Twist基因干扰抑制猴视网膜微血管内皮细胞的迁移

Twist gene interference suppress migration of Rhesus retinal vascular endothelial cells

目的 观察Twist基因干扰对体外培养的猴视网膜微血管内皮细胞（RF/6A）迁移及磷酸化Akt（pAkt）蛋白表达的影响.方法 体外培养的RF/6A细胞系,并构建Twist干扰质粒和对照质粒.分为Twist干扰质粒组、阴性对照组、磷酸盐缓冲液（PBS）组,各组应用脂质体基因转染方法,转入相应的质粒表达载体,采用Transwell小室法检测发生迁移的内皮细胞并计数;铺Matrigel胶,行内皮细胞体外成管实验,观察Twist基因干扰对内皮细胞成管的抑制作用;蛋白质免疫印迹检测Twist基因干扰对RF/6A细胞pAkt蛋白表达的影响.结果 Transwell小室计数结果表明,转染Twist干扰质粒后,迁移的细胞数显著低于阴性对照组和PBS组（F=23.786,P=0.000）.体外成管实验结果表明,Twist干扰质粒组内皮细胞成管数显著低于阴性对照组和PBS组（F=7.159,P=0.014）.Ttwist干扰质粒组的pAkt蛋白表达明显减少.结论 Twist基因干扰可显著抑制视网膜微血管内皮细胞的迁移,可能机制是通过抑制pAkt蛋白的表达发挥作用.

Objective To observe the effect of Twist gene interference on the migration and pAkt protein expression of Rhesus retinal vascular endothelial cell line. Methods The Rhesus retinal vascular endothelial cells （RF/6A） were divided into Twist interference plasmid group, negative control group, and phosphate buffered solution （PBS） group; plasmid vectors were transfected via liposome gene transfection method. Migrated endothelial cells was detected and counted by Transwell chamber assay. Matrigel was used in endothelial-cell tube formation; the inhibitory effect of Twist gene interference on endothelial-cell tube formation was observed. The effect of Twist gene interference on the expression of pAkt protein in RF/6A cells was measured by Western blot. Results The number of migrated endothelial cells in Twist interference plasmid group was lower than that in the negative control and PBS group （F = 23. 786, P =0. 000）. The number of endothelial-cell tubes in Twist interference plasmid group was apparently less than that in the negative control and PBS gorup （F=7. 159,P=0. 014）. The expression of pAkt protein in Twist interference plasmid group decreased markedly. Conclusion Twist gene interference can suppress the migration of retinal endothelial cells via inhibiting the expression of pAkt protein.