摘要
目的探讨K-ras突变多肽致敏树突状细胞(DC)对细胞趋化因子CCL19、CCL22和细胞骨架蛋白fascin-1表达的影响。方法联合应用重组人粒-巨噬细胞集落刺激因子和白细胞介素(IL)-4诱导培养外周血DC,收集培养7d后的DC并分为未负载组(加入RPMI1640培养液50μg/ml)和K-ras负载组(加入K-ras突变多肽50μg/ml).流式细胞仪测定负载前、后DC表面标志CD1a、CD80及CD86;扫描电镜和透射电镜观察负载K-ras突变多肽后的DC形态结构;ELISA法检测2组DC培养上清液中IL-12、CCL19和CCL22的表达;Westernblot法测定2组DC骨架蛋白fascin-1的表达。结果①K-ras突变多肽负载DC后,CD1a、CD80及CD86表面分子表达率明显高于负载前(P<0.01).②扫描电镜下见负载后的DC呈花瓣状、树枝样突起;透射电镜下见负载后的DC形态非常不规则,树枝状或毛刺状的突起明显增多。③K-ras负载组负载后不同时相(6、12、24及48h)的IL-12、CCL19及CCL22的表达水平明显高于未负载组(P<0.01).④K-ras负载组fascin-1蛋白的表达水平也高于未负载组(P<0.01).结论K-ras突变多肽能够促进DC成熟,并使细胞趋化因子和骨架蛋白表达水平增加,能够增强DC游走迁移。
Objective To explore the effect of dendritic cells （DCs） allergized by K-ras mutant peptide on expressions of chemokines CCL19,CCL22,and cytoskeletal protein fascin-1. Methods DCs were derived from peripheral blood in the presence of granuloceyte/macrophage-colony stimulating factor,interleukin （IL）-4 in vitro. The DCs were collected on day 7 after culture,and were divided into non-K-ras mutant peptide group （addition of RPMI 1604 culture solution 50 μg/ml） and K-ras mutant peptide group （addition of K-ras mutant peptide 50 μg/ml）. Phenotype was identified by flow cytometry. The morphological structure was observed by scanning and transmission electron microscopies,respectively. The expressions of IL-12,CCL19,and CCL22 were tested continuously by enzyme-linked immunosorbent assay （ELISA）. The expression of cytoskeletal protein fascin-1 was determined by Western blot. Results ①The expressions of CD1a,CD80,and CD86 after loading K-ras mutant peptide were higher than that before loading K-ras mutant peptide （P0.01）. ②The DCs with petal-like and branch-like profections after loading were observed under scanning electron microscopy; The DCs with irregular shapes,branch-like or burr-like were showed under transmission electron microscopy. ③The expressions of IL-12,CCL19,and CCL22 in the K-ras mutant peptide group were higher than those in the non-K-ras mutant peptide group at different times （6,12,24,and 48 h） after loading K-ras mutant peptide （P0.01）. ④The expression of fascin-1 in the K-ras mutant peptide group was also higher than that in the non-K-ras mutant peptide group （P0.01）. Conclusion K-ras mutant peptide can promote DC to mature and improve the expression of chemokines and cytoskeletal protein which will strengthen DC migration.
出处
《中国普外基础与临床杂志》
CAS
2010年第9期917-921,共5页
Chinese Journal of Bases and Clinics In General Surgery
基金
国家自然科学基金(项目编号:30670624
30870719)~~
