模拟微重力下三维培养对人肝干细胞增殖和分化的影响

发布时间：2018-02-11 09:21

本文关键词： 模拟微重力肝干细胞三维培养旋转式生物反应器微载体　出处：《南京医科大学学报(自然科学版)》2011年08期 　论文类型：期刊论文

【摘要】：目的:探讨微重力环境对肝干细胞增殖及分化的影响。方法:从人肝组织中分离、培养肝干细胞,进行肝干细胞鉴定后,分别进行平面培养、微载体培养、模拟微重力下三维培养。利用倒置相差显微镜、扫描电镜观察不同培养条件下细胞形态学变化;检测上清液中葡萄糖消耗量和乳酸脱氢酶含量以比较细胞增殖和活性情况;荧光定量PCR法检测细胞ALB、EpCAM、CK19、AFP、HNF6、CYP3A4、CYP3A7 mRNA表达变化。结果:模拟微重力环境下,肝干细胞以微载体球面为基底呈三维立体结构生长,形成大小不等细胞团;扫描电镜下见细胞团表面有丰富的细胞外基质及微绒毛;从9 d开始葡萄糖消耗量明显增加,至17 d达高峰,而LDH含量始终保持低水平;培养至21 d细胞仍高表达EpCAM、ALB、CK19、HNF6、CYP3A7,不表达AFP、CYP3A4,符合肝干细胞表型特征,与微载体培养结果相比有统计学差异(P0.05)。结论:模拟微重力环境能促进肝干细胞呈三维立体结构生长,有利于细胞快速增殖并维持细胞活性和表型。
[Abstract]:Objective: to investigate the effect of microgravity on the proliferation and differentiation of hepatic stem cells. Using inverted phase contrast microscope, scanning electron microscope was used to observe the morphological changes of cells in different culture conditions, glucose consumption and lactate dehydrogenase content in supernatant were measured to compare cell proliferation and activity. Fluorescence quantitative PCR assay was used to detect the expression of CYP3A7 mRNA in ALBN EpCAMN CK19. Results: in simulated microgravity environment, liver stem cells grew on the substrates of microcarriers and formed cell clusters of different sizes. Under scanning electron microscope, there were abundant extracellular matrix and microvilli on the surface of the cell mass, and the glucose consumption increased significantly from 9 days to the peak at 17 days, while the content of LDH remained low. The HNF6CYP3A7 was still highly expressed in the cells cultured for 21 days, and no AFPhCYP3A4 was expressed, which was consistent with the phenotypic characteristics of hepatic stem cells. Compared with the results of microcarrier culture, there was a significant difference (P 0.05). Conclusion: the simulated microgravity environment can promote the growth of liver stem cells in three-dimensional structure. It is beneficial to the rapid proliferation of cells and the maintenance of cell activity and phenotype.
【作者单位】：南京医科大学第一附属医院感染病科;
【基金】：江苏省卫生厅面上项目(H200909) 江苏省卫生厅“兴卫工程”开放项目(XK15200901,ZX05200905)
【分类号】：R329.27

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