大鼠肝纤维化模型肝组织及血清中基质金属蛋白酶抑制因子-1的动态变化

Dynamic changes of hepatic structure and serum TIMP-1 in constructing the hepatic fibrosis modle of rats

目的观察二甲基亚硝胺(DMN)构建大鼠肝纤维化模型过程中肝组织及血清中TIMP-1及其相关指标的动态变化。方法1%DMN按10mg/Kg腹腔注射方法建立大鼠肝纤维化模型,第1w连续注药3d,第2、3、4、5、6w各连续2d。分别于第1、2、4、6、8w随机取血及大鼠肝组织。ELISA方法测血清基质金属蛋白酶抑制因子-1(TIMP-1)、基质金属蛋白酶-1(MMP-1)、透明质酸(HA)、层粘连蛋白(LN)、Ⅰ型前胶原(Ⅰ-CP)、Ⅲ型前胶原(Ⅲ-CP)含量。肝组织HE、Masson染色观察肝病理变化、胶原纤维含量,原位杂交观察TIMP-1mRNA表达;免疫组化观察TIMP-1蛋白表达量。结果注药后1w,血清TIMP-1即开始升高,4w后维持高值(131.1±13.16)ng/ml,肝组织内TIMP-1mRNA及蛋白同步增高;1w时,MMP-1含量增高,2w处于高峰(79.3±8.45)ng/ml,随后缓慢降低,8w时含量最低(46.6±6.31)ng/ml,然而仍高出正常对照组(32.1±3.66)ng/ml;1w时,HA含量升幅达对照组2倍,后持续增高,至6w时处高峰(350.8±40.26)ng/ml;LN于1w、2w、4w均小幅递增,6w达高峰(132.8±15.1)ng/ml;CP-I含量注药后1w始较对照组升高,6w处高峰(27.2±3.60)ng/ml;CP-Ⅲ含量用药后小幅升高,6w处于高峰(9.06±1.22)ng/ml。肝胶原纤维百分含量4w小幅递增,4w后,肝胶原纤维显著持续增高,8w时最高。大鼠肝组织病理变化显示:1w可见肝细胞变性及小点状坏死灶,4w时,纤维间隔伴小叶结构紊乱,符合肝纤维化3级,6w小部分3级,大部4级;8w,肝硬化表现。结论DMN构建大鼠肝纤维化模型肝组织及血清相关指标呈动态递进发展,为进一步选择合适时机干预肝纤维化进程提供依据、奠定基础。

Objective Observed the change of hepatic structure and dynamic parameters of serums in various period, in constructing the modle of hepatic fibrosis of rats with dimethylnitrosa-mine （DMN） Methods 1% DMN,abdominal injection,3 times series ir/first week,2 times series in the second,third,fouth,fifth and sixth week;After the first injection,random execute 5 rats differently at the first,second,fouth,sixth and eighth week. Sample blood and liver to detect the content of TIMP- 1 .MMP-1 ,HA,LN ,CP- I .CP-m by ELISA and observe the hepatic structure, and detect colla- gen in liver by Masson, TIMP-1 protein by immunohistochemistry,TIMP-1 mRNA by in situ hybridization. Results Content of TIMP-1 raised from the first week after the first DMN injection, such as expression of TIMP-1 mRNA （ by in situ hybridization） and TIMP-1 protein （by immuno- histochemistry） and continue to the top at the 4th week; content of MMP-1 locateded peak at second week, gradually decrease to the lowest,but still higher than contrast group .Content of HA raised by double times at first week,continue to peak, LN gradually increase to peak at the 6th week; CP-I raise from first week,then continue to the top at the 8th week; CP-re,peak also at the 8th week. Collageb raised gradiuly from the 4th week ,and showed marked difference to contrast group,to peak at 8th week.;One week after injection DMN,hepatic cell showed denaturation and drops of necrosis, the inflammation was appeared with circumference and middle of sinus hepaticus little fibre sedimentation; 4 weeks later, great part of liver showed denaturation of cells and harmo- nized necrosis, the fibro interval and confusion of trabecula were structured with S3 of hepatic fibrisis being classified,6 weeks later,classified to S4. Conclusion The hepatic fibrosis modle of rats are successfully established by abdominal injection of dimethylnitrosamine（DMN）,systematicly observed the change of hepatic structure and dynamic parameters of serums in various period, which provided the experimental models in order to research the hepatic pathological mechanism and hepatic fibrosis treatment.