探讨改良的新生大鼠海马神经元细胞体外培养方法，为新生儿缺血性脑损伤模型等海马神经元相关疾病研究奠定基础。

采用本研究自行设计的改良新生大鼠海马神经元细胞的体外培养方法，进行新生大鼠海马神经元细胞的体外培养。判断新生大鼠原代海马神经元细胞体外培养成功的标准为：荧光显微镜下，可见微管相关蛋白(MAP)2在海马神经元细胞体和树突中表达。该改良方法具体为：①以L-多聚赖氨酸和鼠尾胶原作为神经元细胞体外培养的生长基质，分离出生24 h内新生SD大鼠海马结构。将其运用单一胰蛋白酶水浴振荡消化后，吹打成细胞悬液，将海马神经元细胞以适当的密度种植于含10%胎牛血清DMEM培养基中，贴壁培养4 h后，更换为Neurobasal维持培养基继续培养，以后每3 d更换1/2培养液。②采用抗MAP2-5-异硫氰酸荧光素(anti-MAP2-FITC)免疫荧光法，对所获得的海马神经元细胞进行鉴定。

①新生大鼠海马神经元细胞的体外培养结果显示，新生大鼠海马神经元细胞于培养30 min时，大部分细胞贴壁生长，培养2 h后，细胞贴壁明显，少数细胞开始长出突起。于培养5 d后，神经元突起进一步增多，并形成神经细胞网络，培养7 d后，神经元细胞分化更加成熟，神经元细胞之间的突起联系更加紧密，可见密集的神经细胞网络。海马神经元细胞体外培养至第21天后，神经元细胞开始退化、变性，细胞体皱缩，残留细胞体痕迹，周围光晕消失，折光性减弱，突起融合而粗大杂乱，神经细胞网络粗大老化。②anti-MAP2-FITC免疫荧光法对所获得的海马神经元细胞进行鉴定的结果显示，在所有细胞中，免疫荧光染色MAP2阳性新生大鼠海马神经元细胞纯度达96.3%。

通过上述改良方法培养获得的新生大鼠海马神经元细胞生长状态良好，并具有较高的纯度，可为进一步进行新生儿缺血性脑损伤模型等海马神经元相关疾病的研究提供实验条件。

To establish an improved culture method of rat hippocampus neurons for further research on the hippocampus neuron related diseases such as hypoxia-ischemic brain damage model.

The culture of neonatal rat hippocampus neurons was performed by using improved method of this study. The criteria for judging the success of primary cultured hippocampus neurons in vitro was that the expression of microtubule-associated protein (MAP) 2 in neuronal cell bodies and dendrites could be seen under fluorescence microscope. The improved method was as follows: ① primarily L-polylysine and rat tail collagen were used as the growth medium in vitro culture of neurons, then hippocampal tissues were isolated from the newborn rats (within 24 h of the birth), digested by single trypsin and bath shock, and then made into cell suspension. The suspension was collected and inoculated in the culture of DMEM medium with 10% fetal bovine serum by suitable planting density, and then the neurons were moved into nutrient solution after 4 h. The half of medium was replaced every 3 d. The morphological changes of neurons in different stages were observed under phase-contrast microscope. ②The derivation of cells was identified by immunofluorescent staining with anti-MAP2-fluorescein-5-isothiocyanate (FITC) immunofluorescence assay.

①The results in vitro culture of hippocampus neurons showed a large number of hippocampal neurons began to adhere to the glass slides within 30 min.After 2 h, the adherent wall was obvious, and a few cells began to develop small neuritis. Up to the 5th day, many neurites extended to form dense network. The neuron differentiation was more mature, and the synapses between neurons were more closely linked on the 7th day.Up to the 21st day, hippocampus neurons began to degrade and denature, cell bodies were in a state of shrinkage, and residual traces of cell bodies could be found, halo around cell bodies disappeared, refraction weakened, dendrites were fused and extended into long and thick state, and networks of neurons were thick and aging. ②Anti-MAP2-FITC immunofluorescence assay demonstrated that the rate of positive cells of MAP2 was 96.3%.

The neurons obtained by the above improved method are in good growth state, and have higher purity, which provide the conditions for further study of hippocampus neuron related diseases, such as hypoxia-ischemic brain damage model.