摘要
目的:探讨磷脂酰肌醇3激酶(phosphatidylinositol 3 kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)信号通路在人神经母细胞瘤SK-N-SH(human neuroblastoma,SK-N-SH)细胞增殖和分化中的作用。方法:体外培养SK-N-SH细胞,取对数生长期细胞接种于96孔培养板,将培养板中的细胞分为A组抑制剂LY294002组、B组激动剂类胰岛素生长因子-1(insulin-like growth factors-1,IGF-1)组、C组二甲基亚砜(dimethyl sulfoxide,DMSO)组(LY294002阴性对照组)、D组dd H2O组(IGF-1阴性对照组)和空白组E、F组(培养基组),24 h后A组加入不同浓度的抑制剂LY294002,终浓度为20、40、50、60、100μmol/L,B组换用无血清DMEM继续培养12 h后加入不同浓度激动剂IGF-1,终浓度为25、50、100、150、200 ng/ml,阴性对照组分别加入与LY294002和IGF-1相同浓度的DMSO和dd H2O,空白组为100μl/孔培养基,置于37℃,5%CO2的孵箱中培养,用四甲基偶氮唑盐比色法(MTT)法检测PI3K/AKT特异性抑制剂LY294002和激动剂IGF-1对SK-N-SH细胞增殖影响,倒置相差显微镜下观察细胞形态学分化改变,荧光定量聚合酶链反应(fluorescence quantitative polymerase chain reaction,FQ-PCR)检测SK-N-SH细胞Trk A m RNA的表达与变化。结果:1MTT检测结果显示,LY294002明显抑制SK-N-SH细胞生长,LY294002组(0.39±0.17)细胞增值能力较阴性对照DMSO组(0.78±0.21)减弱,差异有统计学意义(P=0.018)。IGF-1促进SK-N-SH生长,IGF-1组(0.72±0.14)细胞增值能力较阴性对照组(dd H2O组,0.43±0.08)增值能力更强,差异有统计学意义(P=0.004)。2显微镜下观察LY294002组和IGF-1组细胞无明显分化形态学改变。3FQ-PCR结果显示,LY294002组(0.78±0.05)酪氨酸蛋白激酶A(tyrosine kinase receptor A,Trk A)信使核糖核酸(messenger ribonucleic acid,m RNA)的表达与阴性对照组(1.56±0.02)、空白组(1.66±0.15)比较,组间差异有统计学意义(F=81.89,P=0.000)。IGF-1组、dd H2O阴性对照组和空白组3组间的Trk A m RNA的表达水平无统计学差异(F=0.27,P=0.770)。结论:PI3K/AKT信号通路可能参与了SK-N-SH增殖和分化过程,该通路关键基因有望成为临床治疗神经母细胞瘤的分子靶点。
Objectives：To investigate the effects of phosphatidylinositol 3 kinase（PI3K）/protein kinase B（AKT）signal pathway on cell proliferation and differentiation in human neuroblastoma SK-N-SH. Methods：Logarithimic phase SK-N-SH cells were subcultured into96-well plates. The cells in 96-well plates were divided into inhibitor LY294002 group（A）,agonist IGF-1 group（B）,DMSO group（C）,dd H2 O group（D）,blank group（E）and serum group（F）. After 24 h,different concentrations of inhibitor LY294002 were added into group A and the final concentrations were 20,40,50,60,100 μmol/L,respectively. Group B was continued to be cultured with serumfree DMEM for 12 h,then treated with different concentrations（25,50,100,150,200 ng/ml）of agonist IGF-1（which final concentrations were 25,50,100,150,200 ng/ml）. The negative control group was added with DMSO or dd H2 O with the same concentration of LY294002 or IGF-1. The blank group was only added with medium（100 μl/well）. All cells were cultured in appropriate media at 37℃ in humidified,5%CO2incubator. The effects of specific inhibitors LY294002 and PI3K/AKT agonist IGF-1 on cell proliferation were detected by MTT assay. Morphological changes were observed under inverted phase contrast microscope. m RNA expression of Trk A in SK-N-SH cells were detected using fluorescence quantitative polymerase chain reaction（FQ-PCR）. Results：1MTT assay showed that the growth of SK-N-SH cells was dramatically inhibited by LY294002. Cell proliferation ability in LY294002 group（0.39±0.17）was lower than that in negative control DMSO group（0.78±0.21）（F=3.395,P=0.018）. The growth of SK-N-SH cells was promoted by insulin-like growth factors-1（IGF-1）. Cell proliferation ability in IGF-1 group（0.72±0.14）was higher than that in blank dd H2 O group（0.43±0.08）（F=9.480,P=0.004）. 2There was no significant difference in cell differentiation morphology change between LY294002 group and IGF-1 group by microscope observations. 3FQ-PCR results showed that the expression of Trk A m RNA in LY294002 group（0.78±0.05）was lower than that in negative control group（1.56±0.02）and blank group（1.66±0.15）（F=81.89,P=0.000）. There were no statistical difference in the Trk A m RNA expression between negative control group and blank group（P=0.224）. And there were no statistical difference in the Trk A m RNA expression among IGF-1 group,dd H2 O negative control group and blank group（F=0.27,P=0.770）. Conclusion：PI3K/AKT signal pathway may be involved in cell proliferation and differentiation of human neuroblastoma SK-N-SH cells. These findings propose the key gene of PI3K/AKT pathway as an expected molecular target for the therapy of human neuroblastoma.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2015年第8期1089-1094,共6页
Journal of Chongqing Medical University
基金
重庆市卫生局重点课题资助项目(编号:20111069)
国家临床重点专科建设经费资助项目(编号:2013544)
作者简介
雷璐璐，Email：522152570@qq．com，研究方向：儿童实体肿瘤方向。
通信作者：王珊，Email：wangshan778@163．com。
