姜黄素介导的骨髓间充质干细胞外泌体对ACC-M细胞增殖、迁移和侵袭的影响

doi:10.12439/kqhm.1005-4979.2024.01.005

摘要/Abstract

摘要：

目的：探讨姜黄素介导的骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）外泌体对ACC-M细胞增殖和转移的影响及其作用机制。方法：将7.5 µmmol/L的姜黄素与人BMSCs共培养72 h后，分离外泌体并进行鉴定，将外泌体与ACC-M细胞进行共培养，并将ACC-M细胞分为control组（正常培养）、BMSC-Exo组（未经姜黄素处理的BMSCs外泌体与ACC-M细胞共培养）、Cur-BMSC-Exo组（经姜黄素处理的BMSCs外泌体与ACC-M细胞共培养）及Cur组（经7.5 µmmol/L的姜黄素处理细胞）；培养48 h后使用蛋白质印迹法（Western blotting）实验检测外泌体肿瘤易感基因101（tumor susceptibility gene 101，TSG-101）、CD63、CD9和重组人钙连蛋白(calnexin，CANX)及ACC-M细胞上皮钙黏素（E-cadherin）、神经钙黏素（N-cadherin）、波形蛋白（vimentin）和转化生长因子β1（transforming growth factor-β1，TGF-β1)、细胞外信号调节激酶（extracellular signal-regulated kinase，ERK)的蛋白表达情况；使用细胞计数试剂盒-8（cell counting kit-8，CCK8）实验检测ACC-M细胞存活率；使用transwell实验检测ACC-M细胞迁移和侵袭情况；使用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测ACC-M细胞TGF-β1和ERK mRNA相对表达量。结果：与control组和BMSC-Exo组比较，Cur-BMSC-Exo组和Cur组的细胞活力、细胞迁移和侵袭数量及N-cadherin、vimentin、TGF-β1和ERK 表达水平均显著降低（P<0.05），而E-cadherin蛋白水平显著增加（P<0.05）；与Cur-BMSC-Exo组比较，Cur组细胞活力、细胞迁移和侵袭数量及N-cadherin、vimentin、TGF-β1和ERK 表达水平均显著增加（P<0.05），而E-cadherin蛋白水平显著降低（P<0.05）。结论：BMSCs分泌的外泌体可作为姜黄素的载体，抑制ACC-M细胞的增殖和转移并调控TGF-β1/ERK信号通路。

关键词: 姜黄素, 骨髓间充质干细胞, 外泌体, ACC-M, 增殖

Abstract:

Objective: To investigate the effect of curcumin - mediated exosomes of bone marrow mesenchymal stem cells (BMSCs) on proliferation and metastasis of ACC-M cells and its mechanism. Methods: After being co-cultured with 7.5 μmmol/L curcumin and BMSCs for 72 h, exosomes were isolated and identified, and co-cultured with ACC-M cells. ACC-M cells were divided into control group (normal culture), BMSC-Exo group (no curcumin-treated BMSCs exosomes co-cultured with ACC-M cells), Cur-BMSC-Exo group (curcumin-treated BMSCs exosomes co-cultured with ACC-M cells) and Cur group (7.5 μmmol/L curcumin treated cells). Western blotting was used to detect the expression of tumor susceptibility gene 101 (TSG-101), CD63, CD9 and recombinant human calnexin (CANX) proteins in exosome and the expression of E-cadherin, N-cadherin, vimentin, transforming growth factor-β1 (TGF-β1) and p-ERK proteins in ACC-M cells after 48 h of culture. The survival rate of ACC-M cells was determined by cell counting kit-8 (CCK-8) assay. The migration and invasion of ACC-M cells were detected by transwell assay. The relative expression levels of TGF-β1 and ERK mRNA in ACC-M cells were detected by real-time quantitative polymerase chain reaction(RT-qPCR) assay.Results: Compared with control group and BMSC-Exo group, the cell viability, cell migration and invasion number, and the expression levels of N-cadherin, vimentin, TGF-β1 and ERK were significantly decreased in Cur-BMSC-Exo and Cur groups (P<0.05), while E-cadherin protein level was significantly increased (P<0.05); compared with the Cur-BMSC-Exo group, the cell viability, cell migration and invasion numbers, and the expression levels of N-cadherin, vimentin, TGF-β1 and ERK were significantly increased in the Cur group (P<0.05), while E-cadherin protein level was significantly decreased (P<0.05). Conclusion: BMSCs exosomes can act as curcumin carrier to inhibit proliferation and metastasis of ACC-M cells and regulate TGF-β1/ERK signaling pathway.

