不同载体及年龄对大鼠视神经组织体外培养的影响

Effects of different medium and age on optic nerve tissue culture of rats

目的探讨不同载体及年龄对大鼠视神经组织体外培养的影响。方法取出生4d和3月龄SD大鼠的视神经．分别使用鼠尾胶原玻片、多聚赖氨酸玻片和Biocoat小室进行组织培养，观察组织生长情况。培养48h后观察组织块贴壁率，培养5d时使用图像分析仪测量细胞最大迁移距离。使用乳酸脱氢酶（LDH）试剂盒对Biocoat小室组不同年龄大鼠视神经组织培养液LDH活性进行动态比较。HE染色形态学观察，透射电镜观察组织块超微结构变化。结果Biocoat小室组的组织贴壁率明显高于鼠尾胶原组、多聚赖氨酸组：在Biocoat小室中培养的成年和新生大鼠视神经组织的细胞最大迁移距离均明显大于鼠尾胶原组和多聚赖氨酸组：在相同培养载体上．新生大鼠细胞迁移距离均明显高于成年大鼠，差异均有统计学意义（P〈0．05）。视神经组织培养液LDH活性在接种3d后开始下降，成年大鼠LDH水平在培养9d后再次上升，而新生大鼠在培养12d时仍保持较低水平。视神经在培养早期即有细胞从组织块边缘游出，逐渐发出突起。新生大鼠较早出现细胞迁移。随着培养时间延长，组织出现结构紊乱和坏死。新生大鼠组织存活时间较成年大鼠明显延长。结论通过选择适宜的培养条件，视神经可以在体外较长时间存活。

Objective To investigate the effects of different medium and rat age on optic nerve tissue culture of rats. Methods The optic nerves from newborn rats （4 d postbirth） or adult rats （3-month old） were cultured on the rat-tailed collagen slide, poly-L-Lycine （PLL） slide, and Biocoat culture inserts, respectively. Their growth status was dynamically observed under a phase contrast microscope every day. The adherence rate of explant was recorded 48 h after culture. The maximum migration distance was measured by an image analysis system on the 5th day after culture. The activity of lactic dehydrogenase （LDH） in the tissue culture medium was measured dynamically. Morphological observance was carried out by routine HE staining and the ultrastructure of the tissue explants were observed by a transmission electron microscope. Results The tissue adherence rate was higher in the Biocoat insert group than in the rat-tailed collagen slide group or PLL slide group. The maximum migration distance of the tissue explants cultured in the Biocoat insert group was longer than that in the rat-tailed collagen slide group or the PLL slide group. The maximum migration distance of the newborn rats was longer than that of the adult rats under same culture condition（P〈0.05）. The LDH activity in the tissue culture medium began to descend 3 d after culture. The LDH activity in the adult rat group increased again on the 9th day since culture while it remained low level in the newborn rat group even on 12th day since culture. The cell processes showed up from the edge of explants and neuralgia cell migration was observed at the early stage, especially in newborn rats. The optic nerve structure gradually died out with the increase of culture time. The survival time of optic nerve explant from newborn rats was longer than that of adult rats. Conclusion The optic nerve tissue can be cultured for a long time under suitable culture conditions.