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目的:探讨苹果酸舒尼替尼对人肝癌c细胞凋亡的作用及其机制。方法:常规体外培养HepG2细胞,利用MTT法检测舒尼替尼杀伤HepG2细胞的IC50,Western blotting检测药物处理前后HepG2细胞分子靶点蛋白表达,膜联蛋白(Annexin-V)/碘化丙啶(PI)双标法和TUNEL染色法检测舒尼替尼处理前后HepG2细胞凋亡情况,实时荧光定量PCR检测药物处理前后HepG2细胞凋亡基因mRNA的表达情况。结果:舒尼替尼杀伤HepG2细胞的IC50值为(3.22±0.50)μmol/L。以对HepG2细胞无明显抑制作用的剂量1μmol/L舒尼替尼处理HepG2细胞后,细胞内VEGFR1、VEGFR2、PDGFRα、Kit、FLT3蛋白表达均有不同水平下降(均P<0.05),HepG2细胞的凋亡率[(15.18±1.28)%vs(5.90±0.45)%,P<0.05]、凋亡指数(AI)[(23.54±4.73)vs(4.17±0.64),P<0.05]均显著升高。舒尼替尼处理HepG2细胞后,上调促凋亡基因Bax、NOXA、PUMA、P53 mRNA表达水平(均P<0.05),降低抑凋亡基因Bcl-2、X-IAP mRNA表达水平(均P<0.05)。结论:舒尼替尼能够诱导肝癌HepG2细胞凋亡,其机制可能是通过上调促凋亡基因的表达及降低抑凋亡基因表达水平来实现的。
Objective: To investigate the effect of sunitinib malate on human hepatocellular carcinoma cell apoptosis and its mechanism. Methods: The HepG2 cells were cultured in vitro. The IC50 of HepG2 cells was detected by MTT assay. The protein expression of HepG2 cells was detected by Western blotting. The expression of Annexin-V / propidium iodide PI) double staining and TUNEL staining were used to detect the apoptosis of HepG2 cells before and after sunitinib treatment. The expression of apoptosis gene mRNA in HepG2 cells was detected by real-time fluorescence quantitative PCR. Results: The IC50 value of sunitinib against HepG2 cells was (3.22 ± 0.50) μmol / L. The expression of VEGFR1, VEGFR2, PDGFRα, Kit and FLT3 in HepG2 cells treated with 1 μmol / L of sunitinib at a dose of 1 μmol / L decreased at different levels (all P <0.05), and at HepG2 cells Apoptosis rate was significantly higher than that of control group [(15.18 ± 1.28)% vs (5.90 ± 0.45)%, P <0.05] . Sunitinib treatment of HepG2 cells increased the expression of pro-apoptotic genes Bax, NOXA, PUMA and P53 mRNA (all P <0.05), and decreased the expression of Bcl-2 and X-IAP mRNA (all P < 0.05). Conclusion: Sunitinib can induce HepG2 hepatocarcinoma cell apoptosis by up-regulating the expression of pro-apoptotic gene and decreasing the expression of anti-apoptotic gene.