胶原/丝素电纺纳米纤维膜体外培养肝细胞的研究

In vitro Study of Collagen/Silk Nanofiber Scaffold on Hepatocytes

本实验采用静电纺丝技术制备胶原/丝素纳米纤维支架,对支架上培养的人肿瘤肝细胞HepG2进行形态学观察、细胞功能和代谢功能检测。胶原/丝素纳米纤维支架以六氟异丙醇(HFIP)为溶剂通过静电纺丝技术制备,5种支架材料胶原和丝素配比分别为10∶0、7∶3、5∶5、3∶7、0∶10。扫描电镜结果显示制备的纤维平均直径在550～1100 nm之间,随着丝素含量的增加纤维平均直径增加。细胞培养结果显示HepG2细胞在材料表面生长状态良好并与支架材料紧密结合。随培养时间延长,常规培养组细胞在第5 d后逐渐死亡,失去细胞功能,胶原/丝素纳米纤维支架组细胞在4～9 d内能够维持稳定状态,其尿素合成、蛋白分泌与常规培养组有明显差别,其中丝素含量为50%组的细胞状态和细胞功能高于其它组。实验表明胶原/丝素纳米纤维支架材料细胞相容性良好,较之常规培养细胞增殖效果明显,维持功能表达时间延长,有望用于改善人工肝生物反应器中的细胞活性,维持细胞功能表达。

In this work a collagen/silk nanofibrous scaffold was fabricated via electrospinning technique. Human hepatoma cell line HepG2 were seeded on the scaffold. The cell morphology, viability and other functions were investigated. In the scaffold preparation, 1,1,1,3,3,3- hexafluoro- 2-propanol （HFIP） was used as solvent to prepare the collagen/silk nanofiber scaffold in the electrospinning, and the mass ratio of collagen/silk fibroin was 100： 0, 70： 30, 50： 50, 30： 70, 0： 100. The Scanning electron microscopy （SEM） images showed that the average diameter of the fibers ranged from 550 nm to 1100 nm, which was proportionally correlated with SF content. Cell cuhure results showed that HepG2 cells grew well on the surface of the scaffolds and integrated with the scaffold fibers. The collagen/silk nanofiber scaffold supported the cells growing in a steady state whitin 4-9 days. The urea synthesis and protein secretion have significant differences with the ones of control group. Cells growing in the nanofibrous scaffold with SF content of 50% displayed better status and functions than those in the other groups. Compared with cells in the control group, HepG2 cell grows well in the collagen/silk nanofiber scaffold and maintained the functions in longer time.