口腔癌细胞Cal27外泌体对人正常牙龈成纤维细胞的生物学作用

doi:10.7518/hxkq.2021.03.011

华西口腔医学杂志 ›› 2021, Vol. 39 ›› Issue (3): 313-319.doi: 10.7518/hxkq.2021.03.011

口腔癌细胞Cal27外泌体对人正常牙龈成纤维细胞的生物学作用

贾美娥¹(), 李志勇², 徐凯¹, 王怡衡¹, 于菲¹, 何祥一¹()

^1.兰州大学口腔医学院修复科，兰州 730000
^2.中国农业科学院兰州兽医研究所，兰州 730000

收稿日期:2020-04-15 修回日期:2021-02-19 出版日期:2021-06-01 发布日期:2021-05-26
通讯作者: 何祥一 E-mail:jiame12@lzu.edu.cn;hexy@lzu.edu.cn
作者简介:贾美娥，硕士，E-mail：jiame12@lzu.edu.cn
基金资助:
国家自然科学基金(31572522);兰州大学口腔医学科研扶持基金(lzukqky-2019-t01)

Biological effects of exosome derived from Cal27 on normal human gingival fibroblasts

Jia Mei’e¹(), Li Zhiyong², Xu Kai¹, Wang Yiheng¹, Yu Fei¹, He Xiangyi¹()

^1.Dept. of Prosthodontics, School of Stomatology, Lanzhou University, Lanzhou 730000, China
^2.Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China

Received:2020-04-15 Revised:2021-02-19 Online:2021-06-01 Published:2021-05-26
Contact: He Xiangyi E-mail:jiame12@lzu.edu.cn;hexy@lzu.edu.cn
Supported by:
The National Natural Science Foundation of China(31572522);Scientific Research Foundation of School of Stomatology, Lanzhou University(lzukqky-2019-t01)

摘要/Abstract

摘要： 目的

通过将Cal27外泌体（Cal27-exo）与正常人牙龈成纤维细胞（NHGFs）共培养，测定共培养后NHGFs+Cal27-exo的增殖迁移能力及相关蛋白表达情况，探讨Cal27-exo对NHGFs的活化及生物学行为的影响。

方法

采用超速离心法提取Cal27-exo，并用蛋白免疫印迹（Western blot）、透射电子显微镜以及粒径分析对外泌体进行鉴定。将Cal27-exo与NHGFs共培养，检测NHGFs对Cal27-exo的摄取情况，用划痕实验和CCK8检测NHGFs+Cal27-exo的迁移和增殖能力，用实时荧光定量聚合酶链反应检测NHGFs活化相关蛋白金属基质蛋白酶9（MMP-9）、成纤维细胞活化蛋白（FAP）、α平滑肌肌动蛋白（αSMA）和转化生长因子β（TGF-β）的mRNA表达。

结果

超速离心法提取的Cal27-exo中Alix和CD63表达阳性；透射电子显微镜示Cal27-exo呈类圆形双层膜性囊泡；粒径检测示Cal27-exo直径为30~150 nm。共培养后Cal27-exo进入NHGFs内，划痕实验结果示NHGFs+Cal27-exo的迁移能力强于NHGFs，CCK8结果示NHGFs+Cal27-exo的增殖活力强于NHGFs。实时荧光定量聚合酶链反应结果显示，与NHGFs相比，NHGFs+Cal27-exo的MMP-9表达上调，TGF-β、αSMA mRNA表达下调（P<0.05）。

结论

NHGFs+Cal27-exo的增殖活性和迁移能力明显增强，相关蛋白的mRNA水平改变。Cal27-exo能活化NHGFs，对肿瘤的侵袭转移有潜在意义。

关键词: 口腔癌, 外泌体, 细胞增殖, 细胞迁移, 成纤维细胞

Abstract: Objective

The proliferation, migration capacity, and expression of activation-related proteins of NHGFs+Cal27-exo were determined by coculturing Cal27 exosome (Cal27-exo) with normal human gingival fibroblasts (NHGFs) to explore the effects of Cal27-exo on the activation and biological behavior of NHGFs.

Methods

Cal27-exo was extracted using supercentrifugation, and exosomes were identified using Western blot, transmission electron microscopy (TEM), and particle size detection. Cal27-exo was cocultured with NHGFs to detect the uptake of Cal27-exo by NHGFs, and the proliferation and migration capacity of NHGFs+Cal27-exo were detected using CCK8 and wound healing tests, respectively. The expression levels of NHGF activation-related proteins, i.e., matrix metalloproteinase-9 (MMP-9), fibroblast-activating protein (FAP), alpha smooth muscle actin (αSMA), and transforming growth factor-β (TGF-β), were detected using real-time quantitative polymerase chain reaction (qRT-PCR).

Results

Cal27-exo was extracted u-sing supercentrifugation, and Western blot showed the positive expression levels of Alix and CD63. TEM showed that Cal27-exo had a circular double-layer vesicle. The particle size was between 30 and 150 nm. Cal27-exo labeled with PKH67 entered NHGFs after the coculture method. The wound healing test showed that the migration capacity of NHGFs+Cal27-exo was stronger after the scratch compared with that of NHGFs. CCK8 results showed that the proliferation activity of NHGFs+Cal27-exo was enhanced. qRT-PCR results showed that the MMP-9 levels of NHGFs+Cal27-exo were upregulated, whereas the TGF-β and αSMA mRNA levels of NHGFs+Cal27-exo were downregulated (P<0.05).

Conclusion

The proliferation and migration ability of NHGFs+Cal27-exo are enhanced, and the mRNA expression of related proteins is changed. Cal27-exo can activate NHGFs, which suggests that Cal27-exo has potential significance in tumor invasion and metastasis.

Key words: oral cancer, exosome, cell proliferation, cell migration, fibroblast

中图分类号:

R 739.8

贾美娥, 李志勇, 徐凯, 王怡衡, 于菲, 何祥一. 口腔癌细胞Cal27外泌体对人正常牙龈成纤维细胞的生物学作用[J]. 华西口腔医学杂志, 2021, 39(3): 313-319.

Jia Mei’e, Li Zhiyong, Xu Kai, Wang Yiheng, Yu Fei, He Xiangyi. Biological effects of exosome derived from Cal27 on normal human gingival fibroblasts[J]. West China Journal of Stomatology, 2021, 39(3): 313-319.

图/表 6

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口腔癌细胞Cal27外泌体对人正常牙龈成纤维细胞的生物学作用

Biological effects of exosome derived from Cal27 on normal human gingival fibroblasts

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