【作者】 陈刚；

【导师】 徐晓玉；

【作者基本信息】 重庆医科大学 ， 药理学， 2005， 博士

【摘要】 目的:通过观察川芎嗪对大鼠胶原性关节炎滑膜炎症发展,血管生成和血管内皮生长因子表达的影响,以及川芎嗪对大鼠腹腔巨噬细胞受脂多糖刺激后分泌血管内皮生长因子和表达缺氧诱导因子-1 的影响,探讨川芎嗪抗血管生成的机制。 方法: 1. 皮内注射Ⅱ型胶原诱导大鼠胶原性关节炎,给予 100mg/kg?d、50 mg/kg?d 川芎嗪灌胃,连续用药 30 天。以 10mg/kg?d 环孢霉素 A灌胃为阳性对照。检测关节炎指数积分和炎症足垫厚度,X 摄片检测关节结构,透射电镜观察滑膜毛细血管内皮细胞超微结构,免疫组化观察滑膜血管内皮生长因子的表达和滑膜微血管密度,RT-PCR 方法检测血管内皮生长因子 mRNA 的表达,Western Blot 方法检测血管内皮生长因子蛋白的表达。 2. 原代培养大鼠腹腔巨噬细胞,给予 10μg/ml 脂多糖刺激同时,分别加入剂量为 100μg/ml、10μg/ml、1μg/ml 的川芎嗪共同培养,以加入 0.5μg/ml 地塞米松为阳性对照。ELISA 方法检测巨噬细胞培养上清液中血管内皮生长因子的含量,MTT 法检测药物对巨噬细胞增殖的影响,Western Blot 方法检测巨噬细胞中缺氧诱导因子-1α蛋白的表达。 结果: 1. 用药 30 天后,100mg/kg?d 川芎嗪组大鼠关节炎指数为 6.57±1.06,与模型组 7.42±1.59 相比显著降低(P<0.05);50 mg/kg?d更多还原

【Abstract】 AIM:Collagen-induced arthritis were established in Wistar rats to investigate the effects of tetramethylpyrazine on arthritis, angiogenesis and expression of vascular endothelial growth factor in synovium. At the same time, abdominal macrophages were obtained from Wistar rats to investigate the effects of tetramethylpyrazine on the secretion of vascular endothelial growth factor and the expression of hypoxia-inducible factor-1 after the macrophages were stimulated by lipopolysaccharides. METHODS: 1. Collagen-induced arthritis animal models were established in Wistar rats by intradermal injections of chicken collagen typeⅡemulsified with incomplete Freund’s adjuvant. Rats with established collagen-induced arthritis were treated with 100mg/kg?d、50 mg/kg?d and 10mg/kg?d cyclosporine A, ig, continuously for 30 days . The effects of treatment were monitored by measurement of arthritis scores, footpad thickness, pathological characteristics of joint by histopathological techniques and joint roentgenography, the ultrastructure of vascular endothelial cells in synovium by transmission electron microscope investigation, microvessels density by immunohistochemical test as well as the expression of vascular endothelial growth factor in synovium by RT-PCR and Western Blot analysis. 2. Abdominal macrophages were stimulated by 10μg/ml lipopolysaccharides, and 100μg/ml、10μg/ml、1μg/ml tetramethylpyrazine and 0.5μg/ml dexamethasone were added. ELISA method was used to investigate the effect of tetramethylpyrazine on the secretion of vascular endothelial growth factor by macrophages, MTT method was used to investigate the effect of tetramethylpyrazine on the proliferation of macrophages, and Western Blot analysis was used to investigate the effect of tetramethylpyrazine on the expression of hypoxia-inducible factor-1α in macrophages. RESULTS: 1. Compared with collagen-induced arthritis models, 100mg/kg?d tetramethylpyrazine could remarkably reduce the arthritis index from 7.42±1.59 to 6.57±1.06 (P<0.05) after 30 day’ treatment; however, 50 mg/kg?d tetramethylpyrazine had no remarkable effect on the arthritis index, which was 7.14±1.15 (P>0.05). The footpad thickness of 100mg/kg?d tetramethylpyrazine group was 8.46 ± 1.56mm, compared with collagen-induced arthritis models group 10.12 ± 1.89mm, there was significant difference (P<0.05); The footpad thickness of 50mg/kg?d tetramethylpyrazine group was 9.89 ± 2.02mm, compared with collagen-induced arthritis models group, there was no significant difference (P>0.05). Joint roentgenography and transmission electron microscope investigation showed that 100mg/kg?d tetramethylpyrazine also could improve the radiographic findings in the joint and ultrastructure findings in the vascular endothelial cells, however, 50mg/kg?d tetramethylpyrazine had no remarkable effects on them. Immunohistochemical test showed that, compared the model group 50.41±4.78, positive cell percent of the vascular endothelial growth factor in synovium of 100mg/kg ? dtetramethylpyrazine group reduced to 44.37± 5.01, the difference is remarkable (P<0.01), but 50mg/kg?d tetramethylpyrazine had no effect on it, whose positive cell percent of the vascular endothelial growth factor was 47.01±3.71 (P>0.05). At the same time, compared the model group 29.22±5.07, the microvessel density of 100mg/kg?d tetramethylpyrazine group reduced to 24.79±4.22 (P<0.05), but 50mg/kg?d tetramethylpyrazine had no effect on it, whose microvessel density was 27.13±4.73 (P>0.05). RT-PCR and Western Blot analysis showed that 100mg/kg ? d tetramethylpyrazine could reduce the expression of vascular endothelial growth factor mRNA and protein in synovium, the OD value remarkably reduced from 1.10±0.11 to 0.39±0.17 (P<0.01), or from 0.71±0.29 to 0.39±0.14 (P<0.01), but the OD value of 50mg/kg?d tetramethylpyrazine was 1.08±0.09, or 0.68±0.25, there was no significant difference between 50mg/kg?d tetramethylpyrazine group and model group. 2. 100μg/ml and 10μg/ml tetramethylpyrazine, but not 1μg/ml tetramethylpyra更多还原