Vassiliadis Efstathios, Larsen Dorthe Vang, Clausen Rikke Elgaard, Veidal Sanne Skovgård, Barascuk Natasha, Larsen Lise, Simonsen Henrik, Silvestre Toni Segovia, Hansen Christina, Overgaard Trine, Leeming Diana Julie, Karsdal Morten A
Nordic Bioscience, Herlev, Denmark.
Biomark Insights. 2011 Mar 24;6:49-58. doi: 10.4137/BMI.S6347.
The current study utilized a carbon tetrachloride (CCl(4))-induced liver fibrosis model to measure levels of the MMP9-mediated collagen type III degradation fragment CO3-610 (site of cleavage: KNGETGPQGP), during disease progression and regression, and to investigate a potential prognostic role of the biomarker.
72 female Sprague-Dawley rats aged 6 months old were injected with CCl(4) twice a week over different periods of time to induce varying degrees of liver fibrosis. After 4, 6 and 8 weeks of treatment, administration of CCl(4) was stopped. The 6- and 8-week treatment groups were left to regress for a further 6 or 12 weeks at which point they were sacrificed and livers removed and sectioned. Liver fibrosis was quantified using Visiopharm software to analyse Sirius red-stained sections. Serum levels of CO3-610 were measured in all animals using an ELISA assay as described by Barascuk et al.1
Quantitative histology revealed total collagen deposition in the liver increased as fibrosis progressed. In animals treated with CCl(4) for 4 weeks, collagen comprised on average 4.94% of the total tissue in liver sections, while after 6 weeks the mean was 8.25%, and after 8 weeks, 9.11%. During the regression phase, the total collagen deposition gradually decreased to a mean of 6.9% and 5.09% for animals regressing 6 and 12 weeks respectively after 6 weeks treatment, and 6.27% for animals regressed 12 weeks after 8 weeks treatment. CO3-610 values increased progressively in rats treated for 4 weeks (by a mean of 55.0 ng/ml), 6 weeks (mean 61.1 ng/ml) and 8 weeks (mean 70.2 ng/ml). During the regression phase, CO3-610 values rapidly decreased by a mean of 28.9 ng/ml at 6 weeks and 21.6 ng/ml at 12 weeks in animals previously treated for 6 weeks, and by a mean of 19.52 ng/ml in animals treated for 8 weeks and regressed for 12 weeks. CO3-610 levels were statistically significantly correlated with total collagen during disease progression (r = 0.5701, P < 0.0001). No statistically significant correlation was observed during regression (r = 0.2081, P = 0.1138).
Levels of the MMP-9 generated fragment of collagen type III, CO3-610, correlated with the degree of liver fibrosis in rats during the progression phase, but were not correlated with total collagen levels during regression. CO3-610 seems to be produced only under the CCL(4) stimulus, and signifies CO3-610 as a potential marker of progression rather than regression. The corresponding steep elevations in levels of CO3-610 total collagen and collagen type III during liver fibrosis progression underline a potential prognostic capacity of the biomarker.
本研究利用四氯化碳（CCl₄）诱导的肝纤维化模型，测定疾病进展和消退过程中基质金属蛋白酶9（MMP9）介导的III型胶原降解片段CO3 - 610（切割位点：KNGETGPQGP）的水平，并研究该生物标志物的潜在预后作用。
72只6月龄雌性Sprague - Dawley大鼠在不同时间段每周注射两次CCl₄，以诱导不同程度的肝纤维化。治疗4、6和8周后，停止给予CCl₄。6周和8周治疗组再分别继续消退6周或12周，之后处死大鼠，取出肝脏并切片。使用Visiopharm软件对天狼星红染色切片进行分析，以量化肝纤维化程度。按照Barascuk等人所述的酶联免疫吸附测定法（ELISA），检测所有动物血清中CO3 - 610的水平。
定量组织学显示，随着纤维化进展，肝脏中总胶原沉积增加。用CCl₄治疗4周的动物，肝脏切片中胶原平均占总组织的4.94%，6周后平均值为8.25%，8周后为9.11%。在消退期，总胶原沉积逐渐减少，6周治疗后分别消退6周和12周的动物，其总胶原沉积平均值分别降至6.9%和5.09%，8周治疗后消退12周的动物，总胶原沉积平均值为6.27%。在接受4周（平均增加55.0 ng/ml）、6周（平均61.1 ng/ml）和8周（平均70.2 ng/ml）治疗的大鼠中，CO3 - 610值逐渐升高。在消退期，先前接受6周治疗的动物，CO3 - 610值在6周时平均迅速下降28.9 ng/ml，在12周时下降21.6 ng/ml；接受8周治疗并消退12周的动物，CO3 - 610值平均下降19.52 ng/ml。在疾病进展过程中，CO3 - 610水平与总胶原呈显著统计学相关性（r = 0.5701，P < 0.0001）。在消退期未观察到显著统计学相关性（r = 0.2081，P = 0.1138）。
III型胶原的MMP - 9生成片段CO3 - 610的水平在大鼠肝纤维化进展期与肝纤维化程度相关，但在消退期与总胶原水平无关。CO3 - 610似乎仅在CCl₄刺激下产生，表明CO3 - 610是进展而非消退的潜在标志物。肝纤维化进展过程中CO3 - 610、总胶原和III型胶原水平相应的急剧升高突出了该生物标志物的潜在预后能力。
