F213I突变型高雪氏病分子伴侣治疗方法的研究

Preclinical research of a new therapy for Gaucher's disease with F213I mut ation

目的 研究高雪氏病新的分子治疗方法。方法 用β-葡萄糖脑苷脂酶 (β-glucocerebrosidase,β-Glc) (EC3 .2 .1.45)的底物类似物 (glucocerebroside analogue,GCA)作为分子伴侣 ,处理体外培养的不同突变型的高雪氏病患者皮肤成纤维细胞 ,采用荧光酶学手段测定β-Glc活性 ;Western印迹杂交技术分析该酶蛋白表达的量 ;细胞双染确定 β-Glc在细胞内的定位 ;薄层层析法分析葡萄糖脑苷脂的降解情况。结果该酶的底物类似物 GCA可以提高体外培养的 F2 13 I突变型患者成纤维细胞内 β-Glc活性 ;增加该酶蛋白的表达量 ;促进该酶蛋白向溶酶体内转运 ;加速底物葡萄糖脑苷脂 (glucocerebroside,Glc Cer)的降解。结论 低分子量的 GCA作为一种分子伴侣可能为 F2 13

Objective: To design and make trial of a new therapy for Gaucher disease. Methods: A substrate analogue of β-Glc (glucocerebroside analogue, GCA) was used as a molecular chaperon. Normal and mutant skin fibroblasts were cultured with or without GCA. The activity of β-Glc was assayed by fluorescent enzymologic techniques. The amount of β-Glc was determined using Western blot. The β-Glc was localized by double cell stain experiment. The degradation of glucocerebroside was assessed by thin layer chromatography (TLC) experiment using 14C-Serine. Results: It was found that GCA could enhance the activity and amount of β-Glc with F213I mutation. It also promoted the β-Glc with F213I mutation to the lysosome and accelerated the degradation of glucocerebroside. Conclusion: The low molecular compound analogous to β-Glc substrate (GCA) may be a new therapeutic strategy for Gaucher's disease with F213I mutation.