大鼠骨髓Thy-1.1^+干细胞诱导分化为肝干细胞样细胞的体外实验研究

Experimental studies on differentiation of rat bone marrow Thy-1.1^+ stem cells into hepatic stem cell-like cells in vitro

目的:分离培养大鼠骨髓Thy-1·1+干细胞,探讨其肝分化条件及潜能。方法:分离大鼠骨髓细胞,Per-coll密度梯度离心获取骨髓单个核细胞后免疫磁珠分选Thy-1·1+干细胞群,流式细胞术测定分选后细胞的纯度与表型。不同条件下诱导分化,用形态学观察、逆转录聚合酶链反应和免疫细胞化学法判定其所处的肝系分化阶段。结果:免疫磁珠分选Thy-1·1+细胞纯度达94·2%。Thy-1·1+细胞不表达CD34和c-kit;高表达β2微球蛋白和CD45;部分表达Flt-3。Thy-1·1+细胞在HGF/FGF-1/FGF-2诱导下呈多角形上皮状克隆样生长,诱导前后持续表达细胞角质素18,诱导过程中白蛋白的表达逐渐减少,甲胎球蛋白的表达逐渐增强。结论:免疫磁珠可有效分选Thy-1·1+细胞。Thy-1·1+细胞在HGF/FGF-1/FGF-2诱导下呈肝干细胞样细胞表现。

Objective： To isolate and cultivate Thy-1.1 ^＋ cells from rat bone marrow, and investigate the condition and capability of hepatic differentiation. Methods： Rat bone marrow cells were collected from rat tibias and femurs, and mononeuclear cells were isolated by Percoll density gradient centrifugation. After incubation with Thy-1.1^ ＋ monoclonal antibody, the Thy-1.1 ^＋ stem cells were purified by indirect magnetic activated cells sorting. The purity and phenotype of the cells were evaluated by flowcytometry. Thy-1.1 ^＋ cells were induced under different conditions. Morphology, RT-PCR and immunocytochemistry were used to identify the hepatic differentiation grade. Results： The purity of Thy-1.1^ ＋ cells after MACS was 94. 2%. Thy-1.1 ^＋ cells were CD34 and c-kit negative; β2M and CD45 positive; and partially expressed Fit-3. Under HGF/FGF-1/FGF-2 induction, Thy1.1 ^＋ cells were polygonal epithelium in shape and clonal. Expression of CK18 was continuous during the induction process. The expression of ablumin signal decreased and AFP signal increased. Conclusion： High purified Thy-1.1 ^＋ cells could be obtained from rat bone marrow using MACS. Thy-1.1^＋ cells were inclined to differentiat into hepatic stem cell-like cells under the induction of HGF/FGF-1/FGF-2.