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

doi:10.19439/j.sjos.2022.02.001

上海口腔医学 ›› 2022, Vol. 31 ›› Issue (2): 113-119.doi: 10.19439/j.sjos.2022.02.001

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

陈俊^1,2, 孙凯², 潘蕾^2,3, 杜观环², 宋晨成², 陈俊俊², 杨成龙^1,2, 王宇峰^2,*, 唐国瑶^2,*

1.潍坊医学院口腔医学院,山东潍坊 261021;
2.上海交通大学医学院附属第九人民医院口腔黏膜病科,上海交通大学口腔医学院,国家口腔医学中心,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海200011;
3.上海交通大学医学院附属第九人民医院口腔第二门诊部,上海交通大学口腔医学院,上海 201900

收稿日期:2021-05-28 修回日期:2021-09-10 出版日期:2022-04-25 发布日期:2022-05-16
通讯作者: 唐国瑶,E-mail：tanggy@shsmu.edu.cn;王宇峰,E-mail：dr_wangyufeng@126.com。^*共同通信作者
作者简介:陈俊（1992-）,男,在读硕士研究生,E-mail：825800150@qq.com
基金资助:
国家自然科学基金（81970937,81730030）

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

CHEN Jun^1,2, SUN Kai², PAN Lei^2,3, DU Guan-huan², SONG Chen-cheng², CHEN Jun-jun², YANG Cheng-long^1,2, WANG Yu-feng², TANG Guo-yao²

1. School of Stomatology, Weifang Medical University. Weifang 261021, Shandong Province;
2. Department of Oral Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology. Shanghai 200011;
3. Department of 2nd Dental Center, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine. Shanghai 201900, China

Received:2021-05-28 Revised:2021-09-10 Online:2022-04-25 Published:2022-05-16

摘要/Abstract

摘要： 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。

关键词: 角质形成细胞, 热休克蛋白90, 小细胞外囊泡, 超速离心

Abstract: 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.

Key words: Keratinocytes, Heat shock protein 90, Small extracellular vesicles, Ultracentrifugation

中图分类号:

R781.5

陈俊, 孙凯, 潘蕾, 杜观环, 宋晨成, 陈俊俊, 杨成龙, 王宇峰, 唐国瑶. 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[J]. 上海口腔医学, 2022, 31(2): 113-119.

CHEN Jun, SUN Kai, PAN Lei, DU Guan-huan, SONG Chen-cheng, CHEN Jun-jun, YANG Cheng-long, WANG Yu-feng, TANG Guo-yao. Correlation between low expression of Hsp90 protein in keratinocytes and the number of small extracellular vesicles and its potential clinical significance[J]. Shanghai Journal of Stomatology, 2022, 31(2): 113-119.

参考文献

[1] Feng J, Zhou Z, Shen X, et al.Prevalence and distribution of oral mucosal lesions: a cross-sectional study in Shanghai, China[J]. J Oral Pathol Med, 2015, 44(7): 490-494.
[2] Kurago ZB.Etiology and pathogenesis of oral lichen planus: an overview[J]. Oral Surg Oral Med Oral Pathol Oral Radiol, 2016, 122(1): 72-80.
[3] 李晶晶, 宋晨成, 李晨曦, 等. 口腔苔藓样损害中淋巴滤泡样结构表征及免疫细胞组成的实验研究[J]. 上海口腔医学, 2021, 30(2): 113-119.
[4] Abels ER, Breakefield XO.Introduction to extracellular vesicles: biogenesis, RNA cargo selection, content, release, and uptake[J]. Cell Mol Neurobiol, 2016, 36(3): 301-312.
[5] Théry C, Witwer KW, Aikawa E, et al.Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[J]. J Extracell Vesicles, 2018, 7(1): 1535750.
[6] Kalluri R, LeBleu VS. The biology, function,biomedical applications of exosomes[J]. Science, 2020, 367(6478): eaau6977.
[7] Sjöqvist S, Imafuku A, Gupta D, et al.Isolation and characterization of extracellular vesicles from keratinocyte cultures[J]. Methods Mol Biol, 2020, 2109: 35-44.
[8] Lauwers E, Wang YC, Gallardo R, et al. Hsp90 mediates membrane deformation and exosome release[J]. Mol Cell, 2018, 71(5): 689-702. e9.
[9] Chaiyarit P, Jintakanon D, Klanrit P, et al.Immunohistochemical analyses of survivin and heat shock protein 90 expression in patients with oral lichen planus[J]. J Oral Pathol Med, 2009, 38(1): 55-62.
[10] Madrigal-Matute J, López-Franco O, Blanco-Colio LM, et al.Heat shock protein 90 inhibitors attenuate inflammatory responses in atherosclerosis[J]. Cardiovasc Res, 2010, 86(2): 330-337.
[11] Datta R, Bansal T, Rana S, et al.Myocyte-derived Hsp90 modulates collagen upregulation via biphasic activation of STAT-3 in fibroblasts during cardiac hypertrophy[J]. Mol Cell Biol, 2017, 37(6): e006611-16.
[12] Lv J, Zhou D, Wang Y, et al.Effects of luteolin on treatment of psoriasis by repressing HSP90[J]. Int Immunopharmacol, 2020, 79: 106070.
[13] Shao H, Im H, Castro CM, et al.New technologies for analysis of extracellular vesicles[J]. Chem Rev, 2018, 118(4): 1917-1950.
[14] Lane RE, Korbie D, Trau M, et al.Purification protocols for extracellular vesicles[J]. Methods Mol Biol, 2017, 1660: 111-130.
[15] Rashid S, Lee BL, Wajda B, et al.Nucleotide binding and active site gate dynamics for the Hsp90 chaperone ATPase domain from benchtop and high field (19)F NMR spectroscopy[J]. J Phys Chem B, 2020, 124(15): 2984-2993.
[16] Fucikova J, Truxova I, Hensler M, et al.Calreticulin exposure by malignant blasts correlates with robust anticancer immunity and improved clinical outcome in AML patients[J]. Blood, 2016, 128(26): 3113-3124.
[17] Taha EA, Ono K, Eguchi T.Roles of extracellular HSPs as biomarkers in immune surveillance and immune evasion[J]. Int J Mol Sci, 2019, 20(18): 4588-4600.
[18] Büth H, Wolters B, Hartwig B, et al.HaCaT keratinocytes secrete lysosomal cysteine proteinases during migration[J]. Eur J Cell Biol, 2004, 83(11-12): 781-795.
[19] Jeppesen DK, Fenix AM, Franklin JL, et al. Reassessment of exosome composition[J]. Cell, 2019, 177(2): 428-445.e18.
[20] Than UTT, Leavesley DI, Parker TJ.Characteristics and roles of extracellular vesicles released by epidermal keratinocytes[J]. J Eur Acad Dermatol Venereol, 2019, 33(12): 2264-2272.
[21] Peng Q, Zhang J, Zhou G.Circulating exosomes regulate T-cell-mediated inflammatory response in oral lichen planus[J]. J Oral Pathol Med, 2019, 48(2): 143-150.
[22] Joshi BS, de Beer MA, Giepmans BNG, et al. Endocytosis of extracellular vesicles and release of their cargo from endosomes[J]. ACS Nano, 2020, 14(4): 4444-4455.
[23] Ostrowski M, Carmo NB, Krumeich S, et al.Rab27a and Rab27b control different steps of the exosome secretion pathway[J]. Nat Cell Biol, 2010, 12(1): 19-30.

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

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