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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 inhibitory effect of asON on the expression of TIMP-1 gene and protein in the liver of experimental rats with experimental autoimmune liver fibrosis. Methods Four different groups of asON were designed according to the regulatory sequences of TIMP 1 secondary structure genome, structural proteins and coding region sequences. The cells were induced into the hepatic fibrosis rat model by tail vein injection. The expression of collagen type I and collagen Ⅲ was detected by reverse transcription polymerase chain reaction (RT PCR), in situ hybridization, collagen fiber special staining and electron microscopy Observed the impact of asON on liver fibrosis in rats. Results The asON designed by TIMP-1 could be expressed in vivo after being modified by thio and blocked the gene and protein expression of TIMP-1 in hepatic tissue of experimental liver fibrosis rats at the mRNA level, which could promote the expression of Ⅰ , Type Ⅲ collagen degradation (P <0.01). Liver fibrosis pathological grading and electron microscopy showed that asON has a certain effect on the reversal of liver fibrosis. Conclusions Thio-asON designed for TIMP-1 has a good anti-hepatic fibrosis effect in animals, which lays the foundation for the development of a new generation of anti-hepatic fibrosis gene therapy drugs.