芦丁对骨肉瘤生长和转移的体内外抑制作用

doi:10.3969/j.issn.1674-8115.2025.01.003

摘要/Abstract

摘要：

目的·探讨芦丁对骨肉瘤细胞增殖、凋亡、迁移和侵袭的影响及其可能的分子机制。方法·分别用10、20、40 μmol/L芦丁处理人骨肉瘤MG63和U2OS细胞，采用CCK-8法、集落形成实验、流式细胞术、划痕闭合实验和Transwell实验检测芦丁对骨肉瘤MG63和U2OS细胞增殖、凋亡、迁移和侵袭的影响；Western blotting检测细胞增殖抗原Ki67、B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）和Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）的表达水平。取12只BALB/c裸鼠，皮下注射骨肉瘤MG63细胞，构建裸鼠皮下移植瘤模型，并将其随机分为2组（每组6只）：对照组和芦丁40 mg/kg组。芦丁40 mg/kg组腹腔注射芦丁（40 mg/kg），对照组腹腔注射等体积生理盐水，隔日1次，持续4周。每周测量肿瘤体积，4周后处死小鼠取出肿瘤称质量，免疫组织化学检测肿瘤组织中Ki67和血管内皮生长因子（vascular endothelial growth factor，VEGF）的表达，TUNEL检测瘤组织细胞凋亡。结果·与芦丁未处理MG63和U2OS细胞相比，经芦丁20、40 μmol/L处理的MG63和U2OS细胞的增殖率显著降低，凋亡率升高，迁移和侵袭能力降低，且Ki67蛋白显著下调，Bax/Bcl-2比值显著升高，差异有统计学意义（均P<0.05）。此外，芦丁显著抑制骨肉瘤细胞的体内生长，降低肿瘤组织中Ki67和VEGF的表达，促进细胞凋亡（均P<0.05）。结论·芦丁可以抑制骨肉瘤细胞的增殖、迁移、侵袭，并促进细胞凋亡。

关键词: 芦丁, 骨肉瘤, 增殖, 侵袭, 细胞增殖抗原Ki67

Abstract:

Objective ·To investigate the effects of rutin on proliferation, apoptosis, migration and invasion of osteosarcoma cells and its possible molecular mechanisms. Methods ·Human osteosarcoma MG63 and U2OS cells were treated with rutin at concentrations of 10, 20 and 40 μmol/L, respectively. The effects of rutin on proliferation, apoptosis, migration, and invasion of MG63 and U2OS cells were assessed by using CCK-8 assay, colony formation assay, flow cytometry, scratch closure assay, and Transwell assay. The expression levels of cell proliferation antigen Ki67, B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax) proteins were detected by Western blotting. Twelve BALB/c nude mice were subcutaneously injected with osteosarcoma MG63 cells to establish a subcutaneous transplant tumor model. The mice were randomly divided into two groups: a control group and a rutin 40 mg/kg group (6 mice in each group). The rutin 40 mg/kg group was intraperitoneally injected with rutin (40 mg/kg), and the control group was intraperitoneally injected with an equal volume of saline, once every other day for 4 weeks. The tumor volume was measured every week. After 4 weeks, the mice were euthanized, and the tumors were excised and weighed. Immunohistochemistry was used to detect the expression of Ki67 and vascular endothelial growth factor (VEGF) in tumor tissues. TUNEL was used to detect tumor cell apoptosis. Results ·Compared with MG63 and U2OS cells not treated with rutin, MG63 and U2OS cells treated with rutin at 20 and 40 μmol/L showed a significant decrease in proliferation rate, an increase in apoptotic rate, a decrease in migration and invasion abilities, a significant downregulation of Ki67 protein, and a significant increase in Bax/Bcl-2 ratio, with statistically significant differences (all P<0.05). In addition, rutin significantly inhibited the in vivo growth of osteosarcoma cells, reduced the expression of Ki67 and VEGF in tumor tissues, and promoted cell apoptosis (all P<0.05). Conclusion ·Rutin can inhibit the proliferation, migration, and invasion of osteosarcoma cells, and promote apoptosis.

