枸杞糖肽可减轻放射治疗后人牙龈成纤维细胞来源的外泌体导致的成骨抑制

doi:10.12122/j.issn.1673-4254.2024.09.15

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (9): 1752-1759.doi: 10.12122/j.issn.1673-4254.2024.09.15

枸杞糖肽可减轻放射治疗后人牙龈成纤维细胞来源的外泌体导致的成骨抑制

何思齐¹(), 文楠², 陈勋³, 王跃⁴, 张艇⁴, 牟雁东³()

^1.成飞医院，四川成都 610073
^2.宜宾市第一人民医院，四川宜宾 644000
^3.四川省人民医院口腔科，四川成都 610072
^4.卷烟减害降焦四川省重点实验室，四川成都 610100

收稿日期:2024-01-18 出版日期:2024-09-20 发布日期:2024-09-30
通讯作者: 牟雁东 E-mail:634264913@qq.com;muyd@uestc.edu.cn
作者简介:何思齐，硕士，E-mail: 634264913@qq.com
基金资助:
国家自然科学基金(82071168);四川省科技厅基金(2021YFS0009);卷烟减害降焦四川省重点实验室开放基金资助项目(jykf202205)

Lycium barbarum glycopeptide reduces bone loss caused by exosomes derived from human gingival fibroblasts with radiation exposure

Siqi HE¹(), Nan WEN², Xun CHEN³, Yue WANG⁴, Tin ZHANG⁴, Yandong MU³()

^1.Cheng Fei Hospital, Chengdu 610073, China
^2.The First People's Hospital of Yibin, Yibin 644000, China
^3.Stomatology department, Sichuan Provincial People's Hospital, Chengdu 610072, China
^4.Harmful Components and Tar Reduction in Cigarette Key Laboratory of Sichuan Province, Chengdu 610100, China

Received:2024-01-18 Online:2024-09-20 Published:2024-09-30
Contact: Yandong MU E-mail:634264913@qq.com;muyd@uestc.edu.cn
Supported by:
National Natural Science Foundation of China(82071168)

摘要/Abstract

摘要：

目的研究枸杞糖肽（LbGP）对放射后人牙龈成纤维细胞（HGFs）来源的外泌体对成骨抑制的保护作用。方法将HGFs分为对照组、放射组及枸杞糖肽组，通过RT-qPCR、Western blotting及β-半乳糖苷酶染色检测细胞的凋亡、衰老水平以及α-平滑肌肌动蛋白（α-SMA）的差异表达。提取各组细胞分泌的外泌体，通过电镜观察、粒径分析以及Western blotting鉴定外泌体，并分别将各组牙龈成纤维细胞产生的外泌体作用于破骨前体细胞及骨髓间充质干细胞（BMSCs）中，通过Trap染色、茜素红染色、ALP染色、RT-qPCR以及Western blotting检测细胞的破骨及成骨情况。结果衰老细胞在放射组较对照组增多，而在枸杞糖肽组较放射组减少；与对照组相比，放射组Bcl-2/Bax降低（P<0.0001），α-SMA表达水平增加（P<0.05）；与放射组相比，枸杞糖肽组Bcl-2/Bax增多（P<0.05），α-SMA表达水平降低（P<0.01）；与对照组相比，放射组破骨细胞增多，枸杞糖肽组破骨细胞减少；钙结节、碱性磷酸酶表达水平在放射组低于对照组，在枸杞糖肽组高于放射组；成骨相关基因（BMP2、ALP、RUNX2）及成骨相关蛋白（ALP、RUNX2）的表达水平在枸杞糖肽组高于放射组（P均<0.01）。结论枸杞糖肽可通过作用于放射后的牙龈成纤维细胞的外泌体，有效抑制破骨、促进成骨和预防放射性颌骨骨髓炎的发展，这可能为放射性颌骨骨髓炎的治疗提供一种新策略。

关键词: 枸杞糖肽, 外泌体, 放射性颌骨骨坏死, 成骨分化

Abstract:

