大鼠肝纤维化形成中基质金属蛋白酶2、9活性变化的病理意义

The role of changes of MMP-2, 9 activity in the development of liver fibrosis in rats

目的 探讨大鼠肝纤维化形成中基质金属蛋白酶2、9(MMP—2、MMP—9)活性变化及其病理意义。 方法 采用二甲基亚硝胺4周12次腹腔注射制作大鼠肝纤维化模型,分别于造模后1、2、3 d、1、2、4、6、8周作为动态观察时相点;MMP—2和MMP—9活性测定采用酶谱法,透射电镜观察肝组织超微结构,免疫组织化学观察肝窦壁Ⅳ型胶原(C Ⅳ)、层黏连蛋白(LN)和Ⅰ型胶原(C Ⅰ)表达,western blot方法测定肝组织金属组织蛋白酶抑制剂—2(TIMP—2)含量。 结果 造模后第2、3天,MMP—2、MMP—9活性(灰度值)显著增加(2 d:MMP—2正常对照组为54.72±4.56,模型组为70.76±7.63,F=16.27,P<0.05;MMP—9分别为25.71±4.29和51.76±15.33,F=13.38,P<0.05)。肝窦壁C Ⅳ(阳性面积比％)在造模第2、3天直至1周显著减少(2 d:正常对照组为6.06±1.35,模型组为2.86±0.63,F=69.12,P<0.05),4周末则显著增加(正常对照组为6.06±1.35,模型组为8.04±1.50,F=14.42,P<0.05)。MMP—9活性与肝窦壁C Ⅳ表达量呈显著负相关(F=—0.729,P<0.05);肝窦壁LN沉积4周末达峰值;正常肝窦壁无C Ⅰ表达,造模4周后肝窦壁C Ⅰ呈阳性染色;4周模型大鼠可见完整的基底膜形成;肝纤维化后期TIMP—2表达异常增加。 结论 早期MMP—9(主要的)、MMP—2活性升高,降解肝窦内皮细胞?

Objective To study the role of changes of matrix metalloproteinase-2, 9(MMP-2, 9) activity in the development of dimethylnitrosamine(DMN)-induced liver fibrosis in rats. Methods The rat liver fibrosis model was established by peritoneal injection of DMN(at a dose of 10 mg/kg, 3 times a week, for 4 weeks). The dynamic changes of liver fibrosis were observed at different time points(1d, 2d, 3d, 1week, 2weeks, 4weeks, 6weeks and 8weeks). The MMP-2, 9 activity was measured by zymogram method. Liver ultrastructure was observed by electron microscope. The expressions of type Ⅳ collagen(C Ⅳ), laminin(LN), type Ⅰ collagen(CI) and alpha-smooth muscle actin(α-SMA) were examined by immunohistochemistry. The tissue inhibitor of matrix metalloproteinase-2(TIMP-2) content was measured by Western blot method. Results The MMP-2,9 activity(gray value)significantly increased in the 2d and 3d DMN model rats(2d: normal/model group, MMP-2: 54.72±4.56/70.76±7.63, F=16.27, P<0.05; MMP-9: 25.72±4.29/51.76±15.33, F=13.38, P<0.05). The positive staining area percentage of C Ⅳ in the sinusoidal walls decreased in the 2d, 3d and 1weeks model rats(2d: normal/model group, 6.06±1.35/2.86±0.63, F=69.12, P<0.05), but significantly increased in the 4w model rats(normal/model group, 6.06±1.35/8.04±1.50, F=14.42, P<0.05). There was a remarkable negative correlation between the MMP-9 activity and expression of C Ⅳ in the sinusoidal walls (r=-0.729, P<0.05). Positive expressions of LN and C Ⅰ increased, and the strongest positive staining of them displayed in the 4w model rats. The formation of basement membrane was also observed in the 4 weeks model rats. Expression of TIMP-2 significantly increased in the late stage of fibrosis. Conclusions The increase of MMPs activity, especially MMP-9 which degrades the C Ⅳ normally distributed under the sinusoidal endothelium is the important factor in the formation of sinusoidal capillarization. The deposition and reconstitution of LN and new synthetic C Ⅳ, adding the deposition of C Ⅰconstitute the high density basement membrane. The increase of TIMP-2 expression in the late stage of the fibrosis may be one of reasons why natural resolution of DMN-induced liver fibrosis is difficult.