神经干细胞接种密度对其增生和分化的影响

Effect of Cell Density on Proliferation and Differentiation of Human Fetal Neural Stem Cells

为探讨神经干细胞不同接种密度对其增生和分化的影响,取10～12周龄胎儿的大脑皮质用胰酶消化获得单细胞,分别以1×102/mL、1×104/mL和1×106/mL的细胞密度接种,用无血清培养基DMEM/F12、上皮细胞生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)和白血病抑制因子(LIF)培养;用MTT法测OD值并观察神经干细胞的存活和增生情况.从培养基中剔除生长因子,继尔以10%胎牛血清诱导分化,用免疫细胞化学(ICC)方法,研究不同细胞密度培养后神经干细胞的分化能力.结果:在1×106/mL细胞密度下培养,神经干细胞增生活跃,形成神经球,Nestin表达阳性,10%胎牛血清诱导后神经干细胞可分化为神经细胞、星形胶质细胞和少突胶质细胞;低细胞密度(低于1×104/mL)培养不利于神经干细胞的存活和增生,血清诱导未见分化的细胞.提示:适宜的神经干细胞接种密度有利于其增生和分化.

The aim of the study was to investigate the effect of cell density on the survival, proliferation and differentiation of human neural stem cells in the primary culture. Combination of EGF, bFGF and LIF can stimulate the proliferation of human neural stem cells remarkably. When these growth factors were removed, and 10% fetal bovine serum was added into culture media, the human neural stem cells could differentiate into neurons, astrocytes and oligodendrocytes. There were no proliferation and neurospheres formation in low-density ( cells < 10 000/mL) culture. From this experiment, we can draw the conclusion that the human neural stem cells from the fetal brain has the ability of self-renewing and multiple differentiational potential, and moderate cells density will benefit its survival and proliferation.