热休克蛋白B1在U251细胞株及其干细胞中的差异性表达对肿瘤细胞凋亡的影响

Differentially expression of heat shock protein B1 in glioblastoma multiforme U251 cells and stem cells and its effect on tumor cell apoptosis

目的探讨热休克蛋白B1(heat shock protein B1,HSPB1)在U251细胞株及其干细胞中差异性表达对肿瘤细胞凋亡的影响。方法采用无血清培养基培养U251细胞,获得U251干细胞;应用细胞免疫荧光染色法检测CD133和nestin鉴定胶质瘤干细胞;应用iTRAQ技术对U251细胞株及其干细胞进行HSPs差异性分析;应用MTT法、Western blot和RT-PCR观察经卡莫司汀干预后,肿瘤细胞凋亡与HSPB1蛋白、基因表达变化的关系。结果在未经卡莫司汀干预的胶质瘤与胶质瘤干细胞的HSPB1的蛋白比值为0.523;经卡莫司汀干预U251及其干细胞后,HSPB1蛋白含量分别升高了1.4、3.3倍,HSPB1 mRNA分别升高1.5、3.6倍,经相同浓度的卡莫司汀干预后的U251细胞生长受到抑制,而U251干细胞无明显抑制。结论 HSPB1参与U251胶质瘤干细胞化疗抵抗过程,对肿瘤细胞增殖、凋亡起着重要的调控作用。

Objective To determine the effect of differential expression of heat shock protein B1（HSPB1） in glioblastoma multiforme U251 cells and stem cells on tumor cell apoptosis.Methods U251 cells was cultured in a serum-free culture medium to obtain U251 stem cells,which were further identified by immunofluorescence staining of CD133 and of nestin for glioma stem cells.Isobaric tags for relative and absolute quantitation（iTRAQ） technology was used to detect the HSPs difference in U251 cells and stem cells.MTT assay,Western blotting and RT-PCR were employed for the relationship of tumor cell apoptosis with HSPB1 expression at protein and mRNA levels after carmustine intervention.Results Glioma and glioma stem cells showed HSPB1 protein ratio of 0.523 without intervention carmustine.Carmustine intervention increased HSPB1 protein content by 1.4-fold and 3.3-fold,and HSPB1 mRNA by 1.5 times and 3.6 times respectively in U251 and stem cells.Carmustine at the same concentration also inhibited the growth of U251 cells,but not that of U251 stem cells.Conclusion HSPB1 might be involved in the process of chemotherapy resistance in U251 glioma cells,and play an important role in regulating tumor cell proliferation and apoptosis.