鞘氨醇激酶活性测定方法的建立及其初步应用

ESTABLISHMENT OF A METHOD FOR DETERMINING THE SPHINGOSINE KINASE ACTIVITY AND ITS INITIAL APPLICATION

目的:建立生物样品中鞘氨醇激酶(SPK)活性和1-磷酸鞘氨醇(S1P)含量的测定方法。方法:用Flag标记的SPK基因表达载体转染ECV304细胞,用Western blot方法检测转染后SPK基因的表达,用酶促反应、同位素掺入和薄层层析的方法检测SPK的活性。提取细胞或组织的S1P,碱性磷酸酶消化去除磷酸根,然后利用SPK的催化活性和同位素标记的方法对S1P进行定量。结果:转染基因后细胞的SPK表达明显升高,活性显著增强,细胞内S1P的含量也明显增多。肝细胞生长因子(HGF)刺激能增强ECV304细胞SPK的活性和细胞内S1P水平。结论:建立了SPK活性和S1P含量的测定方法。

Aim： To establish the methods for determining the activity of sphingosine kianse（SPK） and the content of sphingosine 1-phosphate（SIP） in biological samples. Methods： The ECV304 cells were transfected with pcDNA3 vector encoding Flag-labeled SPK gene. The expression of SPK was measured by Western blot assay and the activity of SPK was determined by enzymatic reaction, isotope incorporation and thin layer chromatography methods. The SIP in biological samples was extracted, digested by alkaline phosphatase and then catalyzed by SPK, The S1P contents were determined according to the amounts of products. Results： SPK gene transfection could enhance the expression and activity of SPK in cells markedly, and the cellular SIP was also increased obviously. HGF stimulation could increase the activity of SPK and cellular SIP in ECV304 cells. Conclusion： Methods for determining the activity of SPK and the content of SPK in biological samples were established.