摘要
目的:探讨大黄素干预对家兔离体血管平滑肌细胞增殖的作用,并观察其浓度效应剂量。方法:实验于2004-06/12在第三军医大学西南医院药学部完成。选择雄性家兔1只作为动脉平滑肌细胞的动物来源,采用组织贴块法进行主动脉平滑肌细胞的培养。细胞培养成功后分2组:对照组和大黄素组。对照组不加药,大黄素组按剂量分为5个亚组即1.25,2.5,5,10,20mg/L组,每组6孔。选用生长良好的第三四代细胞,加3H-标记的胸腺脱氧核苷酸行液闪计数以表征细胞增殖的程度,细胞增殖测定采用酶标仪上比色(波长为570nm)用吸光度值表示。超过氧化物歧化酶测定采用邻苯三酚自氧化抑制法,直接以每毫升培养液中超过氧化物歧化酶kat表示。脂质过氧化物测定采用改良TBA微量法,以脂质过氧化物的稳定终产物丙二醛浓度(nmol/L)表示。实验结果采用单因素方差分析(方差齐性检验),用SNK法进行均数之间的显著性检验。结果:①大黄素对细胞增殖的影响:随着大黄素剂量的增加,吸光度值逐渐减少,抑制率逐渐增高。大黄素5,10,20mg/L剂量组的吸光度值明显低于对照组(0.594±0.037,0.410±0.014,0.301±0.024,0.730±0.041,P<0.05)。②大黄素对胸腺嘧啶脱氧核苷酸掺入量的影响:随着大黄素剂量的增加,胸腺嘧啶脱氧核苷酸掺入量逐渐减少,抑制率逐渐增高。大黄素5,10,20mg/L剂量组每分液闪计数值明显低于对照组大黄素(33537±3713,29304±4336,21155±1938,73958±2276,t=5.862,7.330,18.688,P<0.05)。③大黄素对超过氧化物歧化酶的影响:大黄素剂量1.25,2.5mg/L剂量组,超过氧化物歧化酶高于对照组,但差异不显著(P>0.05)。5,10,20mg/L剂量组明显高于对照组犤(118.19±3.56),(127.48±4.59),(140.23±4.96),(100.16±9.83)kat/L,t=4.224,6.170,8.914,P<0.05犦。④对丙二醛含量变化的影响:随着大黄素剂量的增加,其水平逐渐减少,当大黄素剂量为5,10,20mg/L时与对照组相比显著减低犤(2.336±0.113),(1.945±0.135),(1.381±0.193),(3.110±0.256)nmol/L,t=6.774,9.857,13.187,P<0.05犦。结论:随着培养液中大黄素浓度的增加,超过氧化物歧化酶活性逐渐增强,同时丙二醛水平逐渐降低,从而抑制血管平滑肌细胞的增殖此,此作用均从5mg/L剂量组开始,并具有浓度依赖性。
AIM： To investigate the effect of emodin on the proliferation of ex vivo rabbit smooth muscle cells（SMCs）, and observe the effective concentration and dose of emodin. METHODS： The experiment was conducted in the Department of Pharmacy, Southwest Hospital, Third Military Medical University from June to December 2004. Aortic SMCs were separated from one male rabbit, then were cultured by means of tissue adhesion. After successful culture, the SMCs were divided into control group and emodin group. The SMCs in the control group were not added with any drugs, and those in the emodin group were subdivided into 5 groups： emodin 1.25 mg/L, 2.5 mg/L, 5 mg/L, 10 mg/L and 20 mg/L groups with 6 wells in each subgroup. The well-proliferating SMCs on the third or fourth generation were incorporated with tritiated thymidine（3H-TdR） to show the proliferation degree of cultured rabbit aortic SMCs by liquid scintillation counter （LSC）. The proliferation of SMCs was detected through enzyme-labeled immunometric assay（wave length： 570 nm） and expressed as absorbance（A） value. The activity of superoxide dismutase （SOD） was assayed by inhibition of pyrogallic acid oxidation, and expressed as SOD per milliliter in culture medium（kat）. The content of lipid peroxidation in culture medium was assayed by modified TBA microdetermination and expressed by the level of malondialdehyde（MDA, the terminal product of lipid peroxidation） as nmol/L. The results were analyzed by one-way analysis of variance（homogeneity of variance test）. RESULTS：①Effect of emodin on the proliferation of aortic SMCs： The A value was increased and the inhibition rate was promoted along with the increasing dose of emodin. The A value in the emodin 5, 10, 20 mg/L groups was significantly lower than that in the control group （0.594±0.037, 0.410±0.014, 0.301±0.024 vs 0.730±0.041, P〈0.05）.②Effect of emodin on the 3H-TdR incorporation： The 3H-TdR incorporation became less and the inhibition rate was promoted as the dose of emodin was increased. The value of liquid scintillation counting per minute in the emodin 5,10, 20 mg/L groups was significantly less than that in the control group （33 537±3 713, 29 304±4 336, 21155±1938 vs 73 958±2 276, t=5.862, 7.330, 18.688, P 〈 0.05）.③Effect of emodin on the activity of SOD： The activity of SOD was insignificantly higher in the emodin 1.25 and 2.5 mg/L groups than in the control group（P 〉 0.05）, but it was significantly higher in the emodin 5, 10, 20 mg/L groups than in the control group [（118.19±3.56）, （127.48 ±4.59）, （140.23±4.96） kat/L vs （100.16±9.83） kat/L, t=4.224, 6.170, 8.914, P 〈 0.05]. ④Effect of emodin on the level of MDA： The level of MDA was depressed along with the increasing dose of emodin. It was significantly decreased in the emodin 5, 10, 20 mg/L groups, as compared with the control group[（2.336±0.113）, （1.945±0.135）, （1.381±0.193） nmol/L vs （3.110±0.256） nmol/L, t=6.774, 9.857, 13.187, P 〈0.05]. CONCLUSION： As the concentration of emodin in culture medium is increased, the activity of SOD is enhanced and the level of MDA is reduced, thus inhibiting the proliferation of MSCs. The effect of emodin appeared dose-dependent above the dose of 5 mg/L.
出处
《中国临床康复》
CSCD
北大核心
2005年第26期134-135,共2页
Chinese Journal of Clinical Rehabilitation
作者简介
李志刚，男，1969年生，重庆市荣昌县人，汉族，1994年黑龙江商学院毕业，学士，主管药师，主要从事临床药理学的研究。Laoda90@163．com
