丝素蛋白膜表面亲水基团对细胞黏附作用的影响

Influence of hydrophilic groups on silk fibroin membrane surface on cell adhesion

目的通过交联剂分别对丝素蛋白膜表面主要基团氨基、羟基及羧基的屏蔽,测定其表面亲水性基团的作用效果。方法以只铺加丝素膜的聚苯乙烯板为对照,采用乙二醇二缩水甘油醚(EGDE)对丝素蛋白膜表面介导细胞黏附的主要基团氨基、羟基和羧基进行屏蔽,确定屏蔽反应的最适反应时间及最适p H值,并采用MTT方法间接检测屏蔽效果。EGDE对羟基、氨基、羧基的屏蔽作用2组间SUVmax比较采用两样本t检验进行统计学分析。结果 EGDE均可以起到屏蔽羟基(0.514±0.020,P<0.01)、氨基(0.583±0.036,P<0.01)、羧基(0.523±0.023,P<0.01)的目的,且对丝素蛋白膜表面亲水性基团屏蔽的最适时间为10 h,屏蔽羧基所选用的缓冲液最适p H为4。通过MTT法检测丝素蛋白膜表面三种亲水性基团(羟基、羧基、氨基)介导细胞黏附作用总和的大小,羟基、羧基、氨基的屏蔽率分别为:16.67﹪、14.05﹪、10.44﹪,而在丝素蛋白膜表面:含羟基氨基酸、酸性氨基酸、碱性氨基酸的数量比例为17:4:2。结论对于单个羟基、羧基、氨基亲水性基团来讲,介导细胞黏附作用的大小为:氨基>羧基>羟基;但三种亲水基团介导细胞黏附作用的总和是:羟基>羧基>氨基。

Objective To measure the effect of shielding of amino, hydroxy or carboxyl groups on the silk fibroin membrane surface by cross-linking on cell adhesion. Methods The amino, hydroxyl or carboxyl groups on silk fibroin membrane surface, which mediate cell adhesion, were shielded by ethylene glycol diglycidyl ether(EGDE)and polystyrene plate covered with silk fibroin membrane served as control. The reaction time and p H were optimized. Effectiveness of shielding was measured by MTT. The shielding effect of EGDE to hydroxy, amino and carboxyl was compared with two groups of max SUV by using two sample test. Results The optimum time of EGDE in shielding hydrophilic groups on silk fibroin membrane surface was 10 h, and the optimum p H of this reagent in shielding carboxyl was 4. At p H 4 and 10 h reaction time, the shielded effect of hydroxyl, amino, or carboxyl by EGDE was 0.514 ± 0.020, 0.583 ± 0.036, 0.523 ± 0.023(P < 0.01)respectively compared with blank control. The total effect of three groups(amino, hydroxyl or carboxyl)-mediated cell adhesion which detected with MTT was 16.67 ﹪, 14.05 ﹪, or 10.44 ﹪ respectively.Conclusion The effect of group-mediated cell adhesion was amino > carboxyl > hydroxyl for a single hydrophilic group. However, the total effect of that was hydroxyl > carboxyl > amino.