Objective To explore the protective effect of Lycium barbanun glycopeptide (LbGP) against osteogenic inhibition induced by exosomes derived from human gingival fibroblasts (HGFs) exposed to radiation. Methods Cultured HGFs with or without LbGP pretreatment were exposed to 8 Gy X-ray radiation, and the changes in cell apoptosis, senescence and α-SMA level were detected using RT-qPCR, Western blotting and β-galactosidase staining. The exosomes secreted by the treated cells were extracted, and after identification by electron microscopy, particle size analysis and Western blotting, the exosomes were added into primary cultured bone mesenchymal stem cells (BMSCs), and osteoclast activity and osteogenesis in the cell cultures were detected by Trap staining, Alizarin red staining, ALP staining, RT-qPCR and Western blotting. Results In cultured HGFs, X-ray radiation significantly increased the percentage of senescent cells, which was obviously lowered by LbGP treatment. X-ray radiation significantly reduced Bcl-2/Bax ratio and increased α-SMA expression in HGFs, and these changes were significantly suppressed by LbGP pretreatment. In rat BMSCs, incubation with the exosomes derived from HGFs with radiation exposure caused a significant increase of osteoclasts, reduced calcium nodules and lowered alkaline phosphatase expression in the cells; The opposite changes were observed in the cells treated with exosomes from LbGP-pretreated HGFs, which also significantly increased the cellular expressions of the osteogenic genes (BMP2, ALP, and RUNX2) and proteins (ALP and RUNX2) as compared with the exosomes from irradiated HGFs. Conclusion LbGP can effectively inhibit osteoclast activity and promote osteogenesis by acting on exosomes secreted by irradiated HGFs, suggesting its potential value for treatment of osteoradionecrosis of the jaw.

Key words: Lycium barbarum glycopeptide, exosomes, osteoradionecrosis of the jaw, osteogenic differentiation

何思齐, 文楠, 陈勋, 王跃, 张艇, 牟雁东. 枸杞糖肽可减轻放射治疗后人牙龈成纤维细胞来源的外泌体导致的成骨抑制[J]. 南方医科大学学报, 2024, 44(9): 1752-1759.

Siqi HE, Nan WEN, Xun CHEN, Yue WANG, Tin ZHANG, Yandong MU. Lycium barbarum glycopeptide reduces bone loss caused by exosomes derived from human gingival fibroblasts with radiation exposure[J]. Journal of Southern Medical University, 2024, 44(9): 1752-1759.

图/表 6

表1 基因引物序列

Tab.1 Primer sequences

Genes

Sequence primers (5'→3')

GAPDH-F

GAPDH-R

BMP2-F

BMP2-R

ALP-F

ALP-R

RUNX2-R

RUNX2-F

CCTCGTCTCATAGACAAGATGGT

GGGTAGAGTCATACTGGAACATG

CTATATGCTCGACCTGTACCG

CACTCATTTCTGAAAGTTCCTCG

CAGAGAAAGAGAAAGACCCCAG

CTGTCACTGTGGAGACGC

AAGAGGGGTAAGACTGGTCATAGG

CAGTATGAGAGTAGGTGTCCCGC

图1 牙龈成纤维细胞及骨髓间充质干细胞的鉴定

Fig.1 Identification of human gingival fibroblasts (HGFs) and rat bone mesenchymal stem cells (BMSCs). A: Morphology of HGFs under optical microscope (a) and immunocytochemical staining showing positive expression of anti-vimentin (b) and negative expression of anti-ck8 (c) in HGFs. B: Morphology of BMSCs under optical microscopy (a) and identification of BMSCs surface markers by flow cytometry (b).

图2 牙龈成纤维细胞的表征

Fig.2 Characterization of HGFs with X-ray radiation and Lycium barbanun glycopeptide (LbGP) treatment. A: β-galactosidase staining. B: Relative protein expression of Bcl-2 and Bax in each group. C: Relative protein expression of α-SMA in each group. *P<0.05,**P<0.01, ****P<0.0001.

图3 牙龈成纤维细胞分泌的外泌体鉴定

Fig.3 Identification of the exosomes derived from HGFs.A: Electron microscopy for observing structure of the exosomes. B: Exosome particle size analysis. C: Exosome concentration distribution map. D: Western blotting for detecting exosomes surface markers CD81 and Tsg101. E: Exosomes under confocal microscope.

图4 破骨及成骨染色结果

Fig.4 Osteoblast and osteogenic staining results of rat BMSCs treated with exosomes from HGFs in different groups. A: Optical microscopy of the osteoclasts and Trap staining. B: Alizarin red staining. C: ALP staining.

图5 骨髓间充质干细胞中成骨相关因子的表达情况

Fig.5 Expression of osteogenesis-related factors in BMSCs treated with exosomes from HGFs in different groups. A: Relative mRNA expression of BMP2, ALP, and RUNX2 in BMSCs in each group. B: Relative protein expressions of RUNX2 and ALP in each group. *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001.

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枸杞糖肽可减轻放射治疗后人牙龈成纤维细胞来源的外泌体导致的成骨抑制

Lycium barbarum glycopeptide reduces bone loss caused by exosomes derived from human gingival fibroblasts with radiation exposure

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