新生大鼠背根神经节的提取及不同形态的神经元模型建立

Extraction of dorsal root ganglia from neonatal rats and establishment of neuron models with different morphologies

目的提取新生SD大鼠背根神经节(DRG)并建立不同形态的神经元模型。方法选取出生3~5天的大鼠乳鼠,处死后置于75%乙醇中消毒,逐步解剖出坐骨神经及脊柱,分离脊柱两侧肌肉,行椎板切开及椎间孔破坏,摘取坐骨神经与脊髓相交处的DRG,经过Ⅰ型胶原酶及胰蛋白酶+EDTA或Ⅰ型胶原酶、胰蛋白酶+EDTA及15%BSA溶液作用后种植于基质胶预先包被的35 mm培养皿中,待其贴壁后交替使用Neurohasal培养基和添加阿糖胞苷抗有丝分裂的Neurohasal培养基,分别获得DRG神经元团和神经元模型。结果提取培养的DRG神经元团及神经元生长状况正常,神经元轴突的生长旺盛并表达神经元特异性蛋白βⅢ-tubulin,阳性率达89.39%,并能存活生长至少30天。结论本实验方法可简单提取DRG并建立两种不同形态的神经元模型,对神经元轴突生长进一步的研究提供了良好的基础。

Objective To extract dorsal root ganglia(DRG)from newborn SD rats and establish neuron models with different morphologies.Methods Three to five-day-old suckling rats were sterilized with 75%ethanol after sacrificed,then the sciatic nerve and spine were gradually dissected.DRG was obtained from the intersection of sciatic nerve and spinal cord by separating bilateral spine muscles,laminotomy and intervertebral foramen destruction.And then DRG was implanted in 35 mm culture dish precoated with matrigel after being treated with type I collagenase+trypsin+EDTA or type I collagenase+trypsin+EDTA+15%BSA solution.Neurohasal medium and Neurohasal medium supplemented with cytarabine against mitosis were used alternately to obtain DRG neuron cluster model and single neuron model,respectively.Results The cultured DRG neuron clusters and single DRG neurons grew normally.Neuronal axons grew vigorously and expressed neuron-specific protein beta III-tubulin,with a positive rate of 89.39%,and could survive for at least 30 days.Conclusions The experimental method extracts DRG and establishes two different neuron models,which provides a great basis for further study of neuron axon growth.