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目的分析高浓度葡萄糖对肝癌细胞株HepG2的生长和增殖、迁移能力及凋亡的影响,及其与内质网应激的关联,探讨可能的作用机制。方法用含不同葡萄糖浓度的DMEM培养HepG2细胞,设对照组(DMEM含糖浓度为5.5 mmol/L)、高糖组(DMEM含糖浓度分别为25、40、100、150、200mmol/L)和甘露醇对照组(DMEM含糖浓度分别为5.5 mmol/L+19.5、34.5、94.5、144.5、194.5 mmol/L甘露醇),用倒置显微镜和MTT观察高浓度葡萄糖对HepG2细胞的生长和增殖状况;Hoechst染色和流式细胞术检测高浓度葡萄糖对HepG2细胞凋亡的影响;Western blot检测凋亡相关蛋白Bcl-2、Bak、Bax、p53、Grim19、Survinin和细胞核因子-κB(NF-κB)的表达变化,并检测Caspase3的活性;细胞划痕检测肝癌细胞株HepG2的生长和迁移能力,Western blot分析丝裂原活化蛋白激酶(MAPK)相关信号通路蛋白PP38/p38和P-JNK/JNK,以及内质网应激相关蛋白BIP和CHOP的表达变化。结果高浓度葡萄糖能明显抑制HepG2细胞的体外增殖且呈剂量依赖性;Hoechst染色和流式结果显示高浓度葡萄糖能诱导HepG2细胞的凋亡,随着葡萄糖浓度的升高,凋亡现象越来越明显,并且能下调抑制凋亡作用的蛋白Bcl-2和Survinin的表达,上调促进凋亡的蛋白Bak、Bax、p53、Grim19、NF-κB的表达,Caspase3的活性也呈上升趋势;细胞划痕结果显示高浓度葡萄糖能明显抑制HepG2细胞的迁移能力;高浓度葡萄糖可明显提高p38和JNK的磷酸化水平,快速的诱导其磷酸化,并能下调内质网应激凋亡抑制因子BIP的表达,上调内质网应激凋亡促进因子CHOP的表达(P<0.05)。结论高浓度葡萄糖可抑制肝癌细胞株HepG2的生长、增殖及迁移,诱导凋亡。其作用机制可能通过启动内质网应激,调节相关凋亡基因以及某些抑癌基因的表达,协同MAPK信号通路发挥作用。
Objective To analyze the effect of high concentration glucose on the growth, proliferation, migration and apoptosis of hepatocellular carcinoma cell line HepG2 and its relationship with endoplasmic reticulum stress, and to explore the possible mechanism. Methods HepG2 cells were cultured in DMEM with different glucose concentrations. The control group (DMEM with a concentration of 5.5 mmol / L), high glucose (DMEM with concentrations of 25, 40, 100, 150 and 200 mmol / L) Mannitol control group (DMEM sugar concentration of 5.5 mmol / L + 19.5,34.5,94.5,144.5,194.5 mmol / L mannitol, respectively), observed by inverted microscope and MTT glucose concentration in HepG2 cells growth and proliferation status; Hoechst staining and flow cytometry were used to detect the effect of high glucose on the apoptosis of HepG2 cells. Western blot was used to detect the expression of apoptosis related proteins Bcl-2, Bak, Bax, p53, Grim19, Survinin and nuclear factor-kappa B And the activity of Caspase3 was detected. The cell scratch was used to detect the growth and migration of hepatoma cell line HepG2. Western blot was used to analyze the MAPK related signal pathway proteins PP38 / p38 and P-JNK / JNK, Endoplasmic reticulum stress related proteins BIP and CHOP expression changes. Results High concentration glucose could inhibit the proliferation of HepG2 cells in vitro in a dose-dependent manner. Hoechst staining and flow cytometry showed that high concentration of glucose could induce apoptosis of HepG2 cells. As the concentration of glucose increased, the apoptosis became more and more Obviously down-regulated the expression of Bcl-2 and Survivin, and upregulated the expression of apoptosis-inducing proteins Bak, Bax, p53, Grim19 and NF-κB, and the activity of Caspase3 also increased. The results showed that high concentration of glucose could significantly inhibit the migration of HepG2 cells; High concentration of glucose can significantly increase the level of p38 and JNK phosphorylation, rapid induction of its phosphorylation, and can downregulate the expression of endoplasmic reticulum stress apoptosis inhibitor BIP , Upregulated the expression of endoplasmic reticulum stress-induced apoptosis-promoting factor CHOP (P <0.05). Conclusion High glucose can inhibit the growth, proliferation and migration of HepG2 hepatoma cell line and induce apoptosis. Its mechanism may be through the activation of endoplasmic reticulum stress, regulating the expression of related apoptosis genes and some tumor suppressor genes, and cooperating with MAPK signaling pathway.