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目的:探讨JAK2/STAT3信号通路在AG490干预下对人肝细胞癌裸鼠移植瘤生长、凋亡的影响以及与相关基因表达之间的关系。方法:建立人肝癌细胞裸鼠移植瘤模型,将BALB/C-nu裸鼠随机平均分成3组,按照实验设计分为对照组(生理盐水)、低浓度AG490组(浓度4 mg/kg的AG490)、高浓度AG490组(浓度为8 mg/kg的AG490),而后按照实验设计分别给予生理盐水或AG490处理裸鼠。观察期间记录各组裸鼠体质量、瘤重、肿瘤体积、计算肿瘤生长抑制率;通过流式细胞法检测细胞周期变化和凋亡情况;蛋白质印迹法检测细胞中p-JAK2、JAK2、p-STAT3、STAT3、MMP-2、Bcl-2、Bcl-xl及Bax蛋白表达的变化;免疫组织化学检测移植瘤组织中p-JAK2和p-STAT3蛋白表达。结果:实验组裸鼠瘤重、肿瘤体积均小于对照组,抑瘤率高于对照组,差异均具有统计学意义(P<0.05)。流式细胞分析结果显示低浓度AG490组(73.98±0.90)%、高浓度AG490组(81.39±1.66)%的细胞周期G0/G1期较对照组(59.30±1.13)%明显增加(P<0.05),同样低浓度AG490组(33.32±1.16)%和高浓度AG490组(52.22±1.06)%的细胞凋亡率较对照组(1.50±0.12)%明显增高(P<0.05)。Western blot结果显示低浓度AG490组(1.073±0.028)、(0.955±0.025)和高浓度AG490组(0.763±0.023)、(0.751±0.037)的p-JAK2,p-STAT3蛋白表达均较对照组(1.265±0.012)、(1.477±0.020)明显降低(P<0.05)。同时下调通路下游蛋白MMP-2,Bcl-2,Bcl-xl和上调Bax表达水平。免疫组化结果显示AG490组移植瘤肿瘤组织p-JAK2和p-STAT3蛋白表达较对照组明显降低(P<0.05)。结论:AG490能够通过抑制JAK2/STAT3信号通路促进人肝癌细胞的凋亡、抑制增殖和侵袭作用达到抑制裸鼠移植瘤的生长,提示JAK2/STAT3通路在肝细胞癌恶性演进过程中发挥了重要作用。
OBJECTIVE: To investigate the effect of JAK2 / STAT3 signaling pathway on the growth and apoptosis of human hepatocellular carcinoma xenografts in nude mice under the intervention of AG490 and its relationship with the expression of related genes. METHODS: BALB / C-nu nude mice were randomly divided into three groups according to the experimental design. They were divided into control group (saline), AG490 group (4 mg / kg AG490 ), AG490 high concentration (AG490 at a concentration of 8 mg / kg), and then treated with normal saline or AG490 nude mice respectively according to the experimental design. The body weight, tumor weight and tumor volume of nude mice in each group were recorded during the observation period, and the rate of tumor growth inhibition was calculated. The cell cycle changes and apoptosis were detected by flow cytometry. The expressions of p-JAK2, JAK2, p- The expressions of STAT3, STAT3, MMP-2, Bcl-2, Bcl-xl and Bax protein were detected by immunohistochemistry. The expressions of p-JAK2 and p-STAT3 protein were detected by immunohistochemistry. Results: The tumor weight and tumor volume of experimental group were less than those of control group, and the tumor inhibition rate was higher than that of control group (P <0.05). Flow cytometry analysis showed that the cell cycle G0 / G1 phase was significantly increased (P <0.05) in the AG490 (73.98 ± 0.90)% and 81.39 ± 1.66 (%) groups compared with the control group (59.30 ± 1.13)% (P <0.05). The apoptotic rate of the same low concentration AG490 group (33.32 ± 1.16)% and high concentration AG490 group (52.22 ± 1.06)% was significantly higher than that of the control group (1.50 ± 0.12)%. The results of Western blot showed that the expressions of p-JAK2 and p-STAT3 protein in AG490 group (1.073 ± 0.028), (0.955 ± 0.025) and AG490 group (0.763 ± 0.023) and (0.751 ± 0.037) 1.265 ± 0.012) and (1.477 ± 0.020), respectively (P <0.05). At the same time downregulation of downstream protein MMP-2, Bcl-2, Bcl-xl and up-regulate Bax expression levels. The results of immunohistochemistry showed that the expression of p-JAK2 and p-STAT3 in tumor tissues of AG490 group was significantly lower than that of the control group (P <0.05). Conclusion: AG490 can inhibit the growth of human xenografts in nude mice by inhibiting the JAK2 / STAT3 signaling pathway and inhibiting the proliferation and invasion of human hepatocellular carcinoma cells, suggesting that JAK2 / STAT3 pathway plays an important role in the malignant progression of hepatocellular carcinoma .