溪黄草水溶性总黄酮对人肝癌裸鼠移植瘤抑制及机制研究

发布时间：2018-06-06 22:11

本文选题：溪黄草水溶性总黄酮 + HepG2细胞　；参考：《广州中医药大学》2017年博士论文

【摘要】：肝细胞癌(Hepatocellular carcinoma,HCC)是一种全球范围内发病率和死亡率都很高的疾病,目前已成为威胁人类生命健康安全的恶性肿瘤,肝细胞癌的主要特点为疾病发展速度快、术后复发率高、癌细胞转移能力强,这给肝细胞癌的有效防治带来了巨大的困难,与化学合成单体药物相比较,传统植物药具有众多的优势,越来越多的植物药被发现具有抗肿瘤活性。因此,对植物药的研究是探索天然的抗肿瘤药物的重要途径。狭基线纹香茶菜Isodon lophanthoides var gerardianus(Benth)Hara 为唇形科多年生草本植物,其地上部分是广东传统中药溪黄草品种之一,为当前溪黄草的主流种植品种,具有利胆退黄、清热祛湿作用,为防治肝胆系统疾病的常用药,实验表明狭基线纹香茶菜的水提液具有良好的抗炎、抗肿瘤、保肝活性,其提取物的抗肿瘤活性研究较多,但其药效物质基础不清楚,我们研究团队前期筛选狭基线纹香茶菜水溶性成分的抗肿瘤活性,发现水溶性总黄酮部位能显著地抑制HepG2细胞活性,预实验结论也显示具有诱导细胞凋亡作用,有可能成为抗肿瘤候选药物,但药理研究基础薄弱,其机制也没有涉及。因此,研究其抑制HepG2细胞体内外活性及作用机制,对于深入开发应用溪黄草具有重要意义。本研究所用溪黄草药材为狭基线纹香茶菜。目的:本论文对溪黄草水溶性总黄酮的富集纯化方法进行了改良,研究溪黄草水溶性总黄酮对人肝癌细胞HepG2生长抑制和诱导凋亡的作用及机制,通过建立荷瘤裸鼠动物模型,探讨研究溪黄草水溶性总黄酮对人肝癌裸鼠移植瘤抑制作用及相关机制。方法:一、溪黄草水溶性总黄酮对人肝癌细胞HepG2生长抑制作用采用细胞增殖抑制实验(MTT法)检测溪黄草水溶性总黄酮对人肝癌细胞HepG2的生长抑制率。通过平皿集落形成试验观察溪黄草水溶性总黄酮对HepG2细胞贴壁生长并形成克隆的能力。流式细胞术检测水溶性总黄酮对HepG2细胞周期分布的影响。二、溪黄草水溶性总黄酮对人肝癌细胞HepG2凋亡的影响通过检测细胞线粒体膜电位,细胞内ROS水平和流式细胞术分析水溶性总黄酮诱导HepG2细胞凋亡情况。三、溪黄草水溶性总黄酮诱导人肝癌细胞HepG2凋亡机制研究电镜检测细胞内自噬体情况,采用RT-PCR法检测溪黄草水溶性总黄酮对各组细胞Bcl-2mRNA、Bax mRNA、Survivin mRNA的基因表达变化、运用Western blot法检测各组的线粒体和胞质 cytochrome c,cytosolic AIF、Bcl-2、Bax、Survivin 蛋白表达,caspase-3蛋白表达。四、溪黄草水溶性总黄酮对人肝癌裸鼠移植瘤抑制作用研究建立荷瘤裸鼠动物模型,给药后观察并测量裸鼠瘤体,计算体积大小,并绘出生长曲线。分离瘤体后,计算抑制率,病理组织学检查,检测外周血甲胎球蛋白变化,TUNEL染色检测各组移植瘤细胞凋亡。五、溪黄草水溶性总黄酮对人肝癌裸鼠移植瘤抑制作用机制研究透射电镜观察瘤组织细胞的自噬小体和细胞超微结构,RT-PCR法检测基因Beclinl、Atg5、CyclinD1、Caspase-3、bax、bcl-2、Survivin、nm23-H1 和 MMP-9 mRNA的表达,Western blot 法检测指标:Beclinl、Atg5、CyclinD1、Caspase-3、bax、bcl-2、Survivin、nm23-H1 和 MMP-9 蛋白表达。结果:一、溪黄草水溶性总黄酮对人肝癌细胞HepG2生长抑制作用MTT法结果显示,水溶性总黄酮对HepG2细胞增殖有明显的抑制作用,呈现浓度依赖性和一定的时间依赖关系。给药后显微镜下观察到细胞出现各期凋亡细胞形态,产生凋亡小体,提示可能与促进HepG2细胞凋亡有关。细胞平皿集落形成实验显示,水溶性总黄酮处理组的细胞集落形成数明显少于溶媒对照组。提示可能与抑制细胞锚定依赖性生长作用有关。细胞周期实验结果显示,总黄酮各浓度组G0/G1期细胞比例均明显高于对照组,而S期细胞比例均明显低于对照组,这说明水溶性总黄酮能有效地将HepG2细胞阻滞于G0/G1期,进而抑制细胞增殖和生长,呈现剂量依赖性。二、溪黄草水溶性总黄酮对人肝癌细胞HepG2凋亡的影响流式细胞术分析结果显示,水溶性总黄酮各浓度组和各时间点的凋亡率均明显高于对照组,呈剂量和时间依赖关系。细胞线粒体膜电位实验显示,水溶性总黄酮和5-FU均能诱导HepG2细胞线粒体膜电位去极化。检测细胞内ROS水平,结果显示,水溶性总黄酮能诱导HepG2细胞内ROS升高。这些提示水溶性总黄酮能通过干扰细胞线粒体膜电位,升高细胞内ROS水平,诱导HepG2细胞凋亡。三、溪黄草水溶性总黄酮诱导人肝癌细胞HepG2凋亡机制研究透射电镜下观察,水溶性总黄酮具有抑制细胞自噬作用;RT-PCR法检测结果显示,抗凋亡蛋白基因bcl-2 mRNA和Survivin mRNA随着水溶性总黄酮浓度升高而减少,呈浓度依赖关系,相反,促凋亡蛋白基因BaxmRNA和Caspase-3mRNA随着药物浓度升高而增加,也呈浓度依赖关系。Western blot法分析结果也进一步证实水溶性总黄酮是通过下调抗凋亡蛋白(bcl-2 and mcl-1)和survivin和上调促凋亡蛋白(Bax)的表达水平诱导HepG2细胞凋亡。WB分析还发现,水溶性总黄酮还增高线粒体膜通透性,向胞质内释放促凋亡因子Cytochrome c和Cytosolic AIF,引起细胞凋亡。四、溪黄草水溶性总黄酮对人肝癌裸鼠移植瘤抑制作用研究各组连续给药21天,绘制肿瘤生长曲线,计算抑瘤率,与模型组相比,水溶性总黄酮高、中剂量药物组和5-FU组均能抑制肿瘤的生长,呈剂量相关性,其中以药物高剂量组、5-FU组和水溶性总黄酮高剂量+5-FU组效果最佳。各给药组与模型组比较,均能显著地降低AFP。而各给药组的肿瘤/体重比结果显示,水溶性总黄酮高剂量+5-FU组动物的基本情况明显好转,说明水溶性总黄酮和5-FU联合应用既抑制了肿瘤生长,又克服了化药的副作用,改善了生活质量。病理组织学检查发现,水溶性总黄酮各组的瘤体中肿瘤细胞发生凋亡,有中度坏死灶,这表明水溶性总黄酮对癌细胞具有促进凋亡、抑制分裂增殖的作用。荷瘤裸鼠胸腹腔未见积液,心、肝、肾、脾、肺、脑等脏器未见癌细胞转移结节及其他异常。Tunel染色发现,水溶性总黄酮组随着浓度的增大凋亡细胞数量增加,显示出一定的量效关系。五、溪黄草水溶性总黄酮对人肝癌裸鼠移植瘤抑制作用机制研究电镜观察水溶性总黄酮抑制肝癌细胞形成自噬体,这与细胞实验中的结论相一致。