摘要
目的:观察低氧培养对大鼠肺动脉平滑肌细胞增殖的影响,以及Na+/H+交换抑制剂HMA对此增殖效应的抑制作用。方法:实验于2004-12/2005-06在第四军医大学病理生理学教研室完成。健康SD大鼠2只,分离培养肺动脉平滑肌细胞,选择3～6代生长良好的细胞在常氧(O2的体积分数为0.21)或低氧(O2的体积分数为0.02)条件下培养,并分别给予0.3,1,3和10μmol/L等不同浓度的HMA(n=8),采用噻唑蓝比色实验和测定细胞总蛋白含量的方法观察细胞增殖情况,同时光镜观察细胞形态并测定培养液上清乳酸脱氢酶活力以反映药物的非特异性细胞毒作用。结果:实验所用细胞样本均进入结果分析。①体积分数为0.02氧浓度较体积分数为0.05氧浓度下培养的大鼠肺动脉平滑肌细胞生长曲线抬高,低氧刺激24h增殖达到高峰。②体积分数为0.05血清培养使此增殖效应更为显著[噻唑蓝光吸收值无血清常氧组(0.238±0.011),无血清低氧组(0.280±0.009),体积分数为0.05血清常氧组(0.313±0.013),体积分数为0.05血清低氧组(0.389±0.011)]。③HMA可以抑制增殖,显著降低噻唑蓝吸光度值[对照组(0.391±0.011),0.3μmol/L组(0.377±0.010),1μmol/L组(0.328±0.012),3μmol/L组(0.289±0.006),10μmol/L组(0.246±0.007)]。④细胞总蛋白含量也显著降低[对照组、0.3μmol/L组、1μmol/L组、3μmol/L组、10μmol/L组分别为(193.30±8.51),(177.63±8.71),(166.84±9.48),(155.72±10.46)和(135.13±10.30)μmol/L]。⑤各浓度处理组细胞形态无改变,培养液上清乳酸脱氢酶活力也无明显变化[对照组、0.3μmol/L组、1μmol/L组、3μmol/L组、10μmol/L组分别为(212.23±8.44),(208.80±6.37),(209.79±7.12),(208.77±7.33),(215.42±7.81)U/L],细胞无明显损伤。结论:0.3～10μmol/L浓度的Na+/H+交换抑制剂HMA可以有效抑制低氧刺激的大鼠肺动脉平滑肌增殖,此作用不是非特异的细胞毒作用所致。
AIM： To observe the effect of hypoxia culture on proliferation of lung arterial smooth muscle cells （ASMC） of rats and the inhibitory action of Na^±/H^± exchange inhibitor HMA on the proliferation.
METHODS： The experiment was performed in the Pathophysiologic Department of Fourth Military Medical University from December 2004 to June 2005. Two healthy SD rats were seperately cultured of lung ASMC. Well-grown cells of 3-6 passages were selected and cultured in normoxia （21%O2） or hypoxia （2%O2） by added with HMA of different concentrations （n=8） respectively at the dose of 1. 3, 10μmol/L. Proliferation of cells were observed by MTT colorimetric assay and determination of total protein level in cells. Meanwhile, cellular morphous were observed with light microscope, activity of lactic dehydrogenase （LDH） in supematant were detected so as to indicate the nonspecific cytotoxicity. RESULTS： All cellular samples used in the experiment were involved in the analysis of results. ①Hypoxia raised Growth curve of pulmonary ASMC of rats cultured with the oxygen concentration of 0.02 was enhanced than that of 0.05 and proliferation reached maximum after stimulated with 24- hour-hypoxia. ②Serum culture with the concentration of 0.05 made the proliferation be more significant [MTT light absorption without serum normoxia group （0.238 ±0.011）, without serum hypoxia group （0.280 ±0.009） ,serum normoxia of 0.05 concentration group（0.313±0.013）,serum hypoxia of 0.05 concentration group （0.389±0.011 ）]. ③HMA can inhibit the proliferation and significantly reduce the MTT light absorption [control group （0.391±0.011 ） .0.3 μ mol/L group （0.377±0.010）, 1 μ mol/L group （0.328±0.012） ,3 μ mol/L group （0.289±0.006）, 10 μ mol/L group（0.246 ±0.007）] ④Total protein level of cells was also reduced obviously [that in the control group 0.3 μ mol/L group,1 μ mol/L group, 3 μ mol/L group and 10 μ mol/L group were respectively： （193.30 ±8.51）, （177.6
出处
《中国临床康复》
CSCD
北大核心
2006年第13期83-86,共4页
Chinese Journal of Clinical Rehabilitation
