聚乳酸微小凹图式上大鼠海马神经干细胞聚集体形成行为的研究

Characterization of cellular aggregate formation by rat hippocampal neural stem cell on poly(L-lactic acid) microwell patterns

目的制备聚乳酸（PLLA）三维微小凹图式并实现图式上新生大鼠海马神经干细胞（NSC）三维聚集体的形成及培养。方法用2只新生1~3 d SPF级SD大鼠体外分离提取海马组织并培养NSC,观察其悬浮神经球和贴壁生长特性;以紫外光光刻和复制模塑法制备3种不同结构尺寸的PLLA微小凹图式;以普通光学显微镜和Image J图像处理软件研究图式上NSC聚集体的形成及大小分布;以免疫细胞荧光技术研究图式上NSC标志物Nestin的表达。结果 NSC在体外能够以悬浮神经球和贴壁生长的2种方式生长。在所研究的3种结构尺寸的图式中,无通道（120-0μm）和通道宽度为20μm（120-20μm）的微小凹图式更利于NSC聚集体的形成（P〈0.01）;而在通道宽度为40μm的图式（120-40μm）上NSC更倾向于沿通道方向生长、迁移,并形成准一维排布的三维神经细胞网络。图式上聚集体Feret’s直径分布均匀,无大的聚集体（直径〉120μm）出现。在细胞聚集体及神经细胞网络形成期间NSC均保持Nestin阳性表达。结论 PLLA微小凹图式可用于体外NSC聚集体形成及神经细胞网络的三维图式化,这为干细胞组织工程及基于神经细胞的微系统提供了有效的研究及应用途径。

Objective To fabricate three-dimensional（3D） poly（L-lactic acid）（PLLA） microwell patterns, and achieve cellular aggregate formation by newborn rat hippocampal neural stem cell（NSC） on patterns. Methods The hippocampal tissues were isolated in vitro from 2 newborn SPF rats aged 1 - 3 days, and NCS was cultured to observe the characteristics of suspension neurospheres and adherent growth. Three structural dimensions of microwell patterns were prepared by UV lithography and replica molding method. The formation and distribution of NSC aggregates were studied by ordinary optical microscope and Image J image processing software. The expression of NSC marker was studied by immunofluorescenee technique. Results NSC grew in vitro in two ways of suspension and adherent. The patterns without channel connection（120-0 μm） or with channel connection of 20 Ixm in width（120-20μm） were induced aggregate formation more efficiently in 3 structural dimensions（P 〈 0.01）; The NSC tended to grow and migrate along the channel on patterns with channel connection of 40 μm in width （120-40 μm）, and formed a quasi-one- dimensional 3-dimensional neural cell network. The Feret＇s diameters of aggregates on patterns were uniform, and no huge aggregates（diameter 〉 120 μm） were observed. The expression of Nestin by NSC on patterns was confirmed during cell aggregates and neuronal network formation by immunofluorescent staining. Conclusion It is demonstrated that PLLA microwell patterns could be used for NSC aggregate formation and 3D neural cell network patterning, which may become the effective approach in stem cell tissue engineering and micro systems based on nerve cells.