两种不同病毒载体携带靶向大鼠金属蛋白酶组织抑制因子（TIMP）-1小干扰RNA抗肝纤维化作用的比较

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（CCI。）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-I和Lenti／siRNA-TIMP-I，同时包装对照病毒AAV／EGFP和Lenti／EGFP。将Wistar大鼠分为对照组、CCl。模型组、AAV／EGFP组、Lenti／EGFP组、AAV／siRNA-TIMP-I和Lenti／siRNA-TIMP-1组，观察CCl。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Westernblot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，CCI．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34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-l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-l和Lenti／siRNA-TIMP-I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-I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。

Comparison between the antifibrotic effects of adeno-associated virus and ientivirus carrying small interfering RNA of TIMP-1 in rat liver fibrosis

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.