本文選題：ARHGAP9　切入點(diǎn)：Rg3　出處：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：高侵襲轉(zhuǎn)移是原發(fā)性肝癌(簡(jiǎn)稱“肝癌”)重要的生物學(xué)特性,也是導(dǎo)致肝癌高死亡率的重要原因。然而,其發(fā)病機(jī)制并不明確,阻礙了肝癌的有效診治。近幾年研究表明,Rho GAP蛋白家族的許多成員,如p190Rho GAP、DLC1和ARHGAP在人肝癌組織中明顯上調(diào)。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn),ARHGAP9基因在肝癌組織中明顯低表達(dá),且低表達(dá)患者相對(duì)于高表達(dá)患者預(yù)后差。ARHGAP9的高表達(dá)還抑制肝癌侵襲轉(zhuǎn)移相關(guān)信號(hào)通路的激活。由此我們推測(cè),ARHGAP9基因與肝癌的發(fā)生發(fā)展密切相關(guān)。人參皂苷Rg3是人參的重要組成部分,對(duì)多種惡性腫瘤均具有抑制作用。由于其顯著的抗癌療效,人參皂苷Rg3的單體已被開發(fā)成我國一類抗癌新藥—參一膠囊。實(shí)驗(yàn)和臨床研究均已證實(shí),人參皂苷Rg3在治療肝癌的復(fù)發(fā)轉(zhuǎn)移中發(fā)揮重要作用。然而,人參皂苷Rg3抗肝癌的作用機(jī)制研究并不深入,缺乏臨床治療依據(jù),限制了人參皂苷Rg3在臨床上的廣泛應(yīng)用。鑒于我們前期發(fā)現(xiàn),ARHGAP9與肝癌的發(fā)生發(fā)展相關(guān),人參皂苷Rg3抗肝癌是否與ARHGAP9基因相關(guān)值得深入探討。因此本課題的研究主要分為兩個(gè)部分:(1)分析ARHGAP9基因在肝癌生長及侵襲轉(zhuǎn)移中的作用;(2)確定人參皂苷Rg3是否通過ARHGAP9基因抑制肝癌細(xì)胞的侵襲遷移。(一)ARHGAP9在肝癌生長轉(zhuǎn)移中的作用研究我們首先通過細(xì)胞轉(zhuǎn)染和慢病毒感染構(gòu)建了ARHGAP9基因干擾的肝癌細(xì)胞株和過表達(dá)的肝癌細(xì)胞株。然后采用CCK-8、流式細(xì)胞術(shù)、Transwell等一系列實(shí)驗(yàn)檢測(cè)分析ARHGAP9基因干擾和過表達(dá)對(duì)肝癌細(xì)胞增殖、周期、凋亡、侵襲和遷移能力的影響。體內(nèi)實(shí)驗(yàn),通過腋下注射ARHGAP9過表達(dá)的Hep G2肝癌細(xì)胞株建立裸鼠皮下成瘤模型,分析ARHGAP9對(duì)裸鼠皮下瘤生長情況的影響。取得的實(shí)驗(yàn)結(jié)果如下:1、ARHGAP9在肝癌細(xì)胞系中的表達(dá)6種肝癌細(xì)胞株中,ARHGAP9基因表達(dá)最高的是MHCC-97L細(xì)胞,其余5種細(xì)胞株中ARHGAP9基因的表達(dá)相對(duì)較低且相互之間無顯著性差異。因此選擇MHCC-97L細(xì)胞進(jìn)行基因干擾研究,選擇Hep G2和MHCC-97H細(xì)胞進(jìn)行基因過表達(dá)研究。2、干擾ARHGAP9基因?qū)Ω伟┘?xì)胞增殖、周期和凋亡的影響干擾ARHGAP9基因顯著促進(jìn)了MHCC-97L細(xì)胞增殖,抑制細(xì)胞凋亡,且S期細(xì)胞比率增加。3、過表達(dá)ARHGAP9基因?qū)Ω伟┘?xì)胞增殖、周期和凋亡的影響過表達(dá)ARHGAP9基因顯著抑制了Hep G2和MHCC-97H細(xì)胞增殖,促進(jìn)了細(xì)胞凋亡,并將細(xì)胞周期阻滯在G0/G1期。4、ARHGAP9對(duì)肝癌細(xì)胞侵襲和遷移的影響干擾ARHGAP9基因顯著促進(jìn)了MHCC-97L細(xì)胞的遷移和侵襲,過表達(dá)ARHGAP9基因顯著抑制了Hep G2和MHCC-97H細(xì)胞的遷移和侵襲。5、ARHGAP9對(duì)裸鼠肝癌生長的影響ARHGAP9過表達(dá)組腫瘤生長速度明顯低于對(duì)照組。從第24天開始,ARHGAP9過表達(dá)組腫瘤體積較對(duì)照組明顯降低(p0.05)。結(jié)論:我們的研究結(jié)果顯示,ARHGAP9基因高表達(dá)顯著抑制肝癌發(fā)展過程。(二)基于ARHGAP9的人參皂苷Rg3對(duì)肝癌細(xì)胞侵襲遷移的影響我們采用CCK-8、Transwell、PCR、Western blot以及基因干擾等實(shí)驗(yàn)觀察不同濃度20(R)-Rg3對(duì)肝癌細(xì)胞侵襲遷移能力以及對(duì)細(xì)胞內(nèi)ARHGAP9表達(dá)的影響。取得的實(shí)驗(yàn)結(jié)果如下:1、人參皂苷Rg3對(duì)肝癌細(xì)胞活力的影響采用CCK-8實(shí)驗(yàn)檢測(cè)不同濃度人參皂苷Rg3對(duì)肝癌細(xì)胞活力的影響。當(dāng)Rg3處理Hep G2和MHCC-97L細(xì)胞24 h后,實(shí)驗(yàn)組(1.25、2.5和5μg/m L)的細(xì)胞活力與對(duì)照組相比無統(tǒng)計(jì)學(xué)差異。2、人參皂苷Rg3抑制肝癌細(xì)胞的侵襲遷移人參皂苷Rg3在濃度為1.25、2.5和5μg/m L時(shí)均顯著抑制了Hep G2和MHCC-97L細(xì)胞的侵襲和遷移。3、人參皂苷Rg3提高肝癌細(xì)胞中ARHGAP9的表達(dá)與對(duì)照組相比,人參皂苷Rg3(2.5和5μg/m L)顯著提高了Hep G2和MHCC-97L細(xì)胞中ARHGAP9蛋白的表達(dá)。4、人參皂苷Rg3通過ARHGAP9抑制肝癌細(xì)胞侵襲遷移沉默ARHGAP9基因后顯著緩解了人參皂苷Rg3(2.5和5μg/m L)對(duì)Hep G2和MHCC-97L細(xì)胞的抗侵襲遷移作用。結(jié)論:我們的研究結(jié)果顯示,人參皂苷Rg3可抑制肝癌細(xì)胞的侵襲遷移,且此過程與人參皂苷Rg3誘導(dǎo)肝癌細(xì)胞內(nèi)ARHGAP9的表達(dá)有關(guān)。
