一种新的神经干细胞抗贴壁培养方法

目的建立一种适合神经干细胞长期、大量体外培养的抗贴壁培养方法.方法制作琼脂糖抗贴壁培养瓶.实验组在琼脂糖抗贴壁培养瓶中培养神经干细胞,对照组在塑料培养瓶中培养.通过BrdU掺入法检测S期标记指数,噻唑蓝（MT）法检测两组细胞的增殖速度.传代培养3个月后,检测两组细胞的分化率,以及实验组细胞的分化潜能.结果实验组细胞在抗贴壁培养瓶中生长旺盛,S期标记指数和细胞增殖速度与对照组无显著性差异.培养3个月后,实验组神经干细胞的分化率为0.64%,对照组的分化率为32.05%,差异有高度显著性（P＜0.01）.实验组神经干细胞仍保持多分化潜能.结论琼脂糖抗贴壁培养法有利于神经于细胞持续增殖并保持未分化状态,适合神经干细胞在体外长期、大量扩增.

A NOVEL ANTI-ATTACHMENT CULTURE SYSTEM FOR NEURAL STEM CELLS

Objective Neural stem cells are inclined to differentiate when attaching to the floor of the culture flask.Current culture method is unsuitable for long-term neural stem cell culture,since it cannot prevent cell clusters from attachment.This experiment was designed to develop a novel anti-attachment culture system for long-term,large-quantity neural stem cell cultivation. Methods Anti-attachment culture flasks were made of 1.5% agarose gel,whose gel strength was greater than(3?200)?g/cm~2.For the experiment group,neural stem cells were cultured in anti-attachment culture flasks.For the control group,they were cultured in plastic culture flasks.In the course of cultivation,dividing cells were labeled by BrdU in order to determine the S-phase labeling index(LI).Meanwhile,the cell proliferating rates of both groups were measured by MTT method.After three-month culture,the differentiation percentages of both groups were investigated by immunocytochemistry.The stem cells' differentiation capacity of the experiment group was also studied. Results Neural stem cells grew rapidly in the agarose gel anti-attachment flasks.The differences of LI and proliferating rates between the experiment group and the control group were insignificant statistically.When the neural stem cells were cultured in vitro for 3 months,the cell differentiation percentage of the experiment group was 0.64%,while that of the control group was 32.05%.The difference between them was highly significant.Moreover,neural stem cells cultured in the anti-attachment flasks still maintained their multipotency.Conclusion Agarose gel anti-attachment culture flask is suitable for long-term, large-quantity cultivation of neural stem cells,since it is in favor of cell proliferation and capable of preventing neural stem cell from differentiating.