【摘要】 为了探究从斑马鱼(Danio rerio)胚胎裂解液中纯化的卵黄脂磷蛋白(lipovitellin, Lv)进入培养的人成纤维细胞的分子途径,该研究用Sephadex G-200色谱柱结合QAE-Sephadex A50阴离子交换色谱柱从斑马鱼0 h胚胎裂解液中分离纯化出Lv。FITC标记的Lv(FITC-Lv)预先分别与Hepes、牛组蛋白、鱼精蛋白、卵磷脂、0 h胚胎胰蛋白酶水解物或者斑马鱼胚胎裂解液孵育,再加入至opti-MEM培养基中培养人成纤维细胞,培养一定时间后,用荧光倒置显微镜观察,发现Hepes和组蛋白可以促进Lv进入细胞。制霉菌素是陷窝介导的内吞作用(caveolae-mediated endocytosis)的抑制剂,氯丙嗪和蔗糖是网格蛋白介导的内吞作用(clathrin-mediated endocytosis)的抑制剂,阿米洛利是巨胞饮作用(macropinocytosis)的抑制剂。为了证实Lv进入细胞的分子途径,在FITC标记Lv与人成纤维细胞的培养体系中,分别加入上述抑制剂。结果发现氯丙嗪和蔗糖可以抑制Lv进入细胞。该研究获得如下结论:纯化的斑马鱼Lv单独不能进入细胞,在组蛋白的协同作用下, Lv通过网格蛋白介导的内吞作用进入细胞。该研究将为进一步研究Lv进入细胞发挥生物学功能奠定基础。更多还原

【Abstract】 To demonstrate the molecular pathway by which Lv(lipovitellin) purified from zebrafish(Danio rerio) embryonic lysate enters cultured human fibroblasts, in this study, purified Lv was obtained from 0 h zebrafish embryo lysate using Sephadex G-200 column combined with QAE-Sephadex A50 anion exchange column. FITClabeled Lv was pre-incubated with Hepes, calf histone, protamine, lecithin, embryonic trypsin hydrolysate or zebrafish embryo lysate. Next, the incubation mixture was added into human fibroblasts cultured using opti-MEM medium. After a certain period of culture, the inverted microscope was used to observe fluorescence intensity. The results showed that Hepes and histone promoted the entry of Lv into cells. The nystatin is the inhibitors of caveolaemediated endocytosis; chlorpromazine and sucrose are the inhibitors of clathrin-mediated endocytosis; amiloride is the inhibitor of macropinocytosis. To confirm the molecular pathway of Lv entry into cells, the above inhibitors were added to the culture systems of Lv and human fibroblasts, respectively. Chlorpromazine and sucrose were found to inhibit the entry of Lv into cells. It has been concluded that the purified zebrafish Lv alone could not enter cells. Under the synergic action of histone, Lv entered cells through clathrin-mediated endocytosis. This study will lay a foundation for further study of Lv entering cells to play biological functions.更多还原