[Abstract]:High invasion and metastasis of primary liver cancer ("cancer") important biological properties, is an important cause of cancer mortality. However, its pathogenesis is not clear, hinder the effective diagnosis and treatment of liver cancer. In recent years, research shows that many members of the Rho family of GAP proteins, such as p190Rho DLC1 and GAP. ARHGAP in human hepatocellular carcinoma was up-regulated. Our previous study showed that ARHGAP9 gene expression in hepatocellular carcinoma tissues was significantly lower, and patients with low expression relative to the activation of high high expression and poor prognosis of.ARHGAP9 also inhibited the invasion and metastasis of HCC related signaling pathway. We conclude that closely related to the occurrence and development of ARHGAP9 gene and hepatocellular carcinoma. Ginsenoside Rg3 is an important part of ginseng, has inhibitory effect on many malignant tumors. Because of its remarkable anticancer effect of ginsenoside Rg3 has been developed into our country A class of new anticancer drug - capsule. The experimental and clinical studies have confirmed that ginsenoside Rg3 plays an important role in the treatment of liver cancer recurrence and metastasis. However, the mechanism of ginsenoside Rg3 on hepatocellular carcinoma is not thorough, the lack of evidence for clinical treatment, limited the wide application of ginsenoside Rg3 in clinic. In view of our early discovery, occurrence and development of ARHGAP9 and hepatocellular carcinoma, ginsenoside Rg3 against hepatocellular carcinoma is associated with the ARHGAP9 gene which is worthy of further exploration. This research is mainly divided into two parts: (1) analysis of ARHGAP9 gene in hepatocellular carcinoma growth and invasion and metastasis; (2) to determine whether the invasion and migration of ginsenoside Rg3 inhibition of ARHGAP9 gene in hepatocarcinoma cells. (a) role of ARHGAP9 in metastasis of hepatocellular carcinoma we begin by cell transfection and lentivirus infection to construct ARHGAP9 gene interference small hepatocellular carcinoma Cell lines and expression in hepatocellular carcinoma cells. Then using CCK-8, flow cytometry, Transwell and a series of experimental detection and analysis of ARHGAP9 gene interference and overexpression on the proliferation of hepatocellular carcinoma cell cycle, apoptosis, and influence the invasion and migration ability. In vivo, through axillary injection of ARHGAP9 over expression of Hep G2 cell line establishment the nude mice tumor model, analysis of the influence of ARHGAP9 on the growth of subcutaneous tumors. The experimental results are as follows: 1, ARHGAP9 expression in hepatocellular carcinoma cells in the 6 hepatoma cell lines, ARHGAP9 gene expression was highest in MHCC-97L cells, expression of the 5 ARHGAP9 genes in cell lines is relatively low and the mutual there was no significant difference between MHCC-97L cells. So the choice of gene interference, gene overexpression of.2 Hep and G2 MHCC-97H cells, the interference of ARHGAP9 gene on proliferation of hepatocellular carcinoma cell cycle, and Effect