凝血酶体外诱导模拟脑室出血后大鼠蛛网膜细胞纤维化的实验研究

An experimental study of simulating fibrosis of rat arachnoid cells after intraventricular hemorrhage by thrombin stimulation in vitro

摘要:

目的 观察凝血酶（thrombin）刺激体外培养大鼠蛛网膜细胞后纤维化程度的变化，探讨其与脑室出血（intraventricular hemorrhage，IVH）后大鼠慢性脑积水形成的关系。方法 采用SD大鼠蛛网膜细胞，经不同浓度凝血酶刺激，在体外模拟脑室出血（IVH）后慢性脑积水大鼠的蛛网膜细胞纤维化的病理生理过程。细胞免疫化学法（immunocytochemistry ，ICC）鉴定传代培养的SD大鼠蛛网膜细胞标志物细胞角蛋白（CK 8/18）和桥粒蛋白（Desmoplakin）的表达。qRT-PCR与Western Blot检测细胞纤维化因子（Col-I、Col-III、TGF-β1、α-SMA）的表达，观察其纤维化程度，同时检测比较各组细胞的表达差异。结果 （1）培养的蛛网膜细胞标志物Cytokeratin和Desmoplakin表达阳性。（2）凝血酶可诱导蛛网膜细胞纤维化，在50u/ml浓度下，蛛网膜细胞纤维化程度最明显。（3）实验组较对照组细胞纤维化程度明显。结论 体外可以稳定培养大鼠蛛网膜细胞。凝血酶诱导的蛛网膜细胞各种纤维化指标明显高于正常组，提示纤维化模型构建成功。构建成功的纤维化蛛网膜细胞有利于下一步深入研究脑室出血后慢性脑积水的机制。

Abstract:

Objective To investigate the change in the fibrosis degree of cultured rat arachnoid cells in vitro after being stimulated by thrombin, and to explore its relationship with the development of chronic hydrocephalus after intraventricular hemorrhage (IVH) in rats.Methods We used different concentrations of thrombin to stimulate the arachnoid cells of Sprague-Dawley (SD) rats, simulating in vitro the pathophysiological process of fibrosis of arachnoid cells in rats with chronic hydrocephalus after IVH. Immunocytochemistry was used to identify the expression of the markers of subcultured SD rat arachnoid cells, i.e., cytokeratin (CK 8/18) and desmoplakin. The mRNA and protein expression of fibrosis factors (Col-I, Col-III, TGF-β1, and α-SMA) in arachnoid cells was measured by quantitative real-time PCR and Western blot, respectively. Those factors were measured for indicating fibrosis degree, and they were compared between different groups.Results (1) Cultured arachnoid cells showed positive expression of the markers cytokeratin and desmoplakin. (2) Thrombin induced fibrosis of arachnoid cells, with the most significant fibrosis degree at 50 u/ml. (3) The experimental group had a higher fibrosis degree than the control group.Conclusions Rat arachnoid cells can be stably cultured in vitro. Thrombin-stimulated arachnoid cells have significantly higher levels of various fibrosis indicators than the control cells, suggesting the successful establishment of fibrosis model. This arachnoid cell fibrosis model can help to further investigate the mechanism of chronic hydrocephalus after IVH.