0.01) and had certain effect on the reverse of hepatic fibrosis. The electron microscopic observation and pathologic classification of hepatic fibrosis models proved this result.Conclusions The asON of TIMP-1 has a good anti-hepatic fibrosis effect. The result lays the groundwork for the development of a new gene therapy drug of anti-hepatic fibrosis.