of ARHGAP9 gene apoptosis significantly promoted MHCC-97L cell proliferation, inhibition of apoptosis, and the cell ratio in S phase increased.3, overexpression of ARHGAP9 gene on proliferation of hepatocellular carcinoma cell cycle and apoptosis, effects of overexpression of ARHGAP9 significantly inhibited the proliferation of Hep G2 cells and MHCC-97H cells, promote cell apoptosis and cell cycle arrest G0/G1.4, the interference effect of ARHGAP9 gene ARHGAP9 on the invasion and migration of hepatocellular carcinoma cells significantly promoted the migration and invasion of MHCC-97L cells, overexpression of ARHGAP9 significantly inhibited the Hep G2 and MHCC-97H cell migration and invasion of.5, the influence of ARHGAP9 on the growth of human hepatocellular carcinoma in nude mice ARHGAP9 overexpression group the tumor growth rate was significantly lower than the control group. From the beginning of the twenty-fourth day, ARHGAP9 overexpression group tumor volume was significantly lower than the control group (P0.05). Conclusion: Our results suggest that high expression of ARHGAP9 gene was significantly inhibited The development process of liver cancer. (two) the effect of ginsenoside Rg3 on hepatocellular carcinoma cell invasion and migration of ARHGAP9 based on Transwell, we use CCK-8, PCR, Western and blot gene interference experiment observation of different concentrations of 20 (R) the effects of -Rg3 on the invasion and migration ability of hepatoma cells on the expression of ARHGAP9 in cells. The experimental results are as follows 1, the effect of ginsenoside Rg3 on the hepatocellular carcinoma cell viability effect on liver cancer cell viability by CCK-8 assay with different concentrations of ginsenoside Rg3. When Rg3 G2 and MHCC-97L Hep cells after 24 h, the experimental group (1.25,2.5 and 5 g/m L) cell viability compared with the control group there was no significant difference in.2, ginsenoside Rg3 inhibition of cancer cell invasion and migration of ginsenoside Rg3 in the concentration of 1.25,2.5 and 5 g/m L significantly inhibited the invasion and migration of.3 Hep G2 and MHCC-97L cells, ginsenoside Rg3 increased ARHGA in liver cancer cells The expression of P9 compared with the control group, ginsenoside Rg3 (2.5 and 5 g/m L) significantly increased the expression of.4 G2 and Hep ARHGAP9 protein in MHCC-97L cells, ginsenoside Rg3 inhibited the migration and invasion of ARHGAP9 gene silencing significantly alleviated the ginsenoside Rg3 by ARHGAP9 cells (2.5 and 5 g/m L) anti invasion effect on Hep G2 and migration of MHCC-97L cells. Conclusion: Our results show that ginsenoside Rg3 can inhibit the invasion and migration of hepatocellular carcinoma cells, and this process with ginsenoside Rg3 induced expression in hepatocellular carcinoma cell line ARHGAP9.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

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