全反式视黄酸对神经干细胞诱导分化和c-myc基因表达的调控

Effects of all-trans retinoic acid on the differentiation of neural stem cells and the expression of c-myc gene

目的:观察全反式视黄酸对神经干细胞的诱导分化情况,及其对c-myc基因、蛋白表达的影响。方法:实验于2005-03/06在南方医科大学高温医学研究室完成。①基础培养液:DMEM/F12中含Hepes15mmol/L,NaHCO32g/L,2%B27,100U/mL青霉素,100U/mL链霉素。神经干细胞培养液:基础培养液+碱性成纤维细胞生长因子20μg/L。神经干细胞分化诱导培养液:基础培养液+体积分数为0.01的胎牛血清。全反式视黄酸储备液:以二甲基亚砜为溶剂,配成浓度为1.0×10-3mol/L全反式视黄酸储备液。②出生2～3d SD大鼠6只,麻醉后开颅取出全脑,机械分离结合无血清培养基进行鼠纹状体神经干细胞的分离培养,取生长状态良好的第3代细胞用于实验。③将神经干细胞克隆接种于12孔培养板中,全反式视黄酸组加入神经干细胞分化诱导培养液+不同浓度全反式视黄酸(0.01,0.1,1.0,10.0μmol/L)。二甲基亚砜对照组加入神经干细胞分化诱导培养液+0.01%二甲基亚砜。各组细胞培养7d后进行免疫组织化学染色鉴定,计数神经元样细胞分化率。RT-PCR分析c-myc基因表达情况,Western blots检测c-myc蛋白的表达。结果:①神经干细胞的原代和传代培养与鉴定:来源于新生大鼠纹状体组织的原代克隆和传代克隆,呈巢蛋白抗原阳性。第3代细胞加入含不同浓度全反式视黄酸的神经干细胞分化诱导培养液,呈神经元特异性烯醇化酶、胶质纤维酸性蛋白抗原阳性。②全反式视黄酸对神经干细胞分化的影响:随着全反式视黄酸浓度(0.01,0.1,1.0,10.0μmol/L)升高,神经元样细胞分化率逐渐增加(F=358.59,P=0.000)。全反式视黄酸浓度为1.0,10.0μmol/L时神经元样细胞分化率基本相似[(34.27±0.81)%,(35.27±0.32)%,P=0.163]。③全反式视黄酸对c-myc基因及其蛋白表达的影响:随着全反式视黄酸作用时间的增加,与二甲基亚砜对照组相比,全反式视黄酸组的c-myc mRNA表达下调(F=20.891,P=0.000),c-myc蛋白表达亦下调(F=43.138,P=0.000)。结论:生理浓度(1.0μmol/L)的全反式视黄酸是诱导神经干细胞向神经元方向分化的适宜剂量。全反式视黄酸可能通过细胞周期调控因子c-myc从而影响神经干细胞的增殖分化。

AIM： To observe the effects of all-trans retinoic acid iatRA） on the differentiation of neural stem cells （NSCs） and the expression of c-myc. METHODS： The experiment was conducted at the Department of Megatemperature Medicine, Southern Medical University from March to June 2005. ①basic culture medium： DMEM/F12 culture medium was made up of Hepes （15 mmol/L）, NaHCO3 （2 g/L）, B27 supplement （2%）, penicillin （100 U/mL） and streptomycin （100 U/mL）. NSCs Culture Medium was composed of basic culture medium plus 20 μg/L basic fibroblast growth factor （bFGF）. NSCs Culture Medium was made up of basic culture medium plus fetal bovine serum （FBS） of 0.01 volume fraction, atRA deposited solution was made up of 0.01% dimethylsulfoxide （DMSO） （solvent） and 1.0×10^-3 mol/L atRA. ②Six two or three days neonatal Sprague-Dawley （SD） rats were sacrificed by pulling off the necks under sterile condition. Mechanical isolation and serum-free culture were used to obtain newborn rat striatum NSCs. The third generation cells ware selected for the later experiments. ③The NSCs clone wei＇e inoculated into 12-well culture plate. The atRA disposed group received NSCs Differiative and Inductive Culture Medium plus various concentrations of atRA （0.01,0.1,1.0,10.0 μmol/L）. DMSO control group received NSCs Differiative and Inductive Culture Medium plus 0.01% DMSO. After 7 days, immunohistochemical staining was performed in each group. Differentiation rate of neuron-like cells was tested. Reverse transcriptase polymerase chain reaction （RT-PCR） was used to the expression of c-myc gene, and Western-blots was adopted to test the expression of c-myc protein. RESULTS： ①culture and identification of primary and generative NSCs： Immunotluorescent detection showed the primary and generative clones derived from neonatal rats striatum expressed nestin antigen. The third generation cells cultured in NSCs Differiative and Inductive Culture Medium containing different concentration of atRA expressed neuron-specific enolase （NSE） antigen positive and Glial Fibrillory Acidic Protein （GFAP） antigen positive.②effect of atRA on the differentiation of NSCs： The differentiation percentage of neuron-like cells increased gradually （F = 358.59, P = 0.000） along with the increase of concentrations of atRA （0.01,0.1,1.0,10.0 μmol/L）. There was no significant difference in the differentiation percentage of neuron-like cells between 1.0 μmol/L atRA disposed group and 10.0 μmol/L atRA disposed group [（34.27±0.81 ）%, （35.27±0.32）%,P = 0.163]. ③effect of atRA on the expression of c-myc gene and protein： As compared with the DMSO control group, the expression of c-myc mRNA in atRA disposed group decreased along with the increase of atRA treatment time （F= 20.891 ,P = 0.000）, as well as c-myc protein（F = 43.138, P = 0.000 ）. CONCLUSION： Physiological concentration of atRA （1.0 μ mol/L） is an optimal concentration in the inducing of differentiation of NSCs. The atRA affects the proliferation and differentiation of NSCs by change of c-myc expression.