摘要
目的 :从大肠杆菌中制备LPS ,经凝胶色谱纯化 ,用纯化的LPS诱导大鼠腹腔巨噬细胞产生TNF ,建立内毒素感染的细胞模型。方法 :收集培养的大肠杆菌 ,洗去菌毛 ,冻干研磨成菌粉 ,经沸水浴提取 ,蛋白酶K消化 ,制成初提物。再用SephadexG -75色谱柱洗脱 ,收集纯化的组分。将纯化的LPS ,稀释成不同的浓度加入长有巨噬细胞的培养板中 ,刺激一定的时间后 ,收集上清液。用ELISA方法检测上清液中的TNF -α含量。结果 :纯化的LPS用紫外/可见光分光光度计扫描 ,呈单一吸收峰 ,并能被鲎试剂特异检出。用2.5μg/ml和5μg/mlLPS诱导巨噬细胞 ,24h后 ,培养上清液中的TNF -α含量分别为(8.13±1.25)pg/ml和(7.50±4.51)pg/ml(x±sn=4)。结论 :本法制备的LPS纯度较高 ,对鲎试剂高度敏感 ,有较好的内毒素活性 ,能满足建立内毒素感染的细胞模型的需要。该法简便、快速。
Objective:To prepare the LPS and to clarify the role of LPS in setting up the cellular model of endotoxin infection. Methods:A method is described for LPS preparation if E coli by removing fimbrial, boiling water extraction and subsequent digestion with proteinaseK.LPS is farther punted by sephadexG-75.Different concentrations of purified LPS were added to macrophage culture plate and supernatant was obtained after a period of stimulation.Values of LPS were determined by ELISA.Results:Single peak of LPS is obtained using Violet spectrophotometer and LPS can be detected by limulus reagents. TNF-α concentrations in macrophage supernatant are 8.13±1.25 and 7.50±4.51 pg/ml(x±s,n=4),respectively after 24 hour induction of LPS with concentration of 2.5 μg/ml and 5 μg/ml, respectively.Conclusions: LPS prepared by our method has high purity, high endotoxin activity and can be detected by limulus reagents. LPS can be used for establishment of cellular model of endotoxin infection.Our method has convinient, rapid advantage and is suitable for small scale preparation of LPS in laboratory.
出处
《天津医科大学学报》
2002年第4期445-447,共3页
Journal of Tianjin Medical University
