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  

Study on the Anti-Cancer Activity of Ganoderma lucidum Water Extract Based on the Three-Dimensional Culture Model of Colorectal Cancer HCT116 Cells

作　　者：潘海涛 张国亮 钱华 王汉波 徐靖 赵建霞 史月姣 李振皓 PAN Haitao;ZHANG Guoliang;QIAN Hua;WANG Hanbo;XU Jing;ZHAO Jianxia;SHI Yuejiao;LI Zhenhao(Zhejiang Shouxiangu Botanical Drug Institute Co.,Ltd.,Hangzhou 311100,China;Wuyi Shouxiangu Traditional Chinese Medicine Co.,Ltd.,Wuyi 321200,China;Zhejiang Shouxiangu Pharmaceutical Co.,Ltd.,Wuyi 321200,China;Zhejiang Engineering Research Center of Rare Medicinal Plants,Wuyi 321200,China)

机构地区：[1]浙江寿仙谷植物药研究院有限公司,杭州311100 [2]武义寿仙谷中药饮片有限公司,浙江武义321200 [3]浙江寿仙谷医药股份有限公司,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

出　　处：《中国现代应用药学》2021年第13期1550-1558,共9页Chinese Journal of Modern Applied Pharmacy

基　　金：浙江省科技计划项目(2017C02011,2019C02100)。

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。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.

关 键 词：灵芝 结肠癌 三维培养 E-CADHERIN INTEGRINΒ1 

分 类 号：R284.1[医药卫生—中药学]