中文摘要：

目的比较2种培养基下海马神经干细胞（neural stem cells，NSCs）的生长特性，进而实现海马NSCs扩增的优化及其与聚乳酸（poly—L—lactide，PLLA）三维微小凹图式的复合。方法分离大鼠胚胎海马细胞并采用Neurobasal为基础的培养基和DMEM／F12为基础的培养基进行扩增。以四甲基氮唑蓝（MTr）比色法及神经球数目统计法评价2种培养基下细胞增殖行为。采用紫外光光刻、硅蚀刻及软光刻技术制备PLLA三维微小凹图式并实现海马NSCs与微小凹图式的复合。结果原代分离的海马细胞呈神经干细胞标志物阳性并能向神经元系和胶质细胞系分化。在30d的扩增时间内，海马NSCs在以Neurobasal为基础的培养基中缓慢扩增聚集成神经球，少见细胞贴壁及分化；在以DMEM／F12为基础的培养基中海马NSCs扩增迅速，但易贴壁和分化。培养第25天时后者神经球数量为前者的4．7倍。微加工制备的微小凹图式结构清晰、稳定，具有高纵横结构比（≥1）。扩增的海马NSCs能在三维微小凹图式上成功复合生长。结论以DMEM／F12为基础的培养基有利于大鼠海马NSCs的扩增，以Neurobasal为基础的培养基利于海马NSCs的纯化。序贯应用2种培养基可有效扩增海马NSCs并实现其与三维微小凹图式的复合。

英文摘要：

Objective To compare the growth behaviors of rat hippocampal neural stem cells （NSCs） cultured in 2 medium conditions to optimize the proliferation condition, and to achieve integration of hippocampal NSCs with our fabricated three-dimensional （3-D） poly-L-lactide （PLLA） microwell patterns. Methods Rat hippocampal ceils, obtained with mechanical separation, were expanded in either Neurobasal-or DMEM/ F12-hased culture medium. Proliferation behaviors of the isolated hippocampal cells were evaluated with neural sphere counting and the methyhhiazolyl tetrazolium （MTT） assay. PLLA microwell patterns were fabricated with UV lithography, silicon etching and soft lithography and then integrated with the expanded NSCs. Results Immunocytochemistry and immunofluorescence showed that the isolated hippocampal cells were positive for neural stem cell marker nestin and were capable of differentiating to the neuronal and glial lineages. In a period of 30 days＇ expanding, hippocampal NSCs proliferated slowly forming neural spheres in Neurobasal-based culture medium, with few cells attached to substrates and differentiated morphologically. In contrast, cells in DMEM/ F12-based medium proliferated rapidly, with significant cell attachment and morphological differentiation. On the day 25 of expanding, neural sphere number in DMEM/F12-based medium was 4.7 times as much as that in Neurobasal-based medium. The microwell patterns were well defined, of high aspect ratio （ ≥ 1 ） and stable under culture conditions. Population expanded hippocampal NSCs were successfully integrated with the 3-D PLLA microwell patterns. Conclusion DMEM/F12-based medium is favorable to population expansion of rat hippocampal cells while Neurobasal-based medium is conducive to purification of hippocampal NSCs. Sequential use of DMEM/F12- and Neurobasal-based medium can thus result in expansion of hippocampal NSCs and successful integration of the NSCs with 3-D PLLA microwell patterns.