Key words: rutin, osteosarcoma, proliferation, invasion, cell proliferation antigen Ki67

中图分类号:

R738.1

李想, 魏鸣, 吴文曦, 罗小琴, 姚彪, 伍思宇. 芦丁对骨肉瘤生长和转移的体内外抑制作用[J]. 上海交通大学学报（医学版）, 2025, 45(1): 20-28.

LI Xiang, WEI Ming, WU Wenxi, LUO Xiaoqin, YAO Biao, WU Siyu. Inhibitory effect of rutin on the growth and metastasis of osteosarcoma in vitro and in vivo[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(1): 20-28.

图/表 7

图1 芦丁对骨肉瘤细胞活力的影响Note： A. Viability of MG63 cells. B. Viability of U2OS cells. C. Viability of hFOB1.19 cells. ①P=0.002, ②P=0.001, ③P<0.001, ④P=0.003, compared with rutin 0 μmol/L group.

Fig 1 Effect of rutin on the viability of osteosarcoma cells

图2 芦丁对骨肉瘤细胞克隆形成的影响Note：A. Representative images of MG63 cell clones and statistics of colony formation rate. B. Representative images of U2OS cell clones and statistics of colony formation rate. C. Western blotting was used to detect the expression of Ki67 protein, and the relative expression of Ki67 in MG63 and U2OS cells was analyzed. ①P=0.005, ②P<0.001, ③P=0.019, compared with the rutin 0 μmol/L group.

Fig 2 Effect of rutin on the colony formation of osteosarcoma cells

图3 芦丁对骨肉瘤细胞凋亡的影响Note： A. Apoptosis was detected by flow cytometry and the apoptosis rate of MG63 cells was analyzed. B. Apoptosis was detected by flow cytometry and the apoptosis rate of MG63 and U2OS cells was analyzed. C. The expression of Bax and Bcl-2 proteins was detected by Western blotting and the Bax/Bcl-2 ratio in MG63 and U2OS cells was calculated. ①P=0.001, ②P<0.001, compared with the rutin 0 μmol/L group.

Fig 3 Effect of rutin on the apoptosis of osteosarcoma cells

图4 芦丁对骨肉瘤细胞迁移的影响Note： A. Migration ability was assessed by scratch closure assay and the scratch closure rate of MG63 cells was analyzed. B. Migration ability was assessed by scratch closure assay and the scratch closure rate of U2OS cells was analyzed. ①P=0.001, ②P<0.001, ③P=0.005, compared with the rutin 0 μmol/L group.

Fig 4 Effect of rutin on the migration of osteosarcoma cells

图5 芦丁对骨肉瘤细胞侵袭的影响Note： A. Transwell assay was used to assess the invasion ability and the number of invasive MG63 cells was analyzed. B. Transwell assay was used to assess the invasion ability and the number of invasive U2OS cells was analyzed. ①P<0.001, compared with the rutin 0 μmol/L group.

Fig 5 Effect of rutin on the invasion of osteosarcoma cells

图6 芦丁对骨肉瘤体内移植瘤生长的影响Note： A. Tumor image. B. Tumor weight. C. Tumor volume. D. The expression of Ki67 detected by immunohistochemistry and statistical analysis of Ki67-positive cell rate. E. The expression of VEGF detected by immunohistochemistry and statistical analysis of VEGF-positive cell rate. F. The tumor cell apoptosis detected by TUNEL and analysis of apoptosis rate. ①P<0.001, compared with the control group.

Fig 6 Effect of rutin on the growth of osteosarcoma xenograft tumors in vivo

图7 小鼠心、肝、脾、肺、肾组织学分析（H-E染色，×200）

Fig 7 Histological analysis of heart, liver, spleen, lung and kidney in mice (H-E staining, ×200)

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