【摘要】：肝癌是最常見(jiàn)的非傳染性的疾病之一,該癌癥的發(fā)生率每年都有升高。當(dāng)前的治療手段如化學(xué)療法等的毒副作用大,因此尋找新型有效、安全的治療藥物是亟待解決的問(wèn)題,而天然化合物是抗癌新藥的主要來(lái)源,逐漸引起了人們的關(guān)注。刺果番荔枝被俗稱作“癌癥殺手”,能導(dǎo)致多種腫瘤細(xì)胞發(fā)生凋亡,然而其抗癌及誘導(dǎo)凋亡的分子機(jī)制仍需要進(jìn)一步研究。本文針對(duì)刺果番荔枝葉片的乙醇提取物,以肝癌HepG2細(xì)胞作為細(xì)胞模型,探究該提取物能否以及如何誘導(dǎo)肝癌細(xì)胞發(fā)生凋亡。主要結(jié)果如下:1.MTT法檢測(cè)到刺果番荔枝的提取物能夠降低肝癌HepG2細(xì)胞和結(jié)腸癌細(xì)胞HCT116的活力,并呈時(shí)間和劑量依賴效應(yīng);流式細(xì)胞術(shù)的結(jié)果顯示,該提取物可以促使SubG1期(指示凋亡細(xì)胞)細(xì)胞數(shù)增多,并具有劑量依賴效應(yīng);TUNEL法結(jié)果顯示,提取物處理(120μg/ml)的細(xì)胞凋亡率明顯較高。2.蛋白質(zhì)組學(xué)技術(shù)結(jié)果:通過(guò)對(duì)比處理組和對(duì)照組的差異蛋白點(diǎn),共找到14個(gè)差異蛋白并進(jìn)行了蛋白身份的鑒定;之后對(duì)14個(gè)蛋白進(jìn)行KEGG通路分析尋找可能的分子通路,從分析結(jié)果中發(fā)現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激信號(hào)通路,并且有三個(gè)蛋白涉及其中:分別為HSP70、GRP94、DPI-5蛋白。3.通過(guò)Western blot實(shí)驗(yàn)鑒定,該提取物增加了Bip和CHOP蛋白的表達(dá)量,提高了PERK和eIF2α的磷酸化水平,并呈現(xiàn)時(shí)間劑量依賴效應(yīng)。當(dāng)通過(guò)si RNA技術(shù)敲低CHOP蛋白的水平時(shí),提取物引起的凋亡受到抑制,有時(shí)間依賴效應(yīng)。siCHOP敲低CHOP的表達(dá),發(fā)現(xiàn)細(xì)胞的凋亡率較處理組明顯減小。4.為了進(jìn)一步研究該提取物誘導(dǎo)肝癌細(xì)胞凋亡的分子機(jī)制,我們?cè)噲D檢測(cè)該提取物是否引起了腫瘤細(xì)胞內(nèi)活性氧(reactive oxygen species,ROS)的升高。通過(guò)DCFH-DA熒光探針定量細(xì)胞內(nèi)的ROS水平,并使用抗氧化劑NAC驗(yàn)證結(jié)果。發(fā)現(xiàn)提取物處理細(xì)胞2 h后,細(xì)胞內(nèi)ROS的水平較對(duì)照組顯著性升高,同時(shí)NAC能顯著性抑制提取物引起的ROS的升高和細(xì)胞凋亡。結(jié)論:1.刺果番荔枝葉片的乙醇提取物可以降低HepG2細(xì)胞的活性,并呈現(xiàn)時(shí)間和劑量依賴效應(yīng),而且可以促使HepG2細(xì)胞凋亡,說(shuō)明該提取物可以用于開(kāi)發(fā)新型抗腫瘤藥物,如肝癌。2.刺果番荔枝葉片的乙醇提取物通過(guò)內(nèi)質(zhì)網(wǎng)應(yīng)激信號(hào)通路引起肝癌細(xì)胞凋亡。3.刺果番荔枝葉片的乙醇提取物還可以通過(guò)ROS水平的升高引起細(xì)胞凋亡,說(shuō)明提取物的抗癌機(jī)制也與ROS升高有關(guān)。
[Abstract]:Liver cancer is one of the most common non-communicable diseases and its incidence increases every year. The current treatment methods such as chemotherapy have great toxic and side effects, so finding new effective and safe treatment drugs is an urgent problem, and natural compounds are the main source of new anticancer drugs, which has gradually attracted people's attention. Annona chinensis is commonly known as "cancer killer", which can induce apoptosis of many kinds of tumor cells. However, the molecular mechanism of its anticancer and apoptosis still needs to be further studied. In this paper, the ethanol extract from the leaves of Annona chinensis was used as a cell model to explore whether and how the extract could induce apoptosis of hepatoma cells. The main results were as follows: 1.MTT assay showed that the extract of Annona chinensis could reduce the activity of HCT116 in HepG2 cells and colon cancer cells in a time-and dose-dependent manner. The results of flow cytometry showed that the extract could increase the number of apoptotic cells in SubG1 phase (indicating apoptotic cells) in a dose-dependent manner, and the TUNEL assay showed that the apoptotic rate of cells treated with the extract (120 渭 g/ml) was significantly higher than that of control group (120 渭 g/ml). Results of proteomics technique: 14 differentially expressed proteins were found and identified by comparing the differentially expressed protein sites between the treatment group and the control group. After that, 14 proteins were analyzed by KEGG pathway to search for possible molecular pathways. Endoplasmic reticulum stress signaling pathway was found from the analysis results, and there were three proteins involved in it: HSP70,GRP94,DPI-5 protein. 3. Western blot assay showed that the extract increased the expression of Bip and CHOP protein, increased the phosphorylation level of PERK and eIF2 偽, and showed a time-dose dependent effect. When the level of CHOP protein was knocked down by si RNA technique, the apoptosis induced by the extract was inhibited, and there was a time-dependent effect. SiCHOP knock down the expression of CHOP and found that the apoptotic rate of the cells was significantly lower than that of the treatment group. 4. In order to further study the molecular mechanism of apoptosis induced by the extract, we try to detect whether the extract can induce the increase of reactive oxygen species (reactive oxygen species,ROS) in tumor cells. DCFH-DA fluorescence probe was used to quantify the intracellular ROS level and the antioxidant NAC was used to verify the results. It was found that the level of ROS in the cells treated with the extract for 2 h was significantly higher than that in the control group, while NAC could significantly inhibit the increase of ROS and apoptosis induced by the extract. Conclusion: 1. The ethanol extract from the leaves of Annona chinensis can reduce the activity of HepG2 cells in a time-and dose-dependent manner, and induce the apoptosis of HepG2 cells, indicating that the extract can be used to develop new anti-tumor drugs, such as liver cancer. 2. Ethanol extract from the leaves of Annona chinensis induced apoptosis of hepatoma cells through endoplasmic reticulum stress signaling pathway. Ethanol extracts from the leaves of Annona chinensis could also induce apoptosis by increasing the level of ROS, indicating that the anticancer mechanism of the extracts was also related to the increase of ROS.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉金娟;曹成亮;丁盼;蔣繼宏;;野莧菜提取物抗腫瘤作用及誘導(dǎo)人肝癌HepG2細(xì)胞凋亡的分子機(jī)制[J];中國(guó)藥理學(xué)通報(bào);2015年11期

