摘要
目的观察以腺相关病毒(AAV)及慢病毒(Lentivirus)为载体含有针对大鼠金属蛋白酶组织抑制因子(TIMP)-1具有较强抑制作用的小干扰RNA(siRNA)对四氯化碳(CCl4)诱导的大鼠肝纤维化模型的干预作用。方法挑选针对大鼠TIMP-1基因具有较强抑制作用的siRNA序列,在体外构建为短发夹表达载体后,将其包装为重组AAV/siRNA-TIMP-1和Lenti/siRNA-TIMP-1,同时包装对照病毒AAV/EGFP和Lenti/EGFP。将Wistar大鼠分为对照组、CCl4模型组、AAV/EGFP组、Lenti/EGFP组、AAV/siRNA-TIMP-1和Lenti/siRNA-TIMP-1组,观察CCl4造模4周后,经H&E及Masson染色评价各组动物的肝纤维化状况,经荧光定量RCR及Western blot方法检测TIMP-1的表达抑制情况。结果 H&E及Masson染色证实,与对照组相比,CCl4模型组、AAV/EGFP组、Lenti/EGFP组肝脏胶原纤维明显增加,纤维化评分多为3-4级,同时肝脏TIMP-1的转录与蛋白表达水平均明显上升;而AAV/siRNA-TIMP-1和Lenti/siRNA-TIMP-1组肝纤维化程度明显减轻,纤维化评分多为2-3级,伴随肝脏TIMP-1的转录与蛋白表达水平均显著抑制。AAV/siRNA-TIMP-1和Lenti/siRNA-TIMP-1组在组织学表现及TIMP-1基因的转录与表达水平上无统计学差异。结论 AAV/siRNA-TIMP-1和Lenti/siRNA-TIMP-1均可有效抑制大鼠肝脏TIMP-1基因表达,进而发挥抗肝纤维化作用。
Objective To construct recombinant adeno-associated virus and lentivirus carrying siRNA of TIMP-1 and to investigate their antifibretic effects on CC14-induced liver fibrosis in ruts. Methods One pair of siRNA which could effectively inhibit expression of the TIMP-1 gene in HSC-T6 was screened and cloned into AAV vector and lentiviral vector to construct the recombinant AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1. AAV/EGFP and Lenti/EGFP as negativecontrol were also obtained. Fifty-eight male Wistar rats were randomly divided into six groups ： control group （ n = 8 ） , CC14 group, AAV/EGFP, Lenti/EGFP, AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups （all n = 10 ）. After the administration of CC14 for four weeks, liver samples were collected for the immunohistochemical staining and detection of TIMP-1 expression. Results Livers from the control rats showed normal lobular structure around vessels （HE and Masson staining）. In contrast, livers from the model, AAV/EGFP and Lenti/EGFP groups showed severe fibrosis, including septal fibrosis, extensive bridging, and fatty degeneration. The expressions of TIMP-1 mRNA and protein were also elevated in the livers from these groups. Compared with the fibrosis model group, the AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups showed good preservation of liver lobular architecture and only mild bridging fibrosis, accompanied by decreased expression of TIMP-1 mRNA and protein. Semi-quantitative analysis of the fibrosis stage indicated that most rats in the model, AAV/EGFP and Lenti/EGFP groups were of $3 and $4 （80%） , while 20% of the rats were of $5. In contrast, most rats （90%） in the AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups were of stages $2 and $3, with only one rat of $4. There was no significant difference between these recombinant virus therapy groups. Conclusions Both AAV/ siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 can suppress the expression of TIMP-1 in rat fibrotic liver, playing an effective antifibrotic role in the rat liver.
出处
《中国比较医学杂志》
CAS
2013年第6期58-62,I0008-I0009,共7页
Chinese Journal of Comparative Medicine
基金
国家自然科学基金资助项目(81000172)
王宝恩肝纤维化基金(20120131)
北京地区高等学校"肝脏保护与再生调节"重点实验室资助
关键词
腺相关病毒
慢病毒
小干扰RNA
金属蛋白酶组织抑制因子-1
大鼠
Adeno-associated virus （AAV）
Lentivirus
Small interfering RNA （siRNA）
Tissue inhibitor ofmetalloproteinase-1 （ TIMP-1 ）
Rat
