文献类型：期刊文献

中文题名：Wnt/β-catenin信号通路介导红景天苷诱导间骨髓充质干细胞向神经细胞定向分化研究

英文题名：Wnt/β-catenin Signal Pathway Mediated Salidroside Induced Directional Differentiation from Mouse Mesenchymal Stem Cells to Nerve Cells

作者：郭超[1];刘润[1];赵红斌[2];秦冠华[1]

第一作者：郭超

机构：[1]甘肃中医学院基础医学院组织胚胎学教研室,兰州730000;[2]兰州军区兰州总医院骨科研究所,兰州730050

第一机构：甘肃中医药大学基础医学院（敦煌医学研究所）

年份：2015

卷号：35

期号：3

起止页码：349

中文期刊名：中国中西医结合杂志

外文期刊名：Chinese Journal of Integrated Traditional and Western Medicine

收录：MEDLINE(收录号：25951643);CSTPCD;;Scopus;北大核心:【北大核心2014】；CSCD:【CSCD2015_2016】；PubMed;

基金：国家自然科学基金面上项目(No.81073156)

语种：中文

中文关键词：红景天苷;骨髓间充质干细胞;Wnt/β-catenin信号通路;神经细胞

外文关键词：Salidroside; mesenchymal stem cell; Wnt/β-catenin signal pathway ; neuron cell

摘要：目的探讨红景天苷诱导小鼠骨髓间充质干细胞(mesenchyma stem cells,MSCs)向神经元细胞定向分化的分子机制。方法以小鼠MSCs系D1细胞为研究对象,实验分为阴性对照组(完全培养液)、阳性对照组(含1 mmol/Lβ-巯基乙醇)、红景天苷组(20 mg/L红景天苷)和阻断组(20 ng/m L DKK1);1×104个/孔细胞接种于6孔板24 h后分组,荧光免疫化学方法观察阴性对照组、阳性对照组和红景天苷组中β-链蛋白(β-catenin)的表达;RT-PCR法检测阴性对照组、阳性对照组、红景天苷组中神经元特异性烯醇化酶(neuronspecific enolase,NSE)、抗微管蛋白(beta 3 classⅢtubulin,β-tubulinⅢ)、孤儿核受体相关因子1(nuclear receptor related factor1,Nurr1)、神经胶质原纤维酸性蛋白质(glial fibrillary acidic protein,GFAP)mRNA和阻断组Wnt3a、β-catenin、低密度脂蛋白受体相关蛋白6(low-density lipoprotein receptor-related protein6,LRP6)、轴蛋白(Axin)mRNA的表达;Western blot分析阴性对照组、阳性对照组和红景天苷组中β-catenin和NSE蛋白的表达。利用胞外Ca2+螯合剂乙二醇双四乙酸[EGTA(胞外Ca2+螯合剂)]、细胞膜Ca2+通道阻断剂硝苯地平(Nifedipine细胞膜Ca2+通道阻断剂)和LY294002(IP3Ks特异抑制剂)分别阻断细胞不同Ca2+信号阻断剂,RT-PCR检测红景天苷组和阻断组中Wnt3a、LRP-6、Axin、糖原合成酶激酶3(glycogen synthase kinase,GSK-3)和β-catenin mRNA的表达;Western blot法分析β-catenin蛋白的表达。结果与阳性对照组比较,红景天苷组β-catenin为强阳性表达,Wnt3a、β-catenin、LRP6、Axin mRNA表达明显上调(P<0.01);NSE、β-tubulinⅢ、Nurr1 mRNA和NSE蛋白表达上调(P<0.01);与阳性对照组和红景天苷组比较,阻断组β-catenin、NSE、Nurr1和β-tubulinⅢmRNA表达降低,β-catenin和NSE蛋白表达下调(P<0.01)。与红景天苷组比较,尼莫地平组、EGTA、LY294002组Wnt3a、LRP-6、β-catenin和Axin mRNA及蛋白表达降低,β-catenin蛋白表达降低(P<0.05,P<0.01)。结论红景天苷通过Wnt/β-catenin和Ca2+信号通路影响MSCs向神经元细胞定向分化。
Objective To explore the molecule mechanism of Salidroside inducing directional dif- ferentiation of mouse mesenchymal stem ceils （MSCs） into neuronal ceils. Methods The mouse multi- potent mesenchymal precursor cell line （D1） was taken as the objective. Cultured MSCs were divided into the negative control group （complete culture solution）, the positive control group （containing 1 mmol/L β- mercaptoethanol）, the Salidroside induced group （20 mg/L Salidroside）, and the blocked group （20 ng/ ml DKK1, a special inhibitor of Wnt/β-catenin signal pathway）. All cells were inoculated in a 6-well plate （1×10^4cells/cm^2） and grouped for 24 h. The expression of β-catenin was detected by fluorescence Im- munochemistry in the negative control group, the positive control group, and the Salidroside induced group. The expression of neuron-specific enolase （ NSE）, beta 3 class II tubulin （ β-tubulin III ）, nuclear receptor related factor 1 （ Nurrl ）, glial fibrillary acidic protein （GFAP） mRNA, Wnt3a, β-catenin, low- density lipoprotein receptor-related protein6 （LRP6）, Axin mRNA were detected using reverse transcrip- tion PCR （RT-PCR）. The expression of β-catenin and NSE protein were analyzed by Western blot in the negative control group, the positive control group, and the Salidroside induced group. Ca^2＋ chelating a- gents （EGTA）, L-type Ca^2＋ channel blocker （Nifedpine）, and IPsKs special inhibitor （LY294002） were used to block Ca^2＋ signal pathway respectively. The expression of Wnt3a, LRP-6, Axin, glycogen syn- thase kinase （GSK-3）, and β-catenin mRNA were detected by RT-PCR. The β-catenin protein expression was analyzed using Western blot. Results Compared with the positive control group, β-catenin protein was strong positively expressed; the expression of Wnt3a,β-catenin, LRP6, Axin, NSE, β-tubulinⅢ, Nurrl mRNA, and NSE protein were obviously up-regulated in the Salidroside induced group （P 〈0.01 ）. Compared with the positive control group and the Salidroside induced group, β-catenin, NSE, Nurrl, and 13-tubulinⅢ mRNA expression decreased; β-catenin and NSE protein expression were also down-regula- ted in the blocked group （P 〈0.01 ）. Compared with the Salidroside induced group, the expression of Wnt3a, LRP-6, β-catenin, and Axin mRNA were down-regulated in the Ca^2＋ signal blocked group and the salidroside induced group （P 〈0.01, P 〈0.05）. Conclusion Salidroside affected directional differentia- tion of MSCs into neuronal cells through Wnt/β-catenin and Ca^2＋ signal pathway.