角质形成细胞低表达Hsp90蛋白与小细胞外囊泡数量的相关性及其临床意义探讨

Correlation between low expression of Hsp90 protein in keratinocytes and the number of small extracellular vesicles and its potential clinical significance

目的:评价角质形成细胞中热休克蛋白90(heat shock protein 90,Hsp90)表达水平与小细胞外囊泡(small extracellular vesicles,sEVs)数量的关系。方法:采用人角质形成细胞株(HaCaT),分为野生型组、短发夹RNA干扰组(shRNA组,Hsp90蛋白抑制剂)和格尔德霉素抑制组(17-AAG组,Hsp90低表达),进行体外培养。以超速离心法收集各组培养体系中的sEVs,利用透射电镜观察其形态学特征;蛋白质免疫印迹法鉴定生物学特性;纳米颗粒跟踪分析检测粒子数量。采用GraphPad Prism 8.0软件包中的t检验(非参数Mann-Whitney U检验)统计分析各组之间sEVs的数量差异。结果:超速离心法获得的HaCaT细胞来源的sEVs符合形态学和生物学鉴定标准,sEVs中Hsp90蛋白无明显表达。shRNA干扰角质形成细胞中的Hsp90AA1表达后,其sEVs数量上升。第5天时,shRNA干扰组的sEVs粒子数为[(177.4±4.18)×10^(8),n=3],与空载质粒组的粒子数[(82.34±4.83)×10^(8),n=3]相比显著升高(P<0.0001)。17-AAG抑制Hsp90蛋白5天后,17-AAG组的粒子数为[(652.5±26.73)×10^(8),n=3],对照组粒子数为[(262.22±5.44)×10^(8),n=3],差异具有统计学意义(P<0.0001)。结论:低表达Hsp90蛋白可促进HaCaT细胞分泌sEVs,sEVs可能与炎症状态下上皮细胞和免疫细胞间的物质传递有关。

PURPOSE:To investigate the correlation between the level of heat shock protein 90(Hsp90)and the amount of small extracellular vesicles(sEVs)in keratinocytes.METHODS:Human keratinocytes(HaCaT)were cultured in vivo and divided into wild-type group,short hairpin RNA interference group(shRNA group,low expression of Hsp90),and 17-Allylamino-17-demethoxygeldanamycin group(17-AAG group,Hsp90 protein inhibitor).sEVs were isolated from culture system by ultracentrifugation,and their morphological characteristics were observed under transmission electron mi-croscopy(TEM).Western blotting was applied to identify the biological characteristics of sEVs.The number of sEVs parti-cles was detected by nanoparticle tracking analysis(NTA).GraphPad Prism8.0 software was used to analyze the difference in the number of s EVs among the groups by t test(non-parametric Mann-Whitney U test).RESULTS:HaCaT-derived sEVs,obtained by ultracentrifugation,were consistent with the criteria of morphological and biological identification.No expression of Hsp90 protein was detected in HaCaT-derived sEVs.When interfered with Hsp90-shRNA,the number of sEVs were significantly increased.On day 5,the sEVs number of shRNA-interfering group was(177.4±4.18)×10^(8)(n=3),while that of vector group was(82.34±4.83)×10^(8)(n=3),and the difference was statistically significant(P<0.0001).After 5days of inhibition with 17-AAG,the sEVs number of 17-AAG group was(652.5±26.73)×10^(8)(n=3)and that of control group was(262.22±5.44)×10^(8)(n=3),the difference was statistically significant(P<0.0001).CONCLUSIONS:Low expression of Hsp90 protein can promote the secretion of sEVs in HaCaT cells.sEVs may be involved in the transfer of molecules between epithelial cells and immune cells.