Lipovitellin Enters Cultured Human Fibroblasts through Clathrin-Mediated Endocytosis
WANG Xiaodie1#, WU Chongguang1,2#, WANG Wenxia1, WANG Yu1, SONG Zhixue1, JI Ning1, WU Peiyuan1, WANG Xiufang1*, LÜ Zhanjun1*
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.
CN
EN