细胞超微结构也出现了明显的形态学改变和大量的凋亡小体。RT-PCR法检测表明水溶性总黄酮是通过下调抗凋亡蛋白基因bcl-2 mRNA和survivinmRNA和上调促凋亡蛋白Bax mRNA和Caspase-3 mRNA的表达水平诱导移植瘤HepG2细胞凋亡。水溶性总黄酮能抑制自噬基因Beclin1mRNA、Atg5mRNA的表达,能抑制CyclinD1 mRNA、MMP-9mRNA基因的表达,且呈现浓度越高越明显的浓度依赖关系,相反,水溶性总黄酮能促进nm23-H1基因的表达,浓度越高越明显。Western blot法分析结果也进一步证实水溶性总黄酮是通过下调抗凋亡蛋白基因bcl-2和Survivin和上调促凋亡蛋白Bax和Caspase-3的表达水平诱导移植瘤HepG2细胞凋亡。水溶性总黄酮还能抑制Beclin1、Atg5、CyclinD1、MMP-9等蛋白的表达,相反,水溶性总黄酮能促进nm23-H1蛋白的表达,结论:本研究以溪黄草水溶性总黄酮为研究对象,应用了多种方法研究了其体内外抑制HepG2细胞的活性及其机制。在体外,水溶性总黄酮有效地将HepG2细胞阻滞于G0/G1期,干扰细胞线粒体膜电位,升高细胞内ROS水平,诱导HepG2细胞凋亡。其机制可能是通过下调抗凋亡蛋白(bcl-2 and mcl-1)和survivin和上调促凋亡蛋白(Bax)的表达水平,促使细胞色素C向胞质内释放,诱导HepG2细胞凋亡,抑制细胞增殖。在体内,水溶性总黄酮能抑制裸鼠移植瘤的生长。其可能机制一方面通过抑制自噬,阻止肿瘤细胞从周围的基质细胞中获取能量和营养物质,从而抑制肿瘤细胞生长,另一方面,通过下调抗凋亡蛋白基因bcl-2和Survivin和上调促凋亡蛋白Bax和Caspase-3的表达水平诱导移植瘤HepG2细胞凋亡,并通过有效地抑制HepG2细胞体内侵袭和转移能力抑制肿瘤生长。这些研究为溪黄草临床应用及进一步开发应用提供了可靠的理论依据。
[Abstract]:Hepatocellular carcinoma (HCC) is a disease with high morbidity and mortality in the world. It has become a malignant tumor that threatens the health and safety of human life. The main characteristics of hepatocellular carcinoma are rapid development of disease, high recurrence rate after operation and strong metastasis of cancer cells. This is an effective prevention and control zone for hepatocellular carcinoma. Compared with chemical synthetic monomers, traditional plant drugs have many advantages, and more and more plant drugs have been found to have anti-tumor activity. Therefore, the study of plant drugs is an important way to explore natural antitumor drugs. The narrow baselines Isodon lophanthoides var gerardianus (Benth) Hara As one of the perennial herbaceous plants of the family lip family, the upper part of the family is one of the traditional Chinese herbs of Guangdong, one of the main varieties of Creek yellow grass. It is the main main plant of the current Herba yellowgrass. It is a common medicine for the prevention and treatment of liver and gallbladder diseases. The experiment shows that the water extracts of the narrow baselines have good anti-inflammatory, anti-tumor and liver preservation activities. The antitumor activity of the extract is not clear, but our research team screened the antitumor activity of the water-soluble components of the narrow baselines, and found that the water soluble total flavonoids could significantly inhibit the activity of HepG2 cells. The pre experimental conclusion also showed that it has the effect of inducing cell apoptosis. Anti tumor drugs, but the pharmacological research foundation is weak and its mechanism is not involved. Therefore, it is of great significance to study its inhibitory activity and mechanism of HepG2 cells in vivo and in depth for the development and application of Creek grass. The purification method was improved to study the effect and mechanism of the water soluble total flavonoids of Creek grasses on the