冬凌草甲素通過(guò)下調(diào)Mcl-1蛋白表達(dá)誘導(dǎo)肝癌細(xì)胞凋亡的機(jī)制研究
發(fā)布時(shí)間：2018-11-06 18:03
【摘要】：目的1.研究冬凌草甲素(Oridonin,Ori)對(duì)人肝癌細(xì)胞的抑制增殖和誘導(dǎo)凋亡作用。2.探討冬凌草甲素誘導(dǎo)人肝癌細(xì)胞凋亡的分子機(jī)制。方法1.不同濃度的冬凌草甲素作用于肝癌細(xì)胞株HCCLM3和HepG2后,CCK-8檢測(cè)冬凌草甲素對(duì)肝癌細(xì)胞的增殖抑制效率;濃度分別為0、5、10、20μM的冬凌草甲素作用于肝癌細(xì)胞株HCCLM3和HepG2后,集落形成實(shí)驗(yàn)觀察肝癌細(xì)胞的克隆形成情況,Annexin-FITC/PI雙染和流式細(xì)胞儀方法檢測(cè)細(xì)胞的凋亡情況。2.濃度分別為0、5、10、20μM的冬凌草甲素作用于肝癌細(xì)胞株HCCLM3和HepG2后,Western Blot檢測(cè)凋亡相關(guān)蛋白的表達(dá);應(yīng)用siRNA技術(shù)在肝癌細(xì)胞株HCCLM3和HepG2中沉默Mcl-1,流式細(xì)胞儀分別檢測(cè)肝癌細(xì)胞株HCCLM3和HepG2對(duì)照組、Oridonin 10μM、siMcl-1 HCCLM3和siMcl-1HepG2、Oridonin 10μM聯(lián)合siMcl-1 HCCLM3和siMcl-1HepG2四組的凋亡率變化。結(jié)果1.CCK-8法檢測(cè)結(jié)果顯示,低濃度的冬凌草甲素對(duì)肝癌細(xì)胞株HCCLM3、HepG2的抑制作用較弱,高濃度的冬凌草甲素對(duì)肝癌細(xì)胞株HCCLM3、HepG2的抑制作用較強(qiáng),其抑制作用呈劑量依賴(lài)性;集落形成實(shí)驗(yàn)結(jié)果表明冬凌草甲素藥物濃度越高,其抑制肝癌細(xì)胞克隆形成能力越強(qiáng);濃度為5μM、10μM和20μM的冬凌草甲素處理肝癌細(xì)胞HCCLM3 24h后,流式細(xì)胞儀檢測(cè)其細(xì)胞凋亡率分別為20.1%、41.5%和74.3%,明顯高于空白對(duì)照組的6.7%,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);濃度為5μM、10μM和20μM的冬凌草甲素處理肝癌細(xì)胞HepG2 24h后,流式細(xì)胞儀檢測(cè)其細(xì)胞凋亡率分別為20.3%、42.6%和69.2%,明顯高于空白對(duì)照組的4.5%,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。2.Western blot檢測(cè)發(fā)現(xiàn)10μM的冬凌草甲素處理HCCLM3和HepG2肝癌細(xì)胞株24 h后,均能使caspase-3、PARP裂解激活;同時(shí)檢測(cè)結(jié)果顯示10μM、20μM的冬凌草甲素作用于兩株肝癌細(xì)胞株24 h后,Bcl-2抑凋亡家族蛋白Mcl-1的表達(dá)明顯受到抑制,而對(duì)Bcl-2抑凋亡家族蛋白中另外兩個(gè)主要蛋白Bcl-2、Bcl-xL則沒(méi)有明顯影響。應(yīng)用siRNA技術(shù)在肝癌細(xì)胞株HCCLM3和HepG2中沉默Mcl-1,流式細(xì)胞儀檢測(cè)結(jié)果發(fā)現(xiàn)在肝癌細(xì)胞株HCCLM3中Oridonin 10μM、siMcl-1、Oridonin 10μM聯(lián)合siMcl-1處理組的凋亡率分別為39.2%、41.6%、43.7%,均明顯高于對(duì)照組(5.3%),差異有統(tǒng)計(jì)學(xué)意義(P0.05),而Oridonin10μM、siMcl-1、Oridonin 10μM聯(lián)合siMcl-1處理組三組間的凋亡率卻無(wú)統(tǒng)計(jì)學(xué)差異;在肝癌細(xì)胞株HepG2中Oridonin 10μM、siMcl-1、Oridonin 10μM聯(lián)合siMcl-1處理組的凋亡率分別為40.3%、42.4%、44.8%,均明顯高于對(duì)照組(4.6%),差異有統(tǒng)計(jì)學(xué)意義(P0.05),而Oridonin 10μM、siMcl-1、Oridonin 10μM聯(lián)合siMcl-1處理組三組間的凋亡率卻無(wú)統(tǒng)計(jì)學(xué)差異;同時(shí)我們發(fā)現(xiàn)與siMcl-1HCCLM3和siMcl-1HepG2組相比,10μM的Oridonin處理siMcl-1 HCCLM3和siMcl-1HepG2后并不能顯著增加凋亡率。結(jié)論1.冬凌草甲素可以抑制肝癌細(xì)胞的增殖并誘導(dǎo)肝癌細(xì)胞凋亡。2.冬凌草甲素抗肝癌機(jī)制可能是通過(guò)下調(diào)Mcl-1的表達(dá)進(jìn)而誘導(dǎo)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.7
本文編號(hào)：2315062
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.7
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