雷公藤甲素含药血清对大鼠成纤维样滑膜细胞增殖及其巨噬细胞移动抑制因子表达的影响

Effect of triptolide drug serum on the proliferation of fibroblast like synoviocytes and the expression of macrophage migration inhibitory factor in vitro

目的:雷公藤甲素是雷公藤抗炎、抗肿瘤、免疫调节和抑制细胞增殖的主要有效成分。观察雷公藤甲素、雷公藤及其与来氟米特配伍的含药血清对大鼠成纤维样滑膜细胞的增殖及巨噬细胞移动抑制因子表达的影响。方法:实验于2006-12/2007-03在解放军白求恩国际和平医院中心实验室完成。①实验分组:雄性Wistar大鼠12只,体质量150～180g,随机数字表法分为6组,分别为雷公藤组、雷公藤甲素组、来氟米特组、雷公藤+来氟米特组、雷公藤甲素+来氟米特组和空白对照组,每组2只。②实验方法:雷公藤组、雷公藤甲素组、来氟米特组、雷公藤+来氟米特组、雷公藤甲素+来氟米特组,分别将雷公藤、雷公藤甲素、雷公藤+来氟米特、雷公藤甲素+来氟米特的含药血清以0.1、0.2、0.3的体积分数干预成纤维样滑膜细胞,空白对照组加入正常鼠血清。③实验评估:四甲基偶氮唑盐比色法观察细胞增殖情况;免疫组织化学染色及细胞ELISA法检测滑膜细胞和细胞上清液巨噬细胞移动抑制因子的表达。结果:纳入大鼠12只,均进入结果分析。①四甲基偶氮唑盐比色法结果显示,含药血清对体外滑膜细胞增殖具有显著抑制作用,单药最佳作用体积分数为0.2。②雷公藤甲素组、雷公藤组、雷公藤甲素+来氟米特组、雷公藤+来氟米特组的含药血清对成纤维样滑膜细胞均具有显著抗增殖效应,以体积分数为0.1雷公藤甲素联合体积分数为0.3来氟米特的含药血清抑制作用最强,与空白对照组相比,差异具有显著性意义(P<0.05)。③对巨噬细胞移动抑制因子蛋白表达的抑制作用则以雷公藤甲素+来氟米特的含药血清最佳,与其他各组相比,差异均有显著性(P<0.05)。结论:雷公藤甲素是雷公藤抑制成纤维样滑膜细胞增殖和抗炎作用的主要有效成分,而雷公藤甲素+来氟米特组的含药血清在抑制成纤维样滑膜细胞增殖及其表达巨噬细胞移动抑制因子等方面显示出明显的协同/增效作用。

AIM： Triptolide is the main effective ingredient of Tripterygium wilfordii for anti-inflammatory, antitumor, immune regulation and cell proliferation suppression. This study observed the effect of Triptolide, Tripterygium wilfordii combined with Leflunomide＇s drug-serum on the proliferation of fibroblast like synoviocytes and the expression of macrophage migration inhibitory factor （MIF） of macrophage in vitro. METHODS： The experiment was performed in the laboratory center of the Bethune Intemational Peace Hospital of Chinese PLA from December 2006 to March 2007. （1）Twelve male Wistar rats of 150-180 g were randomly divided into Tripterygium wilfordii （TW） group, Triptolide （Tri）, Lefiunomide group, TW＋ Leflunomide, Tri＋ Leflunomide and control group with 2 rats in each group. （2）Except control group, 0.1, 0.2 and 0.3 volume fraction corresponding drug-serums were used to interfere fibroblast like synoviocytes, and normal rat serum was given to normal control group.（3）Cell proliferation was observed by Ml-r method. The expression of MIF in the supernate fluid of fibroblast like synoviocytes was measured by ELISA and immunohistochemistry. RESULTS： All 12 rats were involved in the result analysis. （1）MTT method showed that drug-serum could obviously inhibit the proliferation of synoviocytes, and the best effective density of single drug was 0.2. （2）The drug serum of TW group, Tri group, Leflunomide group, TW＋ Leflunomide and Tri＋ Leflunomide showed remarkable inhibitory effect on the proliferation of synoviocytes, and the best concentration was 0.1 Tri combined with 0.3 Leflunomide （P 〈 0.05）. （3）The inhibitory effect of Tri combined with Leflunomide on the expression of MIF protein was the best, which was significantly different from other groups （P 〈 0.05）. CONCLUSION： Triptolide is the main effective component of Tripterygium wilfordii to inhibit the proliferation and cytokines production of fibroblast like synoviocytes. Triptolide combined with Leflunomide show obvious cooperation or synergistic effect on suppressing synoviocyte proliferation and MIF expression of synoviocyte.