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成纤维细胞生长因子21对二乙基亚硝胺诱导L02肝细胞癌变的抑制作用
Inhibitory effect of fibroblast growth factor-21 on the carcinogenesis of L02 cells induced by diethylnitrosamine
文章发布日期:2018年01月05日  来源:  作者:李舒欢,韩继武  点击次数:414次  下载次数:63次

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【摘要】:目的探讨成纤维细胞生长因子(FGF)21对二乙基亚硝胺(DEN)诱导的L02肝细胞癌变的抑制作用。方法培养L02肝细胞,分别用不同浓度的DEN(1、10、20、50、100和150 μmol/L)作用,MTT法测定DEN对L02肝细胞活力的影响,选择适当的DEN刺激浓度(20 μmol/L)进行后续实验。同时,分别采用丙二醛(MDA)及超氧化物歧化酶(SOD)检测试剂盒检测DEN(20 μmol/L)作用细胞和正常L02细胞内的MDA及SOD水平。将L02肝细胞分为模型对照组(20 μmol/L DEN+PBS处理)、FGF21低剂量组(20 μmol/L DEN+1 μmol/L FGF21蛋白)和FGF21高剂量组(20 μmol/L DEN+2 μmol/L FGF21蛋白)。细胞培养12 h后,检测细胞内的 MDA及SOD水平。同时采用Real-time PCR和Western Blot检测β-Klotho(KLB)的表达水平,并用Western Blot检测蛋白激酶B(PKB, 又称AKT)磷酸化水平。计量资料2组间比较采用t检验,多组间比较采用方差分析,进一步两两比较采用LSD-t检验。 结果DEN作用于L02后细胞内MDA水平明显升高,SOD水平显著下降,差异均有统计学意义(t值分别为9.336和16.281,P值分别为0.011和0.004)。与模型对照组相比,FGF-21低剂量组和FGF-21高剂量组细胞内MDA水平下降、SOD水平升高(P值均<0.05),且FGF21高剂量组细胞内MDA水平显著低于FGF21低剂量组,差异有统计学意义(P=0.030),SOD水平则高于FGF21低剂量组,差异有统计学意义(P=0.042)。FGF21高剂量组与FGF21低剂量组细胞内KLB mRNA水平明显高于模型对照组(P值均<0.001),而FGF21高剂量组KLB mRNA水平高于低剂量组,差异有统计学意义(P<0.001),KLB蛋白表达趋势与KLB mRNA相同。模型对照组中磷酸化AKT(p-AKT)水平高于另外2组,同时FGF21高剂量组p-AKT水平低于FGF21低剂量组,差异均有统计学意义(P值均<0.05)。结论DEN可增强L02细胞内氧化应激,FGF21通过上调KLB表达,降低AKT磷酸化水平,抑制DEN诱导L02肝细胞氧化应激,从而抑制肝细胞癌变的发生。
【Abstract】:ObjectiveTo investigate the inhibitory effect of fibroblast growth factor-21 (FGF-21) on the carcinogenesis of L02 cells induced by diethylnitrosamine (DEN). MethodsL02 cells were cultured and treated with different concentrations of DEN (1, 10, 20, 50, 100, and 150 μmol/L). MTT assay was used to measure the influence of DEN on the viability of L02 cells, and an appropriate stimulation concentration of DEN (20 μmol/L) was selected for further study. The malondialdehyde (MDA) and superoxide dismutase (SOD) detection kits were used to measure the levels of MDA and SOD in L02 cells treated by DEN (20μmol/L) and normal L02 cells. Then L02 cells were divided into model control group (treated with 20μmol/L DEN and PBS), low-dose FGF-21 group (20 μmol/L DEN+1 μmol/L FGF-21), and high-dose FGF-21 group (20 μmol/L DEN+2 μmol/L FGF-21). The levels of MDA and SOD were measured after 12 hours of cell culture. Real-time PCR and Western blot were used to measure the expression of βKlotho (KLB), and Western blot was used to measure the level of phosphorylated protein kinase B (p-AKT). The t-test was used for comparison of continuous data between two groups; an analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsThere was a significant increase in the level of MDA and a significant reduction in the level of SOD after L02 cells were treated with DEN (t=9.336 and 16.281, P=0.011 and 0.004). Compared with the model control group, the low- and high-dose FGF-21 groups had a significant reduction in the level of MDA and a significant increase in the level of SOD (P<0.05), and compared with the low-dose FGF-21 group, the high-dose FGF-21 group had a significantly lower level of MDA and a significantly higher level of SOD (P=0.030 and 0.042), and there was a significant difference between two groups. The high- and low-dose FGF-21 groups had significantly higher mRNA expression of KLB than the model control group (P<0.001), and the high-dose FGF-21 group had significantly higher mRNA expression of KLB than the low-dose FGF-21 group (P<0.001), and there was a significant difference between two groups; the protein expression of KLB showed the same trend. The model control group had a significantly higher level of p-AKT than the other two groups (P<0.05), and the high-dose FGF-21 group had a significantly lower level of p-AKT than the low-dose FGF-21 group (P<0.05). ConclusionDEN can increase oxidative stress in L02 cells. By upregulating the expression of KLB, FGF-21 can reduce the level of p-AKT, inhibit oxidative stress in L02 cells induced by DEN, and thus inhibit the development of hepatocellular carcinoma.
【关键字】:肝细胞; 成纤维细胞生长因子; 二乙基亚硝胺; 氧化性应激
【Key words】:hepatocytes; fabroblast growth factors; diethylnitrosamine; oxidative stress
【引证本文】:LI SH, HAN JW. Inhibitory effect of fibroblast growth factor-21 on the carcinogenesis of L02 cells induced by diethylnitrosamine[J]. J Clin Hepatol, 2018, 34(2): 321-326. (in Chinese)
李舒欢, 韩继武. 成纤维细胞生长因子21对二乙基亚硝胺诱导的L02肝细胞癌变的抑制作用[J]. 临床肝胆病杂志, 2018, 34(2): 321-326.

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