摘要
目的:观察人冠状动脉平滑肌细胞表达基质金属蛋白酶3与炎症因子白细胞介素1β的关系。方法:实验于2005-05/2006-08在解放军第三○五医院老年病中心实验室完成。人冠状动脉平滑肌实验用5～7代的细胞,按2.5×108L-1密度接种于25cm2的培养瓶中,24h后换液,生长接近80%融合时进行如下处理:①20μg/L质量浓度白细胞介素1β培养0,2,4,8,24,36h后收集上清夜和细胞。②不同质量浓度(0,5,20,40μg/L)白细胞介素1β处理6h后收集上清液和细胞。采用细胞总RNATRIzol试剂提取RNA,在紫外分光光度计确定总RNA的浓度和纯度,并在12g/L的琼脂糖凝胶中电泳,凝胶成像分析RNA的质量。应用实时荧光定量聚合酶链反应的方法检测细胞内基质金属蛋白酶3基因的表达量。结果:①细胞总RNA质量及纯度:每份样品取4μL稀释至400μL测定其A260nm/A280nm吸光度比值均在1.8～2.0之间,说明所提RNA纯度较高;电泳图片上可见RNA清晰的18S和28S的条带,无弥散,说明RNA质量较好。②目的基因的表达量:同剂量白细胞介素1β作用下,基质金属蛋白酶3基因的表达量在2h时开始发生上调,8h达高峰,而后开始下降;在不同剂量白细胞介素1β作用下,基质金属蛋白酶3基因的表达量在实验剂量范围内随着白细胞介素1β的剂量加大呈上升趋势(r=0.904,P=0.000),20μg/L和40μg/L白细胞介素1β作用下,基质金属蛋白酶3基因的表达量差异无显著性(P=0.154),其余各剂量间基质金属蛋白酶3基因的表达量差异均有显著性(P<0.05)。结论:炎症因子白细胞介素1β能促进冠状动脉平滑肌细胞中斑块稳定相关标记物基质金属蛋白酶3的表达,可能是炎症在急性冠状动脉综合征发生发展中的作用机制之一。
AIM： To observe the association between interleukin-1 β （IL-1β） and matrix metalloproteinase-3 （MMP-3） gene expression in human coronary arterial smooth muscle cells.
METHODS： The experiment was performed in the Central Laboratory of Senile Disease, 305 Hospital of Chinese PLA from May 2005 to August 2006. Human coronary arterial smooth muscle cells of the fifth to seventh passage were seeded in 25 cm^2 tissue culture flasks at 2.5×10^8 cells/L, and the medium was changed 24 hours later. When the cells reached 80% confluence, ①the supernatant and cells were collected after the cells were culture in 20 μg/L IL-113 for 0, 2, 4, 8, 24 and 36 hours; ②the cells were collected after cultured in IL-113 of 0, 5, 20 and 40 μg/L for 6 hours. Total RNA was extracted from the cells with TRIzol Reagent, and the concentration and purity of RNA were detected with violet spectrophotometer. The quality of RNA was analyzed with 12 g/L gelatin imaging system. Real-time quantitative PCR was performed on RNA to detect MMP-3 gene expression in cells.
RESULTS： ①Quality and purity of total RNA： The absorbance ratio of A260nm/A280nm of 4 μL sample diluted to 400 μL was between 1.8 and 2.0, indicating the purity of RNA was high; the bands of 18 S and 28 S were clearly observed at the electropherogram without diffusion, indicating the quality of RAN was better. ②Expressions of target gene： In the same concentration of IL-1β, the gene expression began to up-regulate at 2 hour, which reached the peak at 8 hour, then began to descent; in different concentrations of IL-1β, gene expression was up-regulated with the dose increase of IL-1β （r =0.904, P =0,000）. There were no significant difference in the gene expression between 20 μg/L and 40 μg/L IL-1β （P =0.154）, and there were significant differences among other groups （P 〈 0.05）.
CONCLUSION： Inflammatory factor IL-1β could promote the gene expression of MMP-3 as the stable marker in human coronary arterial smooth muscle cells, and
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第14期2617-2620,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
全军医药卫生"十五"计划课题(04LX048)~~