Key words: curcumin, bone marrow mesenchymal stem cells, exosome, ACC-M, proliferation

中图分类号:

R782

程昊, 吴发印, 刘智丹. 姜黄素介导的骨髓间充质干细胞外泌体对ACC-M细胞增殖、迁移和侵袭的影响[J]. 《口腔颌面外科杂志》, 2024, 34(1): 34-39.

CHENG Hao, WU Fayin, LIU Zhidan. Curcumin-mediated exosomes from bone marrow mesenchymal stem cells inhibits ACC-M cell proliferation, migration and invasion in vitro[J]. 《Journal of Oral and Maxillofacial Surgery》, 2024, 34(1): 34-39.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2024/V34/I1/34

图/表 8

图1 Western blotting检测外泌体标志物的表达

Figure 1 Expression of exosome markers detected by Western blotting

图2 CCK-8检测培养48 h后ACC-M细胞存活率 *表示P<0.05，与BMSC-Exo组比较；#表示P<0.05，与Cur-BMSC-Exo组比较。

Figure 2 Survival rate of ACC-M cells after 48 h of culture detected by CCK-8 assay

图3 Transwell实验检测各组细胞迁移情况 A.细胞迁移情况染色图（×400）；B.细胞迁移数量统计图。*表示P<0.05，与BMSC-Exo组比较；#表示P<0.05，与Cur-BMSC-Exo组比较。

Figure 3 Cell migration in each group detected by transwell experiment

图4 Transwell实验检测各组细胞侵袭情况 A.细胞侵袭情况染色图（×400）；B.细胞侵袭数量统计图。*表示P<0.05，与BMSC-Exo组比较；#表示P<0.05，与Cur-BMSC-Exo组比较。

Figure 4 Cell invasion in each group detected by transwell experiment

图5 各组细胞迁移和侵袭相关蛋白表达情况

Figure 5 Expression of migration and invasion-related proteins in each group

表1 各组细胞迁移和侵袭相关蛋白表达情况（$\bar{x} \pm s$, n=5）

Table 1 Expression of migration and invasion-related proteins in each group ($\bar{x} \pm s$, n=5)

组别	E-cadherin	N-cadherin	vimentin
control组	0.29±0.04	0.72±0.06	0.89±0.08
BMSC-Exo组	0.32±0.06	0.81±0.08	0.89±0.07
Cur-BMSC-Exo组	0.58±0.05^①	0.58±0.04^①	0.44±0.06^①
Cur组	0.40±0.05^①②	0.70±0.05^①②	0.73±0.06^①②

图6 各组细胞TGF-β1/ERK通路相关蛋白表达情况

Figure 6 Expression of TGF-β1/ERK pathway-related proteins in each group

表2 各组细胞TGF-β1/ERK通路相关基因表达情况（$\bar{x} \pm s$, n=5）

Table 2 Expression of TGF-β1/ERK pathway-related proteins in each group ($\bar{x} \pm s$, n=5)

组别	TGF-β1 mRNA	ERK mRNA	TGF-β1 蛋白	p-ERK 蛋白
control组	1.00±0.09	1.00±0.09	1.02±0.10	0.70±0.07
BMSC-Exo组	0.95±0.10	1.13±0.12	0.98±0.11	0.74±0.06
Cur-BMSC-Exo组	0.53±0.05^①	0.63±0.06^①	0.50±0.06^①	0.37±0.07^①
Cur组	0.75±0.08^①②	0.89±0.10^①②	0.72±0.06^①②	0.55±0.04^①②

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