摘要
目的筛选从矽肺患者双肺大容量灌洗回收液(WLLF)中提取人源性肺表面活性物质(HPS)的条件。方法以提取HPS纯品的获得量为指标,采用正交实验设计分析最佳提取条件组合,凝胶色谱纯化HPS。HPS纯品组分测定采用总磷脂含量测定(孔雀绿直接显色法),磷脂组分分析采用高效液相色谱法(HPLC)。结果提取最佳的条件组合:初离心3000×g,15min;3kD孔径透析袋浓缩;再离心10000×g,15min;正丁醇萃取。纯化HPS色谱条件,层析柱:SephadexLH-20凝胶层析柱(1.5cm×40cm)。流动相:氯仿-甲醇(1∶1/v∶v),流速:1.0ml/min;收集220nm滤片以PC为主峰液体。得到HPS纯品12646.76mg。其中磷脂占95.74%。磷脂酰胆碱PC占80.43%。结论HPS中磷脂酰胆碱含量高且磷脂成分齐全。该提纯方法值得推广。
Objectives To study the methods of isolating human pulmonary surfactant （HPS） in large volume whole lung lavage fluid（WLLF） from patients with silicosis. Methods Nine experiments were designed with the orthogonal experiment design to find the best extraction method with the highest HPS net extraction. HPS was purified with the sepharose chromatography. The component of HPS was measured by analytical chemistry（peacock green direct vision）. The components of the lecithin was analyzed with high performance liquid chromatography（HPLC）. Results The best extraction method was combined with ： 1）first centrifugal rotation at 3 000 × g /15 minutes,concentrated with the dialytic molecular aperture at 3 kDalton; 2）second centrifugal rotation at 10 000 × g /15 minutes,and the extraction distillation with n-butanol. 12 646.76 mg of HPS was obtained after the second purification with sepharose chromatography from 8 men,in which the lecithin was 95.74%,and phosphatidyl choline was 80.43%. Conclusions Since the HPS was high in phosphatidyl choline and with complete components of lecithin,this new technique may be suitable to mass-produce HPS from WLLF.
出处
《临床儿科杂志》
CAS
CSCD
北大核心
2008年第9期803-806,共4页
Journal of Clinical Pediatrics
关键词
人肺表面活性物质
双肺大容量灌洗回收液
提取
色谱纯化
human pulmonary surfactant
huge volume of whole lung lavage liquid
primary isolation
chromatogram
