| 引用本文: | 潘海涛,张国亮,钱华,王汉波,徐靖,赵建霞,史月姣,李振皓.基于结肠癌HCT116细胞三维培养模型的灵芝水提物抗肿瘤活性研究[J].中国现代应用药学,2021,38(13):1550-1558. |
| PAN Haitao,ZHANG Guoliang,QIAN Hua,WANG Hanbo,XU Jing,ZHAO Jianxia,SHI Yuejiao,LI Zhenhao.Study on the Anti-Cancer Activity of Ganoderma lucidum Water Extract Based on the Three-Dimensional Culture Model of Colorectal Cancer HCT116 Cells[J].Chin J Mod Appl Pharm(中国现代应用药学),2021,38(13):1550-1558. |
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| 基于结肠癌HCT116细胞三维培养模型的灵芝水提物抗肿瘤活性研究 |
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潘海涛1,2, 张国亮1,2, 钱华3,2, 王汉波2, 徐靖2,4, 赵建霞1,2, 史月姣1,2, 李振皓1,3,2
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1.浙江寿仙谷植物药研究院有限公司, 杭州 311100;2.浙江寿仙谷医药股份有限公司, 浙江 武义 321200;3.武义寿仙谷中药饮片有限公司, 浙江 武义 321200;4.浙江省珍稀植物药工程技术研究中心, 浙江 武义 321200
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| 摘要: |
| 目的 采用结肠癌HCT116细胞的二维（2D）和三维（3D）培养模型研究灵芝水提物（Ganoderma lucidum water extract，GLWE）的抗肿瘤作用。方法 采用紫外-可见光分光光度法测定GLWE中灵芝多糖（Ganoderma lucidum polysaccharides，GLP）、三萜和甾醇的含量，HPLC分析GLP中水溶性单糖的组成和比例；以Matrigel基质胶为基质材料建立体外HCT116细胞3D培养模型，评价GLWE的抗肿瘤及辅助抗肿瘤活性；CCK-8法检测细胞活力；Real-time PCR检测细胞mRNA表达水平；Western blotting检测细胞蛋白表达水平。结果 GLWE中GLP和三萜及甾醇的含量分别为20.92%和7.18%，其中GLP主要由甘露糖、葡萄糖醛酸、葡萄糖、半乳糖、D-木糖、L-岩藻糖组成；GLWE和5-氟尿嘧啶（5-fluorouracil，5-FU）作用48 h可呈浓度依赖性抑制2D和3D培养的HCT116细胞增殖；与2D培养相比，3D培养的HCT116细胞中整合素β1（Integrin β1）和钙黏蛋白（E-cadherin）mRNA的表达显著升高，并对GLWE和5-FU的敏感性降低；在HCT116细胞3D培养模型中，GLWE既可降低CDK2、CDK4和Bcl-2蛋白的表达，升高p21、p27、cleaved caspase-3和cleaved PARP蛋白的表达，还可显著降低Integrin β1和E-cadherin mRNA的表达，并加强5-FU的抗肿瘤活性。结论 GLWE通过抑制细胞周期和诱导细胞凋亡而抑制3D培养的HCT116细胞增殖，并通过抑制Integrin β1和E-cadherin mRNA的表达增强5-FU的抗肿瘤活性。 |
| 关键词: 灵芝 结肠癌 三维培养 E-cadherin Integrin β1 |
| DOI：10.13748/j.cnki.issn1007-7693.2021.13.003 |
| 分类号:R284.1 |
| 基金项目:浙江省科技计划项目（2017C02011，2019C02100） |
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| Study on the Anti-Cancer Activity of Ganoderma lucidum Water Extract Based on the Three-Dimensional Culture Model of Colorectal Cancer HCT116 Cells |
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PAN Haitao1,2, ZHANG Guoliang1,2, QIAN Hua3,2, WANG Hanbo2, XU Jing2,4, ZHAO Jianxia1,2, SHI Yuejiao1,2, LI Zhenhao1,3,2
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1.Zhejiang Shouxiangu Botanical Drug Institute Co., Ltd., Hangzhou 311100, China;2.Zhejiang Shouxiangu Pharmaceutical Co., Ltd., Wuyi 321200, China;3.Wuyi Shouxiangu Traditional Chinese Medicine Co., Ltd., Wuyi 321200, China;4.Zhejiang Engineering Research Center of Rare Medicinal Plants, Wuyi 321200, China
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| Abstract: |
| OBJECTIVE To investigate the anti-cancer effects of Ganoderma lucidum water extract(GLWE) based on the two-dimensional(2D) and three-dimensional(3D) culture of colorectal cancer HCT116 cells.METHODS The content of Ganoderma lucidum polysaccharide(GLP), Ganoderma triterpenes and sterols in GLWE were determined by UV-visible spectrophotometry, and the composition and proportion of water-soluble monosaccharide in GLP were determined by HPLC. An 3D culture model of HCT116 cells in vitro was established using Matrigel as the matrix material, and the anti-cancer and adjuvant anti-cancer activities of GLWE were evaluated. Cell viability was detected by CCK-8 assay. mRNA expression level of the cells was analyzed by Real-time PCR. Western blotting was used to detect cell protein expression level. RESULTS The contents of GLP and Ganoderma triterpenes(contained sterols) in GLWE were 20.92% and 7.18%, respectively, and GLP was mainly comprised of mannose, glucuronic acid, glucose, galactose, D-xylose and L-fucose. GLWE and 5-fluorouracil(5-FU) inhibited the proliferation of both 2D and 3D cultured HCT116 cells in a dose dependent manner after incubation for 48 h. However, compared with the 2D culture, the expression of E-cadherin and Integrin β1 mRNA was significantly increased, and the sensitivity to GLWE and 5-FU was reduced in 3D cultured HCT116 cell. GLWE could not only reduce the expression of CDK2, CDK4 and Bcl-2 proteins, increase the expression of p21, p27, cleaved caspase-3 and cleaved PARP proteins, but also significantly reduce the expression of E-cadherin and Integrin β1 mRNA, and enhance the anti-cancer activity of 5-FU in 3D cultured HCT116 cell. CONCLUSION GLWE significantly inhibit the proliferation of 3D-cultured HCT116 cells by inhibiting cell cycle and inducing apoptosis, and enhanced the anti-cancer activity of 5-FU by inhibiting the expression of E-cadherin and Integrin β1 mRNA. |
| Key words: Ganoderma lucidum colorectal cancer three-dimensional culture E-cadherin integrin β1 |
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