水蛭提取液对培养的大鼠脑皮质微血管内皮细胞分泌组织型纤溶酶原激活物和纤溶酶原激活剂抑制物1的影响

Effects of hirudo extract liquor on tissue-type plasminogen activator and plasminogen activator inhibitor-1 in microvascular endothelial cells from rat cerebral cortex

摘要:

目的 探讨水蛭提取液（HEL）对培养的大鼠脑皮质微血管内皮细胞分泌组织型纤溶酶原激活物（tPA）、纤溶酶原激活剂抑制物1（PAI-1）的影响。方法 建立大鼠大脑皮质微血管内皮细胞培养实验模型。MTT法筛选HEL的有效浓度。检测培养上清液的tPA、PAI-1含量与活性变化，RT-PCR检测经HEL治疗组与生理盐水对照组处理后的微血管内皮细胞tPA 与PAI-1的表达，免疫组化检测两组微血管内皮细胞tPA的表达。结果 HEL在一定浓度范围内（0.25～1 mg/μl）可促进微血管内皮细胞的生长，有剂量依赖关系（P<0.05）。HEL治疗组较生理盐水对照组能促进培养的大鼠脑皮质微血管内皮细胞分泌tPA，同时提高其活性，促进tPA mRNA的表达及tPA免疫活性表达，且呈剂量依赖性表达增强（P<0.01）。结论 HEL在体外能激活内源性纤溶系统。

Abstract:

Objective To study the effect of hirudo extract liquor (HEL) on activities of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), and levels of tPA and PAI-1 in microvascular endothelial cells of the rat cerebral cortex.Methods The experimental model of brain microvascular endothelial cells (BMEC) of Wistar rat cerebral cortex was prepared in vitro. Cell morphology was observed under the inverted phase contrast microscope and cell activity was measured with MTT assay after BMEC exposure to the concentrations of HEL ranging from 0.0625 to 8 mg/μl. The biochemical index, including activitives and levels of tPA and PAI-1 in cultured supernatants, as well as variation of semi-quantification of tPA, PAI-1 mRNA levels were measured in BMEC by reverse transcription polymerase chain reaction (RT-PCR) in the HEL treatment group and the control group normal saline treatment. The activities of tPA and PAI were measured by colorimetric assay. The contents of tPA and PAI-1 were determined using specific ELISA. The expression of tPA protein in BMEC was measured by immunohistochemistry.Results HEL of the concentrations ranging from 0.25 to 1 mg/μl promoted BMEC proliferation in a dose-dependent manner. Compared with the control group, HEL treatnent increased the content, and activity of tPA in cultured supernatants and tPA mRNA expression in BMEC, and also increased the expression of tPA protein immunoreactivity.Conclusions HEL could activate local endogenous fibrinolytic system in vitro.