冬凌草甲素通过下调Mcl-1蛋白表达诱导肝癌细胞凋亡的机制研究

发布时间：2018-11-06 18:03

【摘要】：目的1.研究冬凌草甲素(Oridonin,Ori)对人肝癌细胞的抑制增殖和诱导凋亡作用。2.探讨冬凌草甲素诱导人肝癌细胞凋亡的分子机制。方法1.不同浓度的冬凌草甲素作用于肝癌细胞株HCCLM3和HepG2后,CCK-8检测冬凌草甲素对肝癌细胞的增殖抑制效率;浓度分别为0、5、10、20μM的冬凌草甲素作用于肝癌细胞株HCCLM3和HepG2后,集落形成实验观察肝癌细胞的克隆形成情况,Annexin-FITC/PI双染和流式细胞仪方法检测细胞的凋亡情况。2.浓度分别为0、5、10、20μM的冬凌草甲素作用于肝癌细胞株HCCLM3和HepG2后,Western Blot检测凋亡相关蛋白的表达;应用siRNA技术在肝癌细胞株HCCLM3和HepG2中沉默Mcl-1,流式细胞仪分别检测肝癌细胞株HCCLM3和HepG2对照组、Oridonin 10μM、siMcl-1 HCCLM3和siMcl-1HepG2、Oridonin 10μM联合siMcl-1 HCCLM3和siMcl-1HepG2四组的凋亡率变化。结果1.CCK-8法检测结果显示,低浓度的冬凌草甲素对肝癌细胞株HCCLM3、HepG2的抑制作用较弱,高浓度的冬凌草甲素对肝癌细胞株HCCLM3、HepG2的抑制作用较强,其抑制作用呈剂量依赖性;集落形成实验结果表明冬凌草甲素药物浓度越高,其抑制肝癌细胞克隆形成能力越强;浓度为5μM、10μM和20μM的冬凌草甲素处理肝癌细胞HCCLM3 24h后,流式细胞仪检测其细胞凋亡率分别为20.1%、41.5%和74.3%,明显高于空白对照组的6.7%,差异具有统计学意义(P0.05);浓度为5μM、10μM和20μM的冬凌草甲素处理肝癌细胞HepG2 24h后,流式细胞仪检测其细胞凋亡率分别为20.3%、42.6%和69.2%,明显高于空白对照组的4.5%,差异具有统计学意义(P0.05)。2.Western blot检测发现10μM的冬凌草甲素处理HCCLM3和HepG2肝癌细胞株24 h后,均能使caspase-3、PARP裂解激活;同时检测结果显示10μM、20μM的冬凌草甲素作用于两株肝癌细胞株24 h后,Bcl-2抑凋亡家族蛋白Mcl-1的表达明显受到抑制,而对Bcl-2抑凋亡家族蛋白中另外两个主要蛋白Bcl-2、Bcl-xL则没有明显影响。应用siRNA技术在肝癌细胞株HCCLM3和HepG2中沉默Mcl-1,流式细胞仪检测结果发现在肝癌细胞株HCCLM3中Oridonin 10μM、siMcl-1、Oridonin 10μM联合siMcl-1处理组的凋亡率分别为39.2%、41.6%、43.7%,均明显高于对照组(5.3%),差异有统计学意义(P0.05),而Oridonin10μM、siMcl-1、Oridonin 10μM联合siMcl-1处理组三组间的凋亡率却无统计学差异;在肝癌细胞株HepG2中Oridonin 10μM、siMcl-1、Oridonin 10μM联合siMcl-1处理组的凋亡率分别为40.3%、42.4%、44.8%,均明显高于对照组(4.6%),差异有统计学意义(P0.05),而Oridonin 10μM、siMcl-1、Oridonin 10μM联合siMcl-1处理组三组间的凋亡率却无统计学差异;同时我们发现与siMcl-1HCCLM3和siMcl-1HepG2组相比,10μM的Oridonin处理siMcl-1 HCCLM3和siMcl-1HepG2后并不能显著增加凋亡率。结论1.冬凌草甲素可以抑制肝癌细胞的增殖并诱导肝癌细胞凋亡。2.冬凌草甲素抗肝癌机制可能是通过下调Mcl-1的表达进而诱导凋亡。
[Abstract]:Objective 1. To study the inhibitory effect of oridonin (Oridonin,Ori) on proliferation and apoptosis of human hepatoma cells. 2. To investigate the molecular mechanism of oridonin induced apoptosis in human hepatoma cells. Method 1. The proliferation inhibition efficiency of oridonin on hepatoma cell line HCCLM3 and HepG2 was measured by CCK-8 after different concentrations of oridonin was treated with HepG2. The colony formation of hepatoma cell line HCCLM3 and HepG2 was observed by colony formation assay. The apoptosis of hepatoma cells was detected by Annexin-FITC/PI double staining and flow cytometry. 2. The expression of apoptosis-related proteins in HCCLM3 and HepG2 cells was detected by, Western Blot after the treatment of oridonin at the concentration of 0 ~ 5 ~ 10 ~ 10 渭 M. SiRNA technique was used to detect the apoptosis rate of HCCLM3 and HepG2 cells in HCCLM3 and HepG2, Oridonin 10 渭 Mcl-1 HCCLM3 and siMcl-1HepG2,Oridonin 10 渭 M combined with siMcl-1 HCCLM3 and siMcl-1HepG2, respectively. Results the results of 1.CCK-8 assay showed that the inhibitory effect of low concentration oridonin