微管末端结合蛋白1在Siha细胞自噬时的表达和意义

目的 探讨宫颈癌Siha细胞自噬过程中微管末端结合蛋白1（EB1）基因的表达变化。 方法 Hank平衡盐溶液（HBSS）和秋水仙素处理体外培养的宫颈癌Siha细胞，分别采用实时定量PCR和Western blotting检测EB1基因、LC3和p62的表达，荧光显微镜检测特异性绿色荧光蛋白（GFP)-LC3以证实自噬体形成。结果 宫颈癌Siha细胞随HBSS作用时间延长，LC3 mRNA、EB1 mRNA、LC3-Ⅱ蛋白和EB1蛋白的表达均呈时间依赖性增加，差异具有统计学意义(P<0.05)。EB1 mRNA和LC3 mRNA表达呈正相关，具有统计学意义(P<0.05)。p62 mRNA和蛋白在HBSS作用后的表达呈时间依赖性降低，差异具有统计学意义(P<0.05)，EB1 mRNA和p62 mRNA表达呈负相关，具有统计学意义(P<0.05)。GFP-LC3结果显示，在HBSS作用12 h后，Siha细胞胞质中GFP-LC3强度和数目明显增加，含有GFP信号的细胞数量也明显增加。秋水仙素处理后，LC3、p62和EB1 mRNA及蛋白均未发生显著改变，差异无统计学意义(P>0.05)，但此时几乎检测不到含GFP-LC3荧光信号的细胞。 结论 EB1在饥饿状态Siha细胞中高表达，此时伴随LC3表达增加、p62表达降低以及自噬体形成增加，而用秋水仙素破坏EB1赖以作用的微管时，上述现象消失，充分表明EB1参与细胞自噬的可能。

Expressions and implications of end-binding protein 1 in autophagic Siha cells

Objective To explore the expressions and implications of end-binding protein 1(EB1) in autophagic cervical cancer Siha cells. Method Real-time quantitative PCR and Western blotting were introduced to detect the mRNA and protein expressions of EB1, LC3 and p62 in Siha cells treated with Hank’s balanced salt solution (HBSS) or colchicinein vitro. Specific green fluoresent protein(GFP)-LC3 was then be detected to confirm the autophagosome formation by fluorescence microscopy withFlowCellectTMGFP-LC3 reporter autophagy assay kit. Result The mRNA and protein levels of LC3 and EB1 in cervical cancer Siha cells were increased significantly when prolong the duration of HBSS treatment (P<0.05). EB1 mRNA was positively correlated with LC3 mRNA expression (P<0.05). The expressions of p62 mRNA and protein reduced dramatically after HBSS treated (P<0.05), and was negatively related with the EB1 mRNA (P<0.05). The number of cells with GFP-LC3 and intensity of GFP-LC3 per cells were increased when starvation for 12 hours. However, the mRNA and protein levels of LC3, p62 and EB1 were not significantly changed when the cells treated with colchicine (P>0.05), and we cannot detected the fluoresce signaling of GFP-LC3 in cells at this moment. Conclusion The expression of EB1 in starved cells is dramatically increased, and accompanied with the increased expression of LC3 and autophagosome, decreased expression of p62. However, when microtubules with which EB1 interaction were destructed by colchicine, the above phenomenon disappears. All these results fully indicate the probably roles of EB1 in autophagy.