胚胎大鼠脑室下区神经细胞体外培养和向黑质细胞定向诱导的实验研究

本研究目的在于探寻体外培养和定向诱导胚胎大鼠脑室下区(SVZ)神经细胞向黑质细胞分化的新方法。采用成年大鼠黑质匀浆上清液(HSN)、碱性成纤维细胞生长因子(bFGF)及HSN+bFGF对取自胎龄期12~16d(E12~E16)胚胎大鼠SVZ培养的神经细胞(经nestin抗体免疫组化及透射电镜识别)进行体外诱导培养48h后,以TH单抗免疫组化方法检测其是否向黑质细胞分化;并用荧光分光光度法测定HSN+bFGF诱导后0、24、48hSVZ神经细胞培养液中多巴胺含量。结果显示:(1)HSN+bFGF诱导组,TH单抗免疫组化出现阳性细胞;(2)HSN+bFGF诱导后0、24、48h多巴胺含量对比分析:48h与24h(P<0.01)、48h与0h(P<0.05)相比,多巴胺含量明显增加,而24h与0h相比则无增加趋势(P>0.05)。以上结果表明:(1)用成年大鼠HSN+bFGF能定向诱导胚胎大鼠SVZ神经细胞向黑质细胞分化,诱导而成的黑质细胞已具有多巴胺分泌功能,诱导后24~48h为多巴胺分泌的一个高峰;(2)透射电镜显示胚胎大鼠SVZ神经细胞超微结构是除免疫组化外识别该神经细胞的另一种新方法。

THE INDUCTION OF CULTURED RAT EMBRYONIC SUBVENTRICULAR ZONE NEURONS INTO SUBSTANTIA NIGRAL NEURONS

The objective of this research is to seek a new method to culture and induce the rat embryonic subventricular zone (SVZ) neurons into the substantia nigral neurons. Brain tissues of SVZ were isolated from 12 to 16 d gestation rat embryos and cultured. Identified by the immunohistochemistry of anti-nestin and transmission electron microscope, the cells culture were respectively treated by the homogenate of substantia nigra (HSN) of adult rat, basic fibroblast growth factor (bFGF) and HSN + bFGF. After incubated for 48 h, immunohistochemistry of monoclonal anti TH was applied to determine if the SVZ neurons were to differentiated into the substantia nigral neurons. We also used fluorospectrophotometry technique to assay the content of dopamine in the culture liquid at 0, 24 and 48 h after HSN + bFGF induction. The results showed that: (1) In the induced group that treated with HSN+bFGF, immunohistochemistry of monoclonal anti-TH showed positive cells; (2) The content of dopamine in the culture liquid from 0 to 48 h (P<0.05) and 24 to 48 h (P<0.01) after induced by HSN+bFGF increased significantly. However, the content of dopamine from 0 to 24 h after induction had no tendency of increase (P>0.05). The results revealed that:(1) Using HSN+bFGF can directionally induce the SVZ neurons of embryonic rat to differentiate into substantia nigral neurons; The substantia nigral neurons induced by HSN+bFGF from the neurons of SVZ already had the competence to secrete dopamine. Moreover, during 24 to 48 h after induction, the dopamine secretion had a high peak. (2) Using transmission electron microscope to show the ultrastructure of the SVZ neurons is another method to identify them in addition to immunohistochemistry.