樟芝多糖抑制肿瘤相关成纤维细胞生成的作用研究

doi:10.12092/j.issn.1009-2501.2019.03.005

摘要/Abstract

摘要：

目的: 研究樟芝多糖抑制肝癌细胞条件性培养基诱导骨髓间充质干细胞（MSC）向肿瘤相关成纤维细胞转化的作用。方法: 分离人肝癌细胞HepG2培养基（CM），将骨髓间充质干细胞分为正常组、对照组和实验组，正常组为常规DMEM培养基+10%胎牛血清（FBS），对照组为CM+10%FBS培养，实验组为CM+10%FBS培养+10 mg/L的樟芝多糖培养。应用CCK-8试剂盒检测MSC的细胞活性，BrdU标记-流式细胞术检测细胞增殖率，流式细胞术检测细胞凋亡率，PI染色检测细胞周期变化，Western blot法检测α-平滑肌激动蛋白（α-SMA）、肌间线蛋白（Desmin）、成纤维细胞活化蛋白（FAP）、凝血酶敏感蛋白1（Tsp-1）、成纤维细胞特异蛋白（FSP）的表达以及转化生长因子β1（TGF-β1）、信号转导分子（Smad1、Smad2）的表达水平，免疫荧光染色法检测α-SMA、FAP和FSP的表达水平。结果: 对照组MSC的细胞活力显著高于正常组和实验组（P<0.05），三组细胞的细胞凋亡率无统计学差异（P>0.05），对照组MSC中G0/G1期比例降低，S期比例增高，相比正常组和实验组具有统计学差异（P<0.05），对照组MSC中α-SMA、Desmin、FAP、Tsp-1、FSP的表达水平以及TGF-β1、Smad1、Smad2的表达水平显著高于正常组和实验组（P<0.05），免疫荧光结果也显示对照组中α-SMA、FAP和FSP的水平显著高于正常组和实验组（P<0.05）。结论: 肝癌细胞条件性培养基可以促进MSC向肿瘤相关成纤维细胞转化，而樟芝多糖可以抑制该转化，这是樟芝多糖抗肿瘤作用的机制之一。

关键词: 樟芝多糖, 肝癌, 肿瘤相关成纤维细胞, 转化

Abstract:

AIM: To inveatigate the effect of polysaccharides on the transformation of bone marrow mesenchymal stem cells (MSC) into tumor associated fibroblasts induced by conditioned medium of hepatoma cells. METHODS: The medium of human hepatoma cell HepG2（CM）was isolated, and the bone marrow mesenchymal stem cells were divided into normal group, control group and experimental group. The normal group was treated with conventional DMEM medium +10%FBS, the control group was cultured with CM+10%FBS, and the experimental group was cultured with CM+10%FBS +10 mg/L polysaccharides. The cell viability of MSC was detected by CCK-8 kit, the cell proliferation detected by BrdU labeling and flow cytometry, the apoptosis rate was detected by flow cytometry, and the cell cycle was detected by PI staining. Western blot method was used to detect the expression of α smooth muscle agonist (α-SMA), myocin (Desmin), fibroblast activation protein (FAP), thrombin sensitive protein 1 (Tsp-1), and fibroblast specific protein (FSP). The expression of α-SMA, FAP and FSP were detected by immunofluorescence staining. RESULTS:The cell viability of MSC in the control group was significantly higher than that in the normal group and the experimental group (P<0.05). There was no significant difference of the apoptosis rate among the three groups (P>0.05). In the control group, the proportion of G0/G1 phase decreased, and the proportion of S phase increased in MSC, which was significantly different from that in the normal group and the experimental group (P<0.05). The expression level of α-SMA, Desmin, FAP, Tsp-1, FSP and the expression level of TGF-β1, Smad1 and Smad2 in the control group was significantly higher than those in the normal group and the experimental group (P<0.05). The immunofluorescence results also showed that the level of α-SMA, FAP, FSP and TGF-β1, Smad1 and Smad2 in the control group was significantly higher than those in the normal group and the experimental group (P<0.05). CONCLUSION: Hepatoma cells can promote the transformation of MSC into tumor related fibroblasts, and polysaccharides can inhibit the transformation, which is one of the mechanisms of anti tumor effect of polysaccharides.

Key words: polysaccharides, liver cancer, tumor associated fibroblasts, transformation

中图分类号:

R965.2
R730

顾艳玲，盛泳佳，王瑾. 樟芝多糖抑制肿瘤相关成纤维细胞生成的作用研究[J]. 中国临床药理学与治疗学, 2019, 24(3): 266-271.

GU Yanling, SHENG Yongjia, WANG Jin. Polysaccharide inhibits tumor associated fibroblasts transformation[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2019, 24(3): 266-271.

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