新生大鼠上丘脑组织培养研究

Tissue Culture Study on Superior Colliculus of Newborn Rats

目的 探讨新生大鼠上丘脑组织培养方法，并对其在培养过程中的变化进行观察。方法取出生4d的sD乳鼠上丘脑组织，分别在鼠尾胶原玻片、多聚赖氨酸玻片和Biocoat培养小室上进行组织培养，使用相差显微镜动态观察组织块生长情况。48h后观察组织块贴壁率，在培养5d时使用图像分析仪观察细胞迁移情况，测量最大迁移距离。使用乳酸脱氢酶试剂盒，测量组织培养液中乳酸脱氢酶活性的动态变化。常规HE染色形态学观察，并使用透射电镜观察组织块超微结构。结幂与传统的培养载体相比，上丘脑组织在Biocoat培养小室上进行培养可以获得较高的组织贴壁率和细胞迁移距离。上丘脑组织在接种后3～15d，其培养液中乳酸脱氢酶活性保持在较低水平。培养早期上丘脑组织块边缘出现细胞突起，胶质细胞迁移，随着培养时间延长，细胞出现凋亡，组织结构破坏。结论Biocoat培养小室是进行新生大鼠上丘脑组织培养的理想载体，接种3d后组织生长趋于旺盛，而在2周后多数组织的生长逐渐衰退。

Objective To study the tissue culture technique for superior colliculus explanted from newborn rats and to observe the growth status dynamically. Methods Four-day newborn rats were divided into 3 groups. Superior colliculus tissue culture was carried out on the rat-tailed collagen slide, poly-L-Lycine slide, and Biocoat culture inserts, respectively. Their growth status was dynamically observed with a phase contrast microscope every day. The adherence rate of tissue clusters was recorded 48 h after culture. The maximum migration distance was measured by an image analysis system on the 5th day. The activity of lactic dehydrogenase （LDH） in the tissue culture medium was measured by a LDH kit. Morphological observance was carried out with routine HE staining, and the uhrastructure of the tissue explants was observed with a transmission electron microscope. Results Compared with traditional culture slide, tissue cultured on Biocoat inserts got optimal adherence rate and cell migration distance. The activity of LDH kept low level for the tissues cultured on Biocoat inserts from the 3rd to the 15th day after inoculation. The cell processes showed up from the edge of explants and neuroglial cell migration was observed at the early stage. The transmission electron microscopy showed that there emerged certain apoptotic cells with damaged structures as the culture time increased. Conclusion Biocoat culture insert is favorable for the growth of superior colliculus explants. The tissue growth prospers in 3 d after inoculation, the majority of which declines in 2 weeks.