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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。
Objective To screen for the extraction of human pulmonary surfactant (HPS) from the lungs of large volume lavage fluid (WLLF) in patients with silicosis. Methods The yield of pure HPS was taken as an index. The optimum extraction conditions were analyzed by orthogonal experimental design and the HPS was purified by gel chromatography. Determination of pure components of HPS using total phospholipid content determination (malachite green direct color method), phospholipid component analysis using high performance liquid chromatography (HPLC). Results Extraction of the best combination of conditions: initial centrifugation 3000 × g, 15min; 3kD dialysis bag concentration; and then centrifuged 10000 × g, 15min; n-butanol extraction. Purified HPS chromatographic conditions, column: Sephadex LH-20 gel column (1.5cm x 40cm). Mobile phase: chloroform-methanol (1: 1 / v: v), flow rate: 1.0ml / min; collect 220nm filter with PC as the main peak liquid. HPS pure product 12646.76mg. Among them, phospholipid accounted for 95.74%. Phosphatidylcholine PC accounted for 80.43%. Conclusion HPS has high phosphatidylcholine content and complete phospholipid composition. The purification method is worth promoting.