2 吳芳;安永康;朱艷琴;;內(nèi)質(zhì)網(wǎng)應(yīng)激與腫瘤細(xì)胞凋亡研究進(jìn)展[J];現(xiàn)代腫瘤醫(yī)學(xué);2014年09期

3 張凱強(qiáng);張穎;顧何鋒;劉璐;馬林;;內(nèi)質(zhì)網(wǎng)應(yīng)激與細(xì)胞凋亡研究進(jìn)展[J];口腔醫(yī)學(xué);2013年06期

4 宋洋;袁宜勤;郁潔;;內(nèi)質(zhì)網(wǎng)應(yīng)激PERK凋亡通路研究進(jìn)展[J];中華中醫(yī)藥學(xué)刊;2013年05期

5 張亞宏;甘瑩;郭子華;謝松強(qiáng);;紫草素通過(guò)ROS/p38信號(hào)通路誘導(dǎo)人宮頸癌HeLa細(xì)胞凋亡[J];中國(guó)藥理學(xué)通報(bào);2011年06期

6 眭維國(guó);黃禮玲;陳潔晶;黃河;戴勇;;蛋白質(zhì)組學(xué)技術(shù)在癌癥相關(guān)研究中的應(yīng)用[J];醫(yī)學(xué)綜述;2008年23期

7 熊戴群;羅輝;;細(xì)胞凋亡與腫瘤放射敏感性[J];實(shí)用臨床醫(yī)學(xué);2008年02期

8 張愛(ài)玲;王興龍;;蛋白質(zhì)組學(xué)及其在獸醫(yī)學(xué)中的應(yīng)用[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2008年02期

9 賴斐,謝衡,王金發(fā);松樹(shù)皮提取物Pycnogenol的生物學(xué)和疾病防治功能研究進(jìn)展[J];中草藥;2005年01期

10 陳建白;亦果亦藥的刺果番荔枝[J];云南熱作科技;2002年02期

相關(guān)博士學(xué)位論文 前1條

1 關(guān)麗英;活性氧激活內(nèi)質(zhì)網(wǎng)應(yīng)激和線粒體凋亡通路介導(dǎo)亞硒酸鈉誘導(dǎo)NB4細(xì)胞凋亡的研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2008年

相關(guān)碩士學(xué)位論文 前3條

1 高蓓;阿爾巴斯絨山羊FGF5基因敲除的研究[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2012年

2 溫廣輝;蛋白質(zhì)相互作用網(wǎng)絡(luò)演化模型[D];南京航空航天大學(xué);2008年

3 肖陽(yáng);家蠶細(xì)胞凋亡相關(guān)基因BmIce的功能鑒定[D];西南大學(xué);2007年

，

本文編號(hào)：2390104