|
胚胎小鼠下丘脑培养状态SRIF神经元的发生免疫细胞化学研究 |
| |
ons increased with culture time,with the highest number at 14 DIV,and decreased when the culture time became longer than 14 days.Furthermore,obvious changes took place in the morphology and immunocytochemical reactivity of the neurons as well as the number of monopolar,bipolar and multipolar neurons at different time points.In addition,the highest percentages of neurons with different profiles turned up at different time points.The per-centages of the monopolar neurons were 52.3%,60% and 43.3% at 10,14 and 16 DIV,respectively,whereas the percentages of those bipolar cells represented 30.7%,20.7% and 30.1% at the corresponding time points.As for the multipolar cells,such percentages reached 81.5%,90.1%,and 67.3% at 7,12 and 20DIV,respectively. Conclusions (1)Distinct hypothalamic neurons develop at different speed under culture conditions and change regularly at different culture time;(2)distinct types of SRIF-positive neurons develop at different rates,suggesting that some of the SRIF-positive neurons may have transformed their types and functions,and (3) in vitro development of the SRIF-positive neurons may reflect their in vivo states.
【Key words】 Somatotropin release inhibiting factor; Hypothalamus; Cell culture; Immuncytochemistry
SRIF为环状的14肽，它不仅是一种能够抑制生长激素释放的一种内分泌激素，而且是一种强有力的神经递质或神经调制物。其生理作用广泛而复杂。随着胚胎发育学、细胞生理学、神经内分泌学的飞速发展，作为肽类家族这种兼有激素作用的神经递质，越来越受到学者们的重视，但是关于SRIF神经元发生学方面以原代培养小鼠的下丘脑作为下丘脑细胞的发育过程的研究不多见，国内未见报道。本实验应用小鼠胚胎做下丘脑分散细胞培养，通过不同时期下丘脑神经元的个体生长发育和形态变化以及SRIF阳性神经元的形态特征，为研究在体胚胎期下丘脑SRIF神经元的分泌功能及其调节提供了一个体外途径。
材料和方法
1. 下丘脑神经元细胞培养
本工作以杂系17d的胚胎小鼠下丘脑为实验材料，采用原代培养的方法Richard［1］等并且加以改良。将怀孕17d母鼠引颈处死，无菌条件下将胚胎小鼠的颅骨从中线剥开，将小鼠的整个脑组织全部取出。放在细胞解离液D1-SGH(D1为无钙镁离子Pucks液，SGH为蔗糖-葡萄糖和HEPES缓冲液)中，剔除脑膜，将脑底朝上，然后用虹膜刀分别在水平位沿视交叉和中脑各切1刀，再将切好的脑片水平放置，按文献［1］所述位置再切3刀，切弃周围组织。将分离好的下丘脑剪成碎块，移入0.125%的胰蛋白酶中于37℃5%CO2孵育箱中作用25min。然后于胰酶终止液作用10min，最后用完全培养液［DMEM80%,胎牛血清20%(Sigma)］，制成单细胞悬液，再将细胞浓度调至1×106细胞/ml，然后再将细胞接种在涂有鼠尾胶原的35mm塑料培养皿中，每皿1ml，于37℃，95%空气和5%C上一页 [1] [2] [3] 下一页
上一个医学论文： 去肾上腺大鼠垂体内IL
下一个医学论文： 大鼠上涎核的传出神经纤维与翼腭神经节中的VIP
|
|
|
|
|
|
|
您现在的位置： 绿色健康网 >> 医学论文 >> 基础医学论文 >> 正文