摘要
目的:观察原代培养的大鼠肺成纤维细胞给予不同浓度的千金子提取液后,对肺成纤维细胞增殖的影响以及药物的细胞毒性作用。方法:实验于2003-12/2004-03在军事医学科学院附属医院药剂科临床药理室完成。选取纯系Wistar雌性大白鼠10只,千金子为北京首创大地药业有限公司产品,经鉴定为大戟科属植物续随子的种子。经过传代5代以后的肺成纤维细胞大部分呈梭形,可以用于实验,3个实验各自独立。①肺成纤维细胞增殖检测与分组:大鼠肺成纤维细胞在正规生长培养基中传代培养,48h后处于对数生长期,可进行增殖试验。随机分为1个空白对照组和6个实验组。空白对照组加入空白DMEM,实验组分别加入浓度为500,250,125,62.5,31.25,15.625mg/L的千金子提取液。应用四甲基偶氮唑盐比色法观察瑞香狼毒提取液对细胞增殖的影响。②乳酸脱氢酶活性测定与分组:随机分为1个空白对照组和5个实验组。空白对照组加入空白DMEM,实验组分别加入浓度为125,62.5,31.25,15.625,7.813mg/L的千金子提取液。全自动生化分析仪测定乳酸脱氢酶活性值来观察其细胞毒性。③细胞形态学观察与分组:随机分为1个空白对照组和6个实验组。空白对照组加入空白DMEM,实验组分别加入浓度为500,250,125,62.5,31.25,15.625mg/L的千金子提取液,显微镜下观察细胞形态学变化。结果:实验纳入10只大鼠无脱落。①千金子提取液对肺成纤维细胞增殖的影响:浓度为500,250,125,62.5mg/L的千金子提取液的吸光度值明显低于空白对照组(0.108±0.004,0.089±0.002,0.093±0.005,0.187±0.002,0.285±0.011,P<0.01);浓度为31.25,15.625mg/L的千金子提取液的吸光度值仍低于空白对照组(0.231±0.014,0.218±0.023,0.285±0.011,P<0.05)。②千金子提取液对细胞的毒性作用:浓度为15.625,7.813mg/L的千金子提取液其乳酸脱氢酶吸光度值与空白对照组比较,均无显著性差异(28.91±0.88,26.77±0.26,25.91±0.39,P>0.05),说明千金子提取液在浓度为15.625,7.813mg/L范围内对细胞无毒性作用;而浓度为125,62.5,31.25mg/L的千金子提取液其乳酸脱氢酶吸光度值均显著高于空白对照组(P<0.05),且随着浓度的增加,毒性作用显著增强。③千金子提取液对肺成纤维细胞形态学的影响:细胞传代至第5代以后,所得细胞已经都是梭形呈放射状的成纤维细胞。用药前伸展良好,折光性较弱,有方向性;而加入千金子提取液后肺成纤维细胞数目显著减少,形状不规则,突起变短,细胞排列混乱,细胞内代谢产物增多。结论:千金子提取液对大鼠原代培养的肺成纤维细胞生长增殖有较强的抑制作用,随着给药剂量的增加,对细胞增殖抑制作用增强,呈剂量依赖性抑制。同时在高浓度范围内千金子提取液对细胞具有一定的毒性作用。
AIM： To observe the influence on the proliferation of lung fibroblasts after the treatment of euphorbiac crude extract of different concentrations, and discuss the cytotoxicity of the medicine. METHODS： The experiment was carried out in the clinical pharmacological room of Department of Pharmacy, Affiliated Hospital, Academy of Military Sciences between December 2003 and March 2004. Ten female pure Wistar white rats were used, euphorbiac was produced by Beijing Shouchuang Dadi Pharmaceutical Company, and it was identifed to be the seed of euphorbiae caper. Most of the lung fibroblasts showed the shape of fusiform after 5 passages, and they could be used in 3 independent experiments. ① Detection of the proliferation of lung fibroblasts and grouping： The lung fibroblasts of rats were subcultured in regular growth medium, and at the logarithm growth period after 48 hours, and could be used in the proliferation test. The lung fibroblasts were randomly divided into one a control group and 6 experimental groups, blank DMEM was added in the blank control group and euphorbiac crude extract of 500, 250, 125, 62.5, 31.25 and 15.625 mg/L were added into the experimental groups respectively. The influence of euphorbiae crude extract on cell proliferation was observed with the methyl-thiazol-tetrazolium colorimetry. ② Detection of the activity of lactate dehydrogenase and grouping： The lung fibroblasts were randomly divided into one a control group and 5 experimental groups, blank DMEM was added in the blank control group and euphorbiac crude extract of 125, 62.5, 31.25, 15.625 and 7.813 mg/L were added into the experimental groups respectively. The activity of lactate dehydrogenase was detected with the automatic biochemistry analyzer to observe its cytotoxicity. ③ Cell morphological observation and grouping： The lung fibroblasts were randomly divided into one a control group and 6 experimental groups, blank DMEM was added in the blank control group and euphorbiae crude extract of 500, 250, 125, 62.5, 31.25 and 15.625 mg/L were added into the experimental groups respectively. The cell morphological changes were observed under microscope. RESULTS： All the 10 rats were involved without deletion. ① Influence of euphorbiae crude extract on cell proliferation of lung fibroblasts： The absorbance values in the euphorbiae crude extract of 500, 250, 125, 62.5 mg/L groups were obviously lower than that in the blank control group （0.108±0.004, 0.089±0.002, 0.093±0.005, 0.187±0.002, 0285±0.011, P 〈 0.01）, and those were still lower in the 31.25 and 15.625 mg/L group than in the blank control group （0.231±0.014, 0.218±0.023, 0.285±0.011, P 〈 0.05）. ② The cytotoxicity of euphorbiae crude extract： The absorbance values of lactate dehydrogenase in the euphorbiae crude extract of 15.625 and 7.813 mg/L groups had no significant difference from that in the blank control group （28.91 ±0.88, 26.77±0.26, 25.91 ±0.39, P 〉 0.05）, indicating that euphorbiae crude extract of the concentration ranged 15.625 to 7.813 mg/L had no cytotoxicity. The absorbance values of lactate dehydrogenase in the euphorbiae crude extract of 125, 62.5, 31.25 mg/L groups were all significantly higher than that in the blank control group （P 〈 0.05）, and the cytotoxicity was significantly enhanced with the increase of the concentration. ③ Influence of euphorbiae crude extract on the morphology of the lung fibroblasts： After the 5th passage, all the obtained cells were the radiate fibroblasts with the shape of fusiform. Before medication, the extension was good, light reflection was weaker with directivity. After euphorbiae crude extract was added, the number of lung fibroblasts was significantly decreased, the shape was irregular, processus became shorter, the cells were disorder, and the intracellular metabolic products were increased. CONCLUSION：Euphorbiae crude extract plays a role in inhibiting the growth and proliferation of primarily cultured lung fibroblasts in rats, and the role is enhanced with the increase of the dose in a dose-dependent manner. Meanwhile, euphorbiae crude extract of high concentration has certain cytotoxicity.
出处
《中国临床康复》
CSCD
北大核心
2005年第27期101-103,共3页
Chinese Journal of Clinical Rehabilitation
