本文選題：CXC195 + 肝癌細(xì)胞�。� 參考：《南昌大學(xué)》2016年博士論文

【摘要】：目的:研究CXC195在體外誘導(dǎo)HepG2細(xì)胞凋亡并抑制細(xì)胞增殖。探討PI3K-AKT-mTOR信號(hào)通路和內(nèi)質(zhì)網(wǎng)應(yīng)激共同誘導(dǎo)HepG2細(xì)胞凋亡。為CXC195治療肝癌提供幫助。材料和方法:體外培養(yǎng)HepG2細(xì)胞株作為實(shí)驗(yàn)組,對照組使用L-02細(xì)胞株。化學(xué)反應(yīng)合成CXC195。聯(lián)合臺(tái)盼藍(lán)拒染法和MTT比色法檢測CXC195分別作用HepG2細(xì)胞和L-02細(xì)胞后細(xì)胞的存活情況,流式細(xì)胞術(shù)分析細(xì)胞凋亡,檢測濃度為10μM,25μM,50μM,100μM,150μM,200μM的CXC195作用于HepG2細(xì)胞和L-02細(xì)胞的平均生長存活率。用Western blotting檢測150μM CXC195作用HepG2細(xì)胞后,PI3K-AKT-mTOR信號(hào)通路PI3K、AKT、mTOR及磷酸化蛋白和內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白表達(dá)變化。結(jié)果:(1)CXC195抑制HepG2細(xì)胞生長:臺(tái)盼藍(lán)拒染法和MMT比色法檢測細(xì)胞存活結(jié)果顯示,加入CXC195后,人肝癌細(xì)胞(HepG2)和正常肝細(xì)胞(L-02)的生長存活率均明顯降低。并且,隨著CXC195劑量從10μM逐漸增加到200μM,HepG2細(xì)胞存活率從97.3%逐漸降到42.9%,L-02細(xì)胞存活率從98.5%逐漸降到65.1%。不同劑量CXC195作用相同細(xì)胞生長存活率下降有顯著差異(P0.01),相同劑量CXC195作用于兩組細(xì)胞比較,存活的肝癌細(xì)明顯少于正常肝細(xì)胞L-02。MMT比色法檢測結(jié)果表明,用150μMCXC195處理HepG2細(xì)胞12、24、48、72小時(shí),細(xì)胞存活率也明顯依次下降(P0.01)。流式細(xì)胞術(shù)檢測發(fā)現(xiàn),150μM CXC195作用HepG2細(xì)胞24小時(shí)后,細(xì)胞存活于G0/G1期較多(71.62%vs45.17%,P0.01),S期(17.53%vs41.11,P0.01)和G2/M期(10.85%vs13.72%,P0.05)細(xì)胞比例明顯減少。結(jié)果表明:與正常肝細(xì)胞(L-02)相比,CXC195能更多抑制肝癌細(xì)胞(HepG2)生長,CXC195對肝癌細(xì)胞生長的抑制作用與藥物濃度和藥物作用時(shí)間呈正相關(guān)。并且CXC195主要作用于S期和G2/M期肝癌細(xì)胞HepG2。(2)CXC195誘導(dǎo)HepG2細(xì)胞凋亡:AnnexinV/PI雙染色法標(biāo)記后,通過流式細(xì)胞儀檢測發(fā)現(xiàn),各濃度CXC195誘導(dǎo)人肝癌細(xì)胞(HepG2)凋亡率明顯高于正常肝細(xì)胞(L-02)(P0.01)。隨著CXC195劑量從10μM逐漸增加到150μM,HepG2細(xì)胞凋亡率從5.04%逐漸上升到35.21%,而L-02細(xì)胞凋亡率從4.03%逐漸上升到19.30%。隨著CXC195劑量增加,HepG2細(xì)胞凋亡數(shù)量明顯增多(P0.01),相同劑量CXC195作用于兩組細(xì)胞比較,HepG2細(xì)胞的凋亡率明顯高于對照組L-02細(xì)胞。結(jié)果表明:CXC195誘導(dǎo)肝癌細(xì)胞(HepG2)凋亡作用顯著強(qiáng)于正常肝細(xì)胞(L-02),且隨著藥物濃度增加,誘導(dǎo)凋亡作用逐漸增強(qiáng)。(3)CXC195對HepG2肝癌細(xì)胞PI3K-AKT-mTOR信號(hào)通路的影響:采用Western blotting分析,濃度為150μΜ的CXC195作用HepG2細(xì)胞,Bcl-2/Bax比值、p-PI3K和PI3K、p-AKT和AKT及p-mTOR和mTOR的表達(dá)量均非常明顯少于未加CXC195對照組(P0.01)。聯(lián)合使用通路上PI3K、AKT、mTOR對應(yīng)的抑制劑LY294002,SH-6和雷帕霉素處理5μmol/L后,細(xì)胞凋亡率超過50%,同時(shí)通路相關(guān)蛋白p-PI3K和PI3K、p-AKT和AKT及p-mTOR和mTOR的表達(dá)量較單用150uM CXC195均非常明顯減少(P0.01)。結(jié)果表明:CXC195通過抑制PI3K-AKT-mTOR信號(hào)傳導(dǎo)通路相關(guān)蛋白誘導(dǎo)HepG2細(xì)胞凋亡,降低HepG2細(xì)胞增殖。(4)采用Western blotting檢測發(fā)現(xiàn),在經(jīng)過濃度為150μΜCXC195作用后,HepG2細(xì)胞中GRP78,GRP94、CHOP、p-PERK,PERK,p-eIF2α,eIF2α、ATF4、IRE1α,p-ASK,ASK,p-p38,p38,p-JNK、JNK、Cleaved ATF6和ATF6表達(dá)量明顯高于對照組。說明了內(nèi)質(zhì)網(wǎng)應(yīng)激參與了CXC195誘導(dǎo)的HepG2細(xì)胞凋亡過程。結(jié)論:CXC195能顯著抑制HepG2肝癌細(xì)胞增殖,誘導(dǎo)細(xì)胞凋亡,主要針對S期和G2/M期細(xì)胞。而對正常肝細(xì)胞(L-02)抑制生長和誘導(dǎo)凋亡的作用則明顯較弱。CXC195的這一作用可能是通過抑制PI3K-AKT-mTOR信號(hào)通路蛋白表達(dá)和激活內(nèi)質(zhì)網(wǎng)應(yīng)激來實(shí)現(xiàn)的。這些研究結(jié)果提示CXC195在體外對肝癌細(xì)胞有一定治療效果。
[Abstract]:Objective: To study the apoptosis of HepG2 cells induced by CXC195 in vitro and to inhibit cell proliferation in vitro. To explore the PI3K-AKT-mTOR signaling pathway and endoplasmic reticulum stress to induce apoptosis of HepG2 cells. To provide help for CXC195 in the treatment of liver cancer. Materials and methods: in vitro culture, HepG2 cell lines were used as experimental group, and L-02 cell lines were used to synthesize CXC195. by chemical reaction. The survival of HepG2 cells and L-02 cells was detected by CXC195 and MTT colorimetric assay, and the cell apoptosis was analyzed by flow cytometry. The detection concentration was 10 mu M, 25 mu M, 50 mu M, 100 mu M, 150 micron, and 200 micron M CXC195 acted on the average growth survival rate of HepG2 cells and cells. 