促自噬剂AZD8055对U87起始细胞Notch通路相关蛋白的表达和成球能力的影响

The effect of autophagy inducer AZD8055 on expression of Notch pathway-related proteins and neurosphere formation in U87 initiating cells

目的研究促自噬剂AZD8055对U87细胞株起始细胞Notch通路相关蛋白的表达和成球能力的影响。方法应用免疫磁珠法分选获得U87细胞株CD133阳性的胶质瘤起始细胞（GICs）。采用免疫荧光染色技术鉴定GICs细胞球干性标志物CDl33和Nestin，以及自噬相关蛋白LC3B和p62的表达。以CCK8检测AZD8055对U87起始细胞存活率的影响。采用Western blot检测AZD8055不同浓度处理组Notch1、Notch胞内域、Hes1、p62、LC3B蛋白的表达。通过单细胞成球实验观察AZD8055对GICs成球能力的影响。结果免疫荧光染色结果显示，GICs细胞球中CD133和Nestin呈阳性表达。CCK8检测结果提示，随着AZD8055浓度的增高，U87起始细胞的存活率逐渐降低。Western blot结果提示，与溶媒对照组相比，随着AZD8055浓度的升高，药物处理组的自噬水平增高，而Notchl通路相关蛋白的表达降低，且各浓度组之间的蛋白表达差异均有统计学意义（均P〈0．05）。免疫荧光染色结果提示，经AZD8055处理后U87起始细胞的LC3B表达明显升高，p62表达降低。单细胞成球实验显示，随着AZD8055药物浓度的增高，各组成球数目和细胞球体积递减，且差异有统计学意义（均P〈0．05），说明U87GICs的成球能力降低。结论AZD8055通过诱发自噬抑制了Notch通路相关蛋白的表达，并且AZD8055能够抑制U87GICs的成球能力。

Objective To investigate the effect of autophagy inducer AZD8055 on expression of Notch pathway-related proteins and neurosphere formation in U87 initiating ceils. Methods CD133- positive glioma cells were isolated using magnetic activated cell sorting method. The expression of stem cell markers （CD133 and Nestin） and autophagy related proteins （LC3B and p62 ） were identified via immunofluorescence analysis in neurospheres of glioma-initiating cells （GICs）. The effect of AZD8055 on the survival viability of U87 initiating ceils was detected via CCK8 kit. The expression of Notchl, Notch intracellular domain, Hesl, p62, LC3B were observed using Western blot with different concentrations of AZD8055. The effect of AZD8055 on the ability of neurosphere formation was detected by single-cell neurosphere formation assay. Results High expression of CD133 and Nestin was identified via immunofluorescence analysis in GICs＇ neurospheres. CCK8 results showed that the increase of AZD8055 concentration was correlated with decrease of cell survival rate of U87 initiating cells. Compared with dissolvent group, the experimental groups had elevated level of autophagy and decreased expression of Notchl pathway-related proteins assessed by Western blot. The difference in protein expression between each concentration group had a dose-dependent manner （ all P 〈 0.05 ）. Immunofluorescence analysis indicated that autophagy related protein LC3B was highly expressed while p62 was down-regulated by AZD8055 treatment. In addition, with the increased drug concentrations, the number and capacity of neurospheres were decreased detected by single-cell neurosphere formation assay （ all P 〈 0.05 ） which suggested that the ability of neurosphere formation was inhibited. Conclusion AZD8055 could suppress the expression of Notch pathway-related proteins by inducing autophagy and inhibit the ability of neurosphere formation in U87 initiating cells.