on HCCLM3,HepG2 was weaker than that of high concentration oridonin on HCCLM3,HepG2. The inhibitory effect was dose-dependent. The results of colony formation experiment showed that the higher the concentration of oridonin, the stronger the ability of inhibiting the clone formation of hepatoma cells. After treated with oridonin (10 渭 M) and oridonin (20 渭 M) for 24 hours, the apoptotic rates of hepatoma cells were 41.5% and 74.3% by flow cytometry, respectively, which were significantly higher than those of the control group (6.7%). The difference was statistically significant (P0.05). After treated with oridonin (10 渭 M) and oridonin (20 渭 M) for 24 h, the apoptotic rates of hepatoma cells were detected by flow cytometry (FCM) as follows: 42.6% and 69.2%, respectively, which were significantly higher than those in the control group (4.5%). The difference was statistically significant (P0.05). 2.Western blot assay showed that 10 渭 M oridonin could activate caspase-3,PARP lysis of HCCLM3 and HepG2 hepatoma cells after 24 h treatment. At the same time, the results showed that the expression of Bcl-2 inhibitor family protein Mcl-1 was significantly inhibited after treated with oridonin 10 渭 m or 20 渭 M for 24 h. Bcl-2,Bcl-xL, the other two major proteins in the Bcl-2 family of antiapoptotic proteins, was not significantly affected. The apoptosis rate of HCCLM3 treated with Oridonin 10 渭 Mcl-1 Oridonin 10 渭 M combined with siMcl-1 was 39.2% and 41.6%, respectively, by using siRNA technique in HCCLM3 and HepG2 silence Mcl-1, flow cytometry. 43.7% were significantly higher than the control group (5.3%), the difference was statistically significant (P0.05), but the apoptosis rate of Oridonin10 渭 Mcl-1 Oridonin 10 渭 M combined with siMcl-1 group had no statistical difference. The apoptotic rates of Oridonin 10 渭 Mcl-1 Oridonin 10 渭 M + siMcl-1 treatment group in HepG2 were 40.33.4% and 44.8%, respectively, which were significantly higher than those of the control group (4.6%). The difference was statistically significant (P0.05). However, the apoptosis rate of Oridonin 10 渭 Mcl-1 Oridonin 10 渭 M combined with siMcl-1 group was not significantly different among the three groups. At the same time, we found that 10 渭 M Oridonin treatment with siMcl-1 HCCLM3 and siMcl-1HepG2 did not significantly increase the apoptosis rate compared with siMcl-1HCCLM3 and siMcl-1HepG2 groups. Conclusion 1. Oridonin can inhibit the proliferation of hepatoma cells and induce apoptosis of hepatoma cells. 2. The anti-hepatoma mechanism of oridonin may be by down-regulating the expression of Mcl-1 and inducing apoptosis.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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