Dynamic changes in ultrastructural morphology and serum levels of hyaluronic acid, laminin and collagen in rats with dimethylnitrosamine-induced liver fibrosis
- VernacularTitle:二甲基亚硝胺诱发大鼠肝纤维化模型肝脏超微结构形态学与血清透明质酸、层粘连蛋白、Ⅳ型胶原含量的变化
- Author:
Chunhui LI
;
Lihui PAN
;
Dongming PIAO
;
Zhengri YIN
;
Wenxie XU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;11(4):780-784,封4
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Development of liver fibrosis accompanies many morphological and functional changes. The pathological alterations of dimethylnitrosamine (DMN)-induced liver fibrosis in rats are similar to those of human liver fibrosis.OBJECTIVE: To observe the dynamic changes in morphology and serum hyaluronic acid (HA), laminin (LN), and type Ⅳ collagen in rats with liver fibrosis induced by DMN.DESIGN: Randomized controlled animal trial.SETTING: Laboratory of Teaching and Research Section of Pathology, College of Medicine, Yanbian University.MATERIALS: Eighty 3-month-old male rats of clean grade with 175-200 g body mass were selected, which were provided by the animal center of College of Medicine, Yanbian University. Agents: Dimethylnitrosamine provided by Sigma company, α smooth muscle actin by Dako company, Sirius red by Aldrich chem company, serum hyaluronic acid,laminin and type Ⅳ collagen kit by Sino-American Biotechnology Company, rabbit-anti-rat I g by Dako, Denmak company. Devices: JEM-1200EX transmission electron microscope made in Japan; enzyme linked immuno analyzer made in Japan; and CMTAS multifunction true color pathological image analysis system developed by Beijing University of Aeronautics and Astronautics.METHODS: The experiment was conducted in the College of Medicine, Yanbian University from June 2004 to December 2005. The rats were divided into 2 groups by lot: model group (n =40): The rats were intraperioneally injected with 10 g/L DMN (10 μL/kg) once daily, 3 days a week for 4 weeks; control group (n =40): The matching normal saline was injected at the same period; the blood from the left ventricle was collected and frozen in refrigerator at -70 ℃ before the rats were killed at days 7, 14, 21, and 28 and the liver tissue was removed for electron and light microscope observation. ①The dynamic changes in the content of serum HA, LN and type Ⅳ collagen were measured by enzyme-linked immunoabsorbent assay (ELISA). ②The morphological changes and liver fibrosis grading were examined by HE staining,immunohistochemical Sirius-red staining (liver fibrosis degree was classified into 5 grades: grade 0: no fibrosis; grade 1:fibrosis in portal area; grade 2: fibrotic septa between portal tracts; grade 3: fibrosis septa and structure disturbance of hepatic lobule; grade 4: cirrhosis), meanwhile, the area-density percentage of collagen fibrosis was calculated. ③The hepatic stellate cells were detected with transmission electron microscope and immunohistochemical alpha smooth muscle actin (SMA) staining. ④The correlation between area-density percentage of collagen fibrosis during liver fibrosis formation and the serum levels of HA, LN and type Ⅳ collagen was analyzed.MAIN OUTCOME MEASURES: ①The changes in the serum levels of HA, type Ⅳ collagen and LN during liver fibrosis formation; ②The morphological changes and liver fibrosis grading and area-density percentage of collagen fibrosis; ③Transformation and distribution characteristics of hepatic stellate cells; ④The correlation between area-density percentage of collagen fibrosis and the serum levels of HA, LN and type Ⅳ collagen.RESULTS: Among the 80 rats, 34 of the experimental group were modeled successfully, which were involved in the result analysis with the 40 rats in the control group. ①The levels of serum HA, type Ⅳ collagen and LN of the model group were significantly higher compared with the control group from day 7 to 28 (P ＜ 0.05), especially that on the 28th day. ②In the model group, the portal area of the rats showed hemorrhagic necrosis at day 7 after injection of DMN; at day 14,hemorrhage, necrosis and thin fibrotic septa joining central areas of liver were found; at days 21 and 28, thick septa was found; The area-density percentage of collagen fibrosis of the model group was significantly higher compared with the control group at days 7, 14, 21 and 28 (P ＜ 0.05), especially that on the 28th day. There were significant differences in the liver pathologic grading between the two groups at each time point (P ＜ 0.01); the pathologic grading of the model group at day 7 differed from those at days 14 and 28 (P ＜ 0.01). ③The α-SMA positive cells and a transitional hepatic stellate cell were found under the electron microscopy; typical myofibroblast was observed in the model group at day 21 and 28 under the electron microscopy. ④The area-density percentage of collagen fibrosis was positively correlated with the content of serum HA, LN and type Ⅳ collagen (r=0.707, 0.675, 0.662, P＜ 0.01).CONCLUSTON: There are significantly progressional changes in morphological and serum levels of HA, type Ⅳ collagen and LN in different stages of DMN-induced liver fibrosis in rats, moreover, the area-density percentage of collagen fibrosis is positively correlated with the serum levels of HA, LN and type Ⅳ collagen at different stages.
