摘要
目的探讨人干细胞标志Musashi2(Msi2)在佛波酯(phorbol myristoyl acetate,PMA)诱导急性单核白血病细胞株THP-1分化过程中的表达变化。方法 PMA作用THP-1细胞0、6、12、24 h后,倒置显微镜下观察THP-1细胞形态学改变,计算分化细胞百分数;流式细胞仪检测THP-1细胞表面分化抗原CD11b的表达;定量PCR和Western blot检测Msi2的基因及蛋白表达水平;定量PCR检测Msi2下游信号分子Numb的mRNA水平;定量PCR和Western blot检测Numb下游分子Notch1的mRNA和蛋白表达水平。结果 PMA处理24 h后可诱导THP-1细胞出现巨噬细胞样分化表型,实验组分化细胞的百分比显著高于未处理组(P<0.05)。同时24 h组CD11b表达量(59.30±8.70)较未处理组(0.32±0.08)显著增加(P<0.05)。实验组白血病细胞经PMA处理24 h后,Msi2的mRNA和蛋白相对表达量分别为(0.33±0.08)和(0.50±0.01),显著低于未处理组(P<0.05);PMA处理12 h后Numb的mRNA相对表达量为(2.92±0.33),较未处理组明显升高(P<0.05);而PMA处理24 h后Notch1的mRNA和蛋白相对表达量分别为(0.31±0.03)和(0.23±0.01),较未处理组显著下降(P<0.05)。结论干细胞标志Msi2表达随THP-1细胞分化而下降,Msi2-Numb-Notch1通路可能参与白血病细胞的分化调控。
Objective To determine the alteration of human stem cell marker Musashi2（Msi2） in the differentiation of human myelomonocytic leukemia cell line THP-1 induced by phorbol myristoyl acetate（PMA）.Methods After THP-1 cells were incubated with PMA for 0,6,12 and 24 h respectively,the cell morphological changes were observed under an inverted microscope.The proportion of differentiated cells was calculated.The expression of CD11b,which is a THP-1 cell differential surface marker,was assayed using flow cytometry（FCM）.Real-time PCR and Western blotting were applied to detect the mRNA and protein expression of Msi2 respectively.The expression of Msi2 downstream signal molecule Numb was detected by real-time PCR.The mRNA and protein expression of Numb downstream signal molecule Notch1 were detected by real-time PCR and Western blotting respectively.Results The THP-1 cells treated by PMA for 24 h showed differential phenotype of macrophages.The proportion of differentiated cells in the experimental group was significantly higher than that in the untreated group（P0.05）,and the expression of CD11b significantly increased in the 24 h treatment group（59.30±8.70） compared with that in the untreated group（0.32±0.08）（P0.05）.The mRNA and protein relative expression of Msi2 were（0.33±0.08） and（0.50±0.01） in the THP-1 cells treated by PMA for 24 h,respectively,and were significantly lower than that in the untreated group（P0.05）.Numb mRNA relative expression was 2.92±0.33 after PMA treatment for 12 h,and was significantly higher than that in the untreated group（P0.05）.However,when THP-1 cells were treated by PMA for 24 h,the mRNA and protein relative expression of Notch1 were 0.31±0.03 and 0.23±0.01,respectively,which decreased significantly compared with that in the untreated group（P0.05）.Conclusion The expression of stem cell marker Msi2 reduces significantly during THP-1 cell differentiation,indicating Msi2-Numb-Notch1 pathway may regulate the differentiation of leukemia cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2013年第1期34-37,共4页
Journal of Third Military Medical University
基金
重庆市科委自然科学基金(CSTC2010BB5363)~~
