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

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）×10⁸, n=3],与空载质粒组的粒子数[（82.34±4.83）×10⁸, n=3]相比显著升高（P<0.000 1）。17-AAG抑制Hsp90蛋白5天后,17-AAG组的粒子数为[（652.5±26.73）×10⁸, n=3],对照组粒子数为[（262.22±5.44）×10⁸,n=3],差异具有统计学意义（P<0.000 1）。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。

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

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 microscopy (TEM). Western blotting was applied to identify the biological characteristics of sEVs. The number of sEVs particles was detected by nanoparticle tracking analysis (NTA). GraphPad Prism8.0 software was used to analyze the difference in the number of sEVs 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)×10⁸(n=3), while that of vector group was (82.34±4.83)×10⁸(n=3), and the difference was statistically significant (P<0.0001). After 5 days of inhibition with 17-AAG, the sEVs number of 17-AAG group was （652.5±26.73）×10⁸(n=3) and that of control group was (262.22±5.44)×10⁸(n=3), the difference was statistically significant (P<0.000 1). 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.