松乳菇多糖治疗原发性肝癌的细胞实验及机制

Cell experiment and mechanism of lactarius deliciosus polysaccharide in the treatment of primary hepatocellular carcinoma

目的:探讨松乳菇多糖(lactarius deliciosus gray polysaccharide,LDG-A)对原发性肝癌的治疗作用,并分析其机制。方法:人肝癌细胞株HepG2培养至对数期时,以1.5×10^6 个/孔接种至6孔板,随机分为4组,每组5个复孔,细胞贴壁后,空白组、实验1,2,3组分别用磷酸盐缓冲液(PBS),和50,150,300 μg/mL LDG-A干预。对比干预24,48,72 h细胞增殖抑制率和凋亡率;对比干预72 h增殖相关基因血管内皮生长因子(vascular endothelial growth factor,VEGF),磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K),肝细胞黏附分子1(hepatocyte adhesion molecule 1,hepaCAM1)和凋亡相关基因B淋巴细胞瘤-2基因(B lymphoma-2 gene,Bcl-2),Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax),活化型含半胱氨酸天冬氨酸蛋白水解酶-3(cleaved cysteinyl aspartatespecific proteinase-3,cleaved-caspase-3) mRNA和蛋白相对表达量。结果:实验2组增殖抑制率最高、实验3组其次、实验1组最低,两两比较差异均有统计学意义(P<0.05);实验2组凋亡率最高、实验3组其次、实验1组更低、空白组最低,两两比较差异均有统计学意义(P<0.05);3剂量组增殖抑制率、凋亡率均随时间延长呈显著升高趋势(P<0.05),空白组凋亡率随时间延长变化不明显(P>0.05);实验2组VEGF,PI3K,Bcl-2 mRNA和蛋白相对表达量最低、实验3组其次、实验1组更高、空白组最高,两两比较差异均有统计学意义(P<0.05);实验2组HepaCAM1,Bax,Cleaved-caspase-3 mRNA和蛋白相对表达量最高、实验3组其次、实验1组更低、空白组最低,每2组间比较差异均有统计学意义(P<0.05)。结论:LDG-A可抑制人肝癌细胞株HepG2的增殖、促进其凋亡,其中浓度为150 μg/mL时效果最佳,推测与下调VEGF,PI3K,Bcl-2 mRNA和蛋白表达、上调hepaCAM1,Bax,cleaved-caspase-3 mRNA和蛋白表达有关。

Objective: To investigate of Lactarius deliciosus gray polysaccharide (LDG-A) therapeutic effect on primary hepatocellular carcinoma and to analyze its mechanism. Methods: When the human hepatocarcinoma cells HepG2 was cultured to the logarithmic phase, 1.5×10^6 /well were subdivided into 6-well plates. The cells were randomly divided into 4 groups. Each group had 5 replicate wells. After adherent growth, the blank group, experiment 1, 2, 3 groups were treated with phosphate buffered saline (PBS), 50, 150, and 300 μg/mL LDG-A, respectively. The rates of proliferation inhibition and apoptosis of the cells after 24, 48, and 72 hours of intervention were compared. The mRNA and protein relative expression of proliferation-related genes vascular endothelial growth factor (VEGF), phosphatidylinositol 3-kinase (PI3K), hepatocyte adhesion molecule 1 (hepaCAM1) and apoptosis-related genes B lymphoma-2 gene (Bcl-2), Bcl-2 associated X protein (Bax), cleaved cysteinyl aspartate specific proteinase-3 (cleaved-caspase-3), after 72 hours intervention were compared. Results: The proliferation inhibition rate of the experiment 2 group was the highest, the experiment 3 group was the second, the experiment 1 group was the lowest, and there were significant differences between each 2 groups (P<0.05). The apoptosis rate of the experiment 2 group was the highest, the experiment 3 group was the second, the experiment 1 group was slightly lower, the control group was the lowest, and there were significant differences between each 2 groups (P<0.05). The proliferation inhibition rates and the apoptosis rates of the 3 experiment groups were significantly increased with time (P<0.05). The apoptosis rate of the control group did not change significantly with time (P>0.05). The mRNA and protein relative expressions of VEGF, PI3K and Bcl-2 of the experiment 2 group were the lowest, the experiment 3 group was the second, the experiment 1 group was slightly higher, the blank group was the highest, and there were significant differences between each 2 groups (P<0.05). The mRNA and protein relative expressions of hepaCAM1, cleaved-caspase-3 and Bax of the experiment 2 group were the highest, the experiment 3 group was the second, the experiment 1 group was slightly lower, the blank group was the lowest, and there were significant differences between each 2 groups (P<0.05). Conclusion: LDG-A can inhibit the proliferation of human hepatocarcinoma cells HepG2 and promote its apoptosis, of which 150 μg/mL LDG-A best, and it is presumed to be related to down-regulate the mRNA and protein expressions of VEGF, PI3K, Bcl-2 and up-regulate the mRNA and protein expressions of hepaCAM1, cleaved-caspase-3 and Bax.