150 mu The expression of PI3K, AKT, mTOR and phosphorylated protein and endoplasmic reticulum stress related protein expression in PI3K-AKT-mTOR signaling pathway after the action of XC195 on HepG2 cells. Results: (1) CXC195 inhibits HepG2 cell growth: trypan blue stain method and MMT colorimetric assay show cell survival results, after adding CXC195, human hepatoma cells (HepG2) and normal hepatocyte (L-02) birth The long survival rate decreased significantly, and as the dose of CXC195 increased from 10 to 200 M, the survival rate of HepG2 cells decreased from 97.3% to 42.9%. The survival rate of L-02 cells decreased from 98.5% to 65.1%. at different doses of CXC195, and there was a significant difference in the survival rate of the same cells (P0.01). The same dose of CXC195 was used in the two groups of cells. The survival rate of liver cancer was less than that of normal hepatocyte L-02.MMT colorimetric assay. The results showed that the survival rate of HepG2 cells was decreased in 12,24,48,72 hours with 150 mu MCXC195 (P0.01). Flow cytometry found that the cells survived more G0/G1 (71.62%vs45.17%, P0.01) after 24 hours of HepG2 cells with 150 M CXC195. The proportion of phase (17.53%vs41.11, P0.01) and G2/M (10.85%vs13.72%, P0.05) cells decreased significantly. The results showed that CXC195 could inhibit the growth of hepatoma cells (HepG2) more than normal hepatocytes (L-02). The inhibitory effect of CXC195 on the growth of hepatoma cells was positively correlated with the drug concentration and the time of drug action. And CXC195 was mainly used in S phase and G2. HepG2. (2) CXC195 induced apoptosis of HepG2 cells in phase /M: after AnnexinV/PI double staining, the apoptosis rate of human hepatoma cells (HepG2) induced by CXC195 was significantly higher than that of normal liver cells (L-02) (P0.01). The apoptosis rate of CXC195 was gradually increased from 5.04% to 150 micron. Up to 35.21%, while the apoptosis rate of L-02 cells increased from 4.03% to 19.30%., with the increase of CXC195 dose, the number of apoptotic HepG2 cells increased significantly (P0.01). The same dose of CXC195 acted on the two groups of cells, and the apoptosis rate of HepG2 cells was significantly higher than that of the control group L-02 cells. The results showed that the apoptosis effect of CXC195 induced hepatoma cells (HepG2) was significant. Stronger than normal hepatocytes (L-02), and with the increase of drug concentration, the induction of apoptosis gradually increased. (3) the effect of CXC195 on the PI3K-AKT-mTOR signaling pathway of HepG2 hepatoma cells: Western blotting analysis, the CXC195 action of HepG2 cells with a concentration of 150 micron, Bcl-2/Bax ratio, p-PI3K and PI3K. The apoptosis rate of PI3K, AKT, mTOR corresponding to LY294002, SH-6 and rapamycin treated with 5 u mol/L was less than that of the control group (P0.01). The results show that CXC195 can induce HepG2 cell apoptosis by inhibiting PI3K-AKT-mTOR signal transduction pathway related proteins and reduce the proliferation of HepG2 cells. (4) Western blotting detection was used to detect GRP78, GRP94, CHOP, HepG2 cells in HepG2 cells. The expression of Cleaved ATF6 and ATF6 was significantly higher than that in the control group. It showed that endoplasmic reticulum stress participated in the apoptosis process of HepG2 cells induced by CXC195. Conclusion: CXC195 can significantly inhibit the proliferation of HepG2 hepatoma cells and induce apoptosis, which is mainly aimed at S phase and G2/M phase cells, but the inhibitory effect of normal liver cells (L-02) on growth and induction of apoptosis is obvious. This effect of weak.CXC195 may be achieved by inhibiting the expression of PI3K-AKT-mTOR signaling pathway protein and activation of endoplasmic reticulum stress. These results suggest that CXC195 has a certain therapeutic effect on liver cancer cells in vitro.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7

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