growth inhibition and induction of apoptosis of human hepatoma cell HepG2. By establishing a tumor bearing nude mouse model, the inhibitory effects and related mechanisms of water soluble total flavonoids of Creek grass on human hepatoma in nude mice were investigated and studied. The inhibitory effect of ketone on the growth of human hepatoma cell HepG2 using cell proliferation inhibition test (MTT method) to detect the growth inhibition rate of water soluble total flavonoids of Creek grasses on human hepatoma cell HepG2. The ability of water soluble total flavonoids of Creek grass to observe the growth and clone of HepG2 cells was observed by the plate colony formation test. Flow cytometry was used to detect water solubility. The effect of total flavonoids on the cycle distribution of HepG2 cells. Two, the effect of water soluble total flavonoids on HepG2 apoptosis in human hepatoma cells by detecting cell mitochondrial membrane potential, intracellular ROS level and flow cytometry analysis of water soluble total flavonoids to induce apoptosis in HepG2 cells. Three, the water soluble total flavonoids of Creek hay induced human hepatoma cells Hep The mechanism of G2 apoptosis was studied by electron microscopy, and RT-PCR method was used to detect the changes in the expression of Bcl-2mRNA, Bax mRNA, Survivin mRNA, and cytochrome c of each group by Western blot method. Protein expression. Four, study on the inhibitory effect of water soluble total flavonoids on human hepatoma xenografts in nude mice, to establish a nude mouse model of nude mice, to observe and measure the tumor in nude mice after administration, to calculate the size, and to draw the growth curve. After the separation of the tumor, the inhibition rate, pathological examination, detection of the changes of peripheral blood alpha fetoglobulin and TUNEL staining were detected. Detection of the apoptosis of transplanted tumor cells in each group. Five, the mechanism of the inhibitory effect of water soluble total flavonoids on human hepatoma xenografts in nude mice, the autophagic corpuscles and ultrastructure of the tumor tissue cells were observed by transmission electron microscopy, and the expression of Beclinl, Atg5, CyclinD1, Caspase-3, Bax, Bcl-2, Survivin, nm23-H1 and MMP-9 mRNA were detected by the transmission electron microscope. N blot assay: Beclinl, Atg5, CyclinD1, Caspase-3, Bax, Bcl-2, Survivin, nm23-H1 and MMP-9 protein expression. Results: 1. The inhibitory effect of water soluble total flavonoids on the growth of human hepatocellular carcinoma cells showed that the water-soluble total flavonoids had a significant inhibitory effect on the proliferation of cells, showing a concentration dependence and certain degree of inhibition. Time dependence. The cell morphology of cell apoptotic cells appeared at various stages and apoptotic bodies were observed under microscopically, suggesting that it may be related to the promotion of HepG2 cell apoptosis. Cell colony formation experiment showed that the number of colony formation in the water soluble total flavonoids treatment group was less than that of the medium control group. The cell cycle test showed that the proportion of G0/G1 phase cells in the total flavonoids was significantly higher than that of the control group, while the proportion of S cells was significantly lower than that of the control group. This indicated that the water-soluble total flavonoids could effectively block the HepG2 cells in the G0/G1 phase, and then inhibit the proliferation and growth of the cells, and showed a dose dependence. Two The effects of water soluble total flavonoids on the apoptosis of human hepatoma cell HepG2 were analyzed by flow cytometry. The results showed that the apoptosis rate of water soluble total flavonoids and each time point were significantly higher than those of the control group, showing a dose and time dependence. The cell mitochondrial membrane potential experiment showed that the water-soluble total flavonoids and 5-FU could induce HepG2 cells. Mitochondrial membrane potential depolarization. Detection of intracellular ROS level, the results show that water-soluble total flavonoids can induce the increase of ROS in HepG2 cells. These suggest that water soluble total flavonoids can induce apoptosis of HepG2 cells by interfering with cell mitochondrial membrane potential and increasing intracellular ROS level. Three, the water soluble total flavonoids of Creek hay induced the apoptosis of human hepatoma cells HepG2 The results of RT-PCR assay showed that the anti apoptotic protein gene Bcl-2 mRNA and Survivin mRNA decreased with the increase of the concentration of water soluble total flavonoids, which showed a concentration dependence. On the contrary, the apoptotic protein gene BaxmRNA and Caspase-3mRNA were increased with the concentration of drugs. The results of.Western blot analysis showed that water soluble total flavonoids also induced.WB analysis by down regulating the expression level of anti apoptotic protein (Bcl-2 and Mcl-1) and Survivin and up regulation of apoptotic protein (Bax), and found that the water soluble total flavonoids also increased the membrane permeability of mitochondrial membrane. Apoptotic factor Cytochrome C and Cytosolic AIF were released in the cytoplasm. Four, the inhibitory effect of water soluble total flavonoids on human hepatoma xenografts in nude mice was administered for 21 days. The tumor growth curve was plotted and the tumor suppressor rate was calculated. Compared with the model group, the total water-soluble flavonoids were high, the medium dose drug group and the 5-FU group were all inhibited. The growth of the tumor was dose-