人胎儿肝干细胞在聚酸酐-氨基葡聚糖三维载体内的生长活性

Growth activity of fetal liver stem cells in polyanhydride-three-dimensional vector-glucan

目的:观察聚酸酐-氨基葡聚糖三维载体材料对胎儿肝干细胞黏附及增殖的影响。方法:采用改进的两步胶原酶灌注消化法加percoll液不连续密度梯度离心的方法,分离胎儿肝干细胞。选取胎儿肝干细胞的第3代细胞,种植于聚酸酐-氨基葡聚糖三维载体材料上。倒置显微镜下观察细胞的黏附和生长状况;计算细胞贴壁率、细胞增殖活力、计数细胞数。取细胞-载体进行组织学切片,苏木精-伊红染色光镜下观察细胞在载体中生长情况。在细胞培养第7天进行免疫荧光化学染色和流式细胞仪检测标志物表达。结果:聚酸酐-氨基葡聚糖三维载体能促进肝干细胞在材料内黏附并保持其在机体内的形态。载体材料内的肝干细胞功能活跃,在材料表面和三维空间内部培养的肝干细胞均能持续增殖。经过连续10d共同培养,聚酸酐-氨基葡聚糖三维载体对干细胞无毒性,人胎儿肝干细胞可以很好的贴附于聚酸酐-氨基葡聚糖三维载体支架上,细胞增殖活力良好,标志物持续表达,培养7d得到的细胞数量增多19.7%。结论:聚酸酐-氨基葡聚糖三维载体能促进肝干细胞的增殖,有可能作为肝干细胞的载体应用于肝脏组织工程。

OBJECTIVE： To observe effects of polyanhydride-three-dimensional vector-glucan material on the fetal liver stem cell adhesion and proliferation. METHODS： The two-step collagenase perfusion digestion and bliquid percoll discontinuous density gradient centrifugation was used to isolate fetal liver stem cells. Fetal liver stem cells at the third passage were incubated on the polyanhydride-three-dimensional vector-glucan material. Inverted microscope was utilized to observe cell adhesion and growth status. Cell adherent rate, proliferation activity were calculated, and cell number was counted. Cell-vector was obtained for tissue section. Using hematoxylin-eosin staining, cell growth in the vector was observed under the optical microscope. At 7 days, immunofluorescence staining and flow cytometry were used to determine marker expression. RESULTS： Polyanhydride-three-dimensional vector-glucan promoted grow and adhesion of liver stem cells. There was the active function of the liver stem cells within carrier materials. In the three-dimensional surface and the internal culture, liver stem cell proliferation was sustained. After 10 days, the polyanhydride common culture-three-dimensional vector-glucan on stem cells was non-toxic, and human fetal liver stem cells could be attached to the polyanhydride-three-dimensional vector-glucan stent. The cell proliferation was better and dynamic sustained expression of markers. 7-days training received 19.7 percent increase in the number of cells. CONCLUSION： Polyanhydride-three-dimensional vector-glucan promotes the proliferation of liver stem cells, and liver stem cells can be used as the vector in liver tissue engineering.