Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, 17 Changle Western Road, Xi'an, Shaanxi Province 710032, China.
Stem Cell Rev Rep. 2011 Mar;7(1):94-102. doi: 10.1007/s12015-010-9119-4.
Although the stem cells are commonly isolated by FACS or MACS, they are very expensive and these is no specific marker for liver stem/progentior cells (LSPCs). This paper applied a convenient and efficient method to enrich LSPCs. The fetal liver cells (FLCs) were firstly enriched by Percoll discontinuous gradient centrifugation (PDGC) from the rat fetal liver. Then the FLCs in culture were purified to be homogeneous in size by differential trypsinization and differential adherence (DTDA). Flow cytometric analysis revealed more than half of the purified FLCs expressed alternative markers of LSPCs (CD117, c-Met, Sca-1, CD90, CD49f and CD133). In other words, the purified FLCs were heterogeneous. Therefore, they were sequentially layered into six fractions by Percoll continuous gradient centrifugation (PCGC). Both CD133 and CD49f expressed decreasingly from fraction 1 to 6. In fraction 1 and 2, about 85% FLCs expressed CD133, which were revealed to be LSPCs by high expressions of AFP and CK-19, low expressions of G-6-P and ALB. To conclude, the purity of CD133(+) LSPCs enriched by combination of PDGC, DTDA and PCGC is close to that obtained by MACS. This study will greatly contribute to two important biological aspects: liver stem cells isolation and liver cell therapy.
虽然干细胞通常通过 FACS 或 MACS 分离，但它们非常昂贵，而且没有用于肝干细胞/祖细胞（LSPCs）的特定标记物。本文应用了一种方便有效的方法来富集 LSPCs。首先通过不连续的 Percoll 梯度离心（PDGC）从大鼠胎肝中富集胎肝细胞（FLC）。然后通过差异胰蛋白酶消化和差异贴壁（DTDA）将培养物中的 FLC 纯化至大小均匀。流式细胞术分析显示，纯化的 FLC 中有一半以上表达 LSPCs 的替代标记物（CD117、c-Met、Sca-1、CD90、CD49f 和 CD133）。换句话说，纯化的 FLC 是异质的。因此，它们通过连续的 Percoll 梯度离心（PCGC）被顺序分层为六个部分。CD133 和 CD49f 的表达从第 1 部分到第 6 部分逐渐减少。在第 1 部分和第 2 部分，约 85%的 FLC 表达 CD133，通过 AFP 和 CK-19 的高表达、G-6-P 和 ALB 的低表达，证实它们是 LSPCs。总之，通过 PDGC、DTDA 和 PCGC 联合富集的 CD133(+)LSPCs 的纯度接近 MACS 获得的纯度。这项研究将极大地促进两个重要的生物学方面：肝干细胞的分离和肝细胞治疗。
