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目的探讨外源性S100A6对黑色素瘤B16细胞增殖、凋亡的影响及其机制。方法采用MTT、台盼蓝拒染、Hoechst、Western blot、免疫细胞化学、免疫荧光和荧光素酶活性分析法检测S100A6对B16细胞增殖、凋亡、β-catenin的水平和分布、β-catenin/TCF4转录活性及经典Wnt信号途径(即Wnt/β-catenin信号)的下游靶基因c-myc表达的影响。结果 GST-hS100A6可抑制B16细胞的增殖,且呈时间-剂量依赖性;GST-hS100A6作用72 h后,B16细胞的凋亡率较GST对照组增加1.43倍;携带S100A6基因的重组腺病毒AdS100A6作用72 h后,B16细胞中β-catenin的表达量较对照腺病毒AdGFP组增加43.9%;GST-hS100A6可使B16细胞中β-catenin的表达增加,且以胞核增加为主;GST-hS100A6作用后,B16细胞的β-catenin/TCF4转录活性增至GST对照组的7.4倍,且该信号途径的靶基因之一c-myc的表达也增加70.4%。上述结果与GST对照组之间的差异均有统计学意义(P均<0.05或0.01)。结论 S100A6具有抑制黑色素瘤增殖、促进凋亡和上调其Wnt/β-catenin信号途径活性的作用,且上调Wnt/β-catenin信号途径活性可能是S100A6对黑色素瘤抑制性作用的机制之一。
Objective To investigate the effects of exogenous S100A6 on proliferation and apoptosis of melanoma B16 cells and its mechanism. Methods The proliferation, apoptosis, β-catenin level and distribution of S100A6 in B16 cells were detected by MTT, trypan blue exclusion, Hoechst, Western blot, immunocytochemistry, immunofluorescence and luciferase activity assay. TCF4 transcriptional activity and the downstream target gene c-myc expression of the canonical Wnt signaling pathway (ie, Wnt / β-catenin signaling). Results GST-hS100A6 inhibited the proliferation of B16 cells in a time-and dose-dependent manner. After treated with GST-hS100A6 for 72 h, the apoptosis rate of B16 cells was 1.43 times higher than that of GST-hS100A6 cells. The recombinant adenovirus AdS100A6 carrying S100A6 gene After 72 h, the expression of β-catenin in B16 cells was increased by 43.9% compared with the control adenovirus AdGFP group. The expression of β-catenin in B16 cells was increased by GST-hS100A6 with the increase of nucleus; GST-hS100A6 , The β-catenin / TCF4 transcriptional activity of B16 cells was increased to 7.4 times that of the GST control group, and the expression of c-myc, one of the target genes of the signaling pathway, was also increased by 70.4%. The difference between the above results and GST control group was statistically significant (P <0.05 or 0.01). Conclusion S100A6 can inhibit melanoma proliferation, promote apoptosis and up-regulate the activity of Wnt / β-catenin signaling pathway. Up-regulation of Wnt / β-catenin signaling pathway activity may be one of the mechanisms of S100A6 inhibitory effect on melanoma.