Phenotypic Characterization of Retinoic Acid Differentiated（实验处理和结果）····

（PLOS one 3区 荷兰神经科学研究所）
蛋白印迹分析：将细胞接种在24孔板里，第八天，细胞在70ml RIPA (25 mM Tris-HCl pH 7.4 (Sigma), 150 mM NaCl (Sigma), 1% NP40 (AppliChemicals), 1% sodium deoxycholate (Sigma), 0.1% SDS and 16Complete Protease Inhibitor (Roche))缓冲液在冰上孵育。采集细胞裂解液，超声检测，用双辛酸蛋白试剂盒计算蛋白浓度。在含有10%十二烷基硫酸钠的负载缓冲液中加热·····
DAT和NAT的检测：DAT和NAT活动RA-诱导分化的SH-SY5Y细胞用不同浓度(0003毫米3.0毫米)的选择性DAT抑制剂1 - (2 - (bis (4-fluorophenyl)甲氧基)乙基)4 -(3苯丙基)哌嗪(GBR 12909)或与不同浓度(0,0,0001毫米1毫米)的选择性NAT抑制剂去郁敏(DMI)培养基。
结果：

Figure 4. Expression of dopamine synthesis and turnover markers in noRA and RA differentiated SH-SY5Y cells at culture day 8. Selection of genes is based on NCBI Gene data base and IPA. A. Microarray expression values of these molecular markers are presented for the noRA (black circle) and RA (gray square) treated cells at D8. Red line characterizes the cut-off at 2log intensity of 7 demarcating expression versus no expression of the mRNA in these cells (equivalent to 26background levels). SLC18A2 (VMAT2) was not detected on the microarray, but confirmed to be expressed by qPCR analysis (red marking). Significantly regulated genes between the two conditions are marked with an ‘’. B. Immunocytochemistry of two dopamine synthesis and transport proteins TH and VMAT2 expressed by the RA treated cells at D8. C. Immunocytochemical detection of DA in noRA and RA SH-SY5Y cells. Images, taken with the same laser intensity, consist of a z-stack projection of the cell layer.
RA诱导分化的SY5Y呈多巴胺能样神经递质表型
SY5Y细胞表达多巴胺能样神经元标志物：我们监测了多巴胺能神经元早期和晚期的标志物的表达。在培养的第八天，SH-SY5Y细胞表达了26中标记物中的5种，这些标记物在多巴胺能神经元发育的早期有作用。其中两种差异显著，但总体来讲，差异并不显著。作者认为RA诱导的SY5Y不表达多巴胺能神经元的早期标志物。

Figure 3. Expression of early stage dopaminergic markers. Based on Smidt and Burbach [50], expression of early stage DAergic markers in noRA (black circle) and RA (gray square) differentiated SH-SY5Y cells on culture day 8 as measured by microarray analysis. The red line represents the cutoff between detected gene expression (2log expression .7) and undetected gene expression (2log expression #7). Significantly regulated genes between the two conditions are marked with an ‘’.
SY5Y细胞合成DA：成熟的DAergic细胞应该产生DA，并表达对DA合成、转运和周转至关重要的分子。SY5Y表达所有的的DA合成酶和降解酶。我们没有观察到RA和noRA细胞在其他DA合成和代谢标志物如COMT、GCH1、MAO-B、PTS、TH和SLC18A2 (VMAT2)表达水平上的显著变化。RA分化SH-SY5Y细胞在蛋白水平上证实了VMAT2和TH的表达，这两个基因在DA合成和转运中起中心作用(图4B)。因此，SH-SY5Y细胞表达产生DA所需的所有分子，大部分不依赖于RA处理。DA含量免疫组化分析显示，noRA和RA处理后细胞均产生DA，且RA处理后DA免疫反应活性增强。MAO-A水平的降低可能是RA处理细胞DA水平升高的原因。我们的数据明确显示RA分化SH-SY5Y细胞合成并储存DA，从而具有daergic样表型。
成熟多巴胺能神经元标志物和神经递质受体在SY5Y中的表达
总的来看，RA诱导分化的SY5Y与未分化的SY5Y二者在多巴胺能神经元标志物和受体上的表达并无差别。最终，多巴胺能神经元标志物的表达和RA的诱导无关。
其他神经递质表型（组胺能、胆碱能、谷氨酰胺能）在RA诱导的SY5Y细胞中表达并不显著。

Figure 5. Expression of mature SN pars compacta DAergic neuron markers and neurotransmitter receptors. Gene levels are indicated in noRA (black circle) and RA (gray square) differentiated SH-SY5Y cells on culture day 8 as measured by microarray analysis. Graph shows expression of DAergic markers known to be expressed by mature SN DAergic neurons [50], and neurotransmitter receptors known to be expressed by SN DAergic neurons such as GABA receptor subunits and chloride chanels [51–53], glutamate receptor subunits [54–56] and nicotinic and muscaric acetylcholine receptor subunits [57]. Red line characterizes the cut-off at 2log intensity of 7 determining the expression and no expression of the mRNA in these cells (equivalent to 26background levels). Significantly regulated genes between the two conditions are marked with an ‘*’.
讨论：
我们发现，RA治疗SH-SY5Y细胞诱导分化程序，促进一般的神经元样状态和主要的DAergic特征。RA诱导后的SY5Y细胞表达部分多巴胺能神经元的特征，在一定程度上与多巴胺能神经元相似，但与成熟的多巴胺能神经元存在差距。
RA抑制了SY5Y向胆碱能、去甲肾上腺素能神经表型的表达。