蛋白聚糖NG2中硫酸软骨素糖胺聚糖链调节稳定转染NG2的U251细胞迁移能力

CS-GAG of proteglycan NG2 modulate the migration of NG2 transfected U251 cells

应用PCR反应使编码蛋白聚糖NG2的核苷酸在3064 3065位置的AG突变成为GC,构建突变型NG2/S999A.用表达野生型NG2和突变型NG2/S999A及空白表达载体分别稳定转染人成胶质细胞瘤U251.应用WesternBlot方法鉴定转染后的U251细胞表达野生型及突变型NG2的状态,证实突变型NG2A999稳定转染的U251细胞株表达的NG2缺失硫酸软骨素葡糖胺聚糖链(CS GAG).比较了3种U251细胞株的迁移,表明蛋白聚糖NG2中CS GAG链影响细胞的迁移能力.

An AG to GC switch at nucleotide positions 3064-3065 encoding the wild type NG2 proteoglycan was made by sequential PCR reactions using overlapping primers, resulting in the change of serine to alanine at amino acid position 999 in the NG2 core protein. Expression constructs pcDNA3.1+/NG2 and pcDNA3.1+/NG2/S999A were made. Transfection of wild-type and mutant forms of NG2 and expression vector (pcDNA3.1+)in U251 cell were performed with LipofectAmine. The NG2 transfectants were isolated, identified by Western Blot. The NG2/S999A transfected U251 cells lack the chondroitin sulfate glycosaminoglycan ((CS-GAG) chain.) Cell migration assays were performed with these three U251 transfectants. The result suggests that NG2 plays a role in promoting the cell migration. Furthermore, it indicates that the CS-GAG of NG2 could modulate this process.