小鼠垂体巢蛋白阳性细胞在体外具有集落生长能力

In vitro colony formation capacity of Nestin-immunoreative cells from mouse pituitary

目的研究小鼠垂体前叶中巢蛋白（Nestin）阳性细胞在体外培养环境中的自我增殖能力。方法建立小鼠垂体Nestin阳性细胞克隆生长培养条件,观察次代细胞在其中的生长情况。应用野生型细胞和增强型绿色荧光蛋白（EGFP）细胞混合培养,以及BrdU渗核实验鉴定次代Nestin阳性细胞培养过程中新生细胞群的来源。结果次代Nestin阳性细胞在克隆生长培养基中能够自我增殖。野生型细胞和EGFP细胞混合培养实验表明,新生细胞群由纯野生型细胞或纯EGFP细胞构成。BrdU渗核实验显示,24h内新生细胞群中有13%的细胞由分裂产生（n=20）。结论部分小鼠垂体前叶Nestin阳性细胞在体外培养环境中具有克隆生长能力。

Objective To study the self-renewal potential of Nestin-immunoreactive cells in mouse anterior pituitary. Methods Primary Nestin-immunoreactive cells were isolated from mouse anterior pituitary and enriched in specific culture condition. In order to analyze colony forming potential of Nestin-immunoreactive cells,secondary generation Nestin-immunoreactive mouse anterior pituitary cells were cultured in colony formation medium. For further confirming single cell origin of new colonies,cells isolated from both wild type and EGFP mice were mixed in culture condition as to trace the source of newly generated colonies. In addition,BrdU incorporation assay was utilized to verify that colonies were formed by cell proliferation,and then to evaluate proliferative rate of cells in colonies. Results Cells derived from primary culture condition with Nestin—immunoreactive cell morphology were detected to express nestin by immuncytochemistry. In conoly culture condition with very low cell density,1% of secondary generation Nestin-immunoreactive cells were capable of forming colonies in vitro. Mixture culture of EGFP and wild type cells showed that colony derivatives under this condition were either homogenous EGFP cells or homogenous wild type cells,wherease there was no mixed colony detected. Moreover,BrdU incorporation assay demonstrated that within 24 hours 13% cells from colonies were generated from cell division （n=20）. Conclusion Mouse anterior pituitary contains Nestin-immunoreative cells. These cells are colony-forming cells,indicating that at least part of Nestin-immunoreative cells has self-renewal capacity,which is one of most important features of stem cells.