CAJ | 학술논문

目的体外原代培养大鼠小脑颗粒神经元,为研究慢性砷暴露对小脑神经元的毒性作用提供实验方法.方法取生后5～7天Wistar仔鼠,体式显微镜下分离小脑皮层,0.25％胰蛋白酶消化、DNA Ⅰ酶洗涤制成单细胞悬液,两次差速贴壁后接种在多聚赖氨酸包被的培养板内,相差镜下观察大鼠小脑颗粒神经元成长、发育变化及突触形成.采用神经元特异性烯醇化酶(NSE)免疫荧光技术鉴定神经元.结果培养后第24小时,相差显微镜下可见大鼠小脑颗粒神经元贴壁,呈网状排列；第2～3天,神经元胞体由椭圆形变成圆形,轮廓逐渐清晰,细胞伸出突起,突起逐渐延长,细胞间通过突起连接,形成了稀疏的神经元突触网络；第4～6天,细胞体积进一步增大,细胞间通过广泛的突触联系,神经元清晰饱满,形成了复杂的神经元网络.共聚焦显微镜下,可见大量含NSE的神经元.结论成功地进行了大鼠小脑颗粒神经元的原代培养,该方法可为今后研究慢性砷暴露对小脑细胞的毒性作用提供实验依据.
목적 체외원대배양대서소뇌과립신경원,위연구만성신폭로대소뇌신경원적독성작용제공실험방법.방법 취생후5～7천Wistar자서,체식현미경하분리소뇌피층,0.25％이단백매소화、DNA Ⅰ매세조제성단세포현액,량차차속첩벽후접충재다취뢰안산포피적배양판내,상차경하관찰대서소뇌과립신경원성장、발육변화급돌촉형성.채용신경원특이성희순화매(NSE)면역형광기술감정신경원.결과 배양후제24소시,상차현미경하가견대서소뇌과립신경원첩벽,정망상배렬；제2～3천,신경원포체유타원형변성원형,륜곽축점청석,세포신출돌기,돌기축점연장,세포간통과돌기련접,형성료희소적신경원돌촉망락；제4～6천,세포체적진일보증대,세포간통과엄범적돌촉련계,신경원청석포만,형성료복잡적신경원망락.공취초현미경하,가견대량함NSE적신경원.결론 성공지진행료대서소뇌과립신경원적원대배양,해방법가위금후연구만성신폭로대소뇌세포적독성작용제공실험의거.
Objective To establish a stable primary culture of rat cerebellar granule neurons in vitro for further study the toxic effects of chronic arsenic exposure on cerebellar cells.Methods Cerebellar cortices were taken from brain of Wistar rat 5-7 day old after born under stereoscopic microscope.Single cell suspension was acquired after digestion and washing with trypsin (0.25％) and DNase Ⅰ solution,respectively.Granule cells were purified from other cells by differential velocity adherence method for two times.Rat cerebellar granule neurons were seeded in culture plate pre-coated with poly-L-lysine.Neurons growth,development and synaptic connections were observed daily.The neurons were identified by neuron specific enolase (NSE) immunofluorescence technique.Results The neurons were affixed to the culture plate in 24 hours,in reticular arrangement observed under contrast microscope.Granule cells gradually turned round from oval and outlines became clearer in 2-3 days.In 4-6 days,there were a wide range of synaptic connections among the neurons and a mature nerve cell network formed.A large quantity of cerebellar granule neurons was seen by NSE identification.Few bigger cells such as purkinjes cells and glial cell outlines were also seen in the same visual field.Conclusions This is a successful primary culture method for acquirement of rat cerebellar granule neurons.The method can provide experimental basis for future studies the toxic effects of chronic arsenic exposure on cerebellar cells.