体外诱导分化多巴胺能神经元的分子特征

Molecular characteristics of in vitro differentiated dopaminergic neurons

背景:一系列研究表明,腹侧中脑多巴胺能前体细胞可在体外经人工诱导分化为大量多巴胺能神经元,有望用于移植治疗帕金森病,但是其分子特征尚不明了。目的:观察体外培养得到的多巴胺能神经元标记基因的表达情况。设计、时间及地点:观察性实验,于2005-12/2008-12在苏州大学附属第二医院神经外科脑肿瘤研究室以及上海生物芯片有限公司完成。材料:健康成年近交系Wistar孕鼠(>36代),体质量约300g,用于中脑细胞体外培养。方法:取材培养胎鼠腹侧中脑细胞,先用碱性成纤维细胞生长因子进行扩增,而后用L-抗坏血酸-2-磷酸酯倍半镁盐诱导其分化。分别于诱导前和诱导后第4天,第7天抽提RNA后利用Affymetrix基因芯片检测多巴胺能神经元标记基因的表达变化。主要观察指标:①多巴胺能前体细胞诱导分化后形态变化。②常见多巴胺能神经元标记基因在诱导分化前后过程中的表达。结果:大鼠腹侧中脑多巴胺能前体细胞可在体外被大量诱导分化成酪氨酸羟化酶阳性细胞,并且不同程度地表达多种多巴胺能神经元标记基因乙醛脱氢酶1A1亚家族、钙结合蛋白1、多巴脱羧酶、多巴胺转运体、G蛋白门控内向整流钾通道、囊泡单胺转运体2。随着细胞诱导分化的进行,这些标记基因表达的变化趋势也不尽相同,其中显著上调的标记基因为钙结合蛋白1,而显著下调的标记基因为G蛋白门控内向整流钾通道。结论:大鼠腹侧中脑多巴胺能前体细胞可在体外大量诱导分化成酪氨酸羟化酶阳性的主要源自中脑腹侧被盖区(A10)的多巴胺能神经元。

BACKGROUND： A series of studies have demonstrated that ventral mesencephalic dopamine precursors may differentiate into considerable dopaminergic neurons （DNs） available for cell transplantation for Parkinson＇s disease. However, the detailed molecular characteristics of these cells remain unclear. OBJECTIVE： To observe the expression of main marker genes of DNs cultured in vitro. DESIGN, TIME AND SETTING： This observation experiment was performed at the Brain Tumor Laboratory in the Second Affiliated Hospital of Soochow University and Shanghai Biochip Company from December 2005 to December 2008. MATERIALS： Inbred pregnant Wistar rats （F＋36） weighing 300 g approximately were used for in vitro culture of mesencephalic cells. METHODS： Ventral mesencephalic cells from inbred pregnant Wistar rats were cultured with basic fibroblast growth factor, and were induced to differentiate in vitro with L-Ascorbic acid-2-phosphate sesquimagnesium salt. Total RNA were extracted before and 4, 7 days after the induction. Affymetrix microarrays were utilized to detect the expression changes of main marker genes of DNs. MAIN OUTCOME MEASURES： Morphologic changes of differentiated ventral mesencephalic precursors; Expression of DN-related common marker genes before and after the induced differentiation. RESULTS： Rat ventral mesencephalic precursors differentiated into numerous tyrosine hydroxylase-positive cells, which expressed multiple marker genes of DNs, such as AHD2, calcium binding protein 1 （Cab1）, DDC, DAT, Drd2, Girk2, VMAT-2, with variously degrees and with different tendencies. Among those genes, only Cab1 was significantly up-regulated and only Girk2 was significantly down-regulated. CONCLUSION： Rat ventral mesencephalic precursors can be induced to differentiate into plenty of tyrosine hydroxylase-positive DNs, mainly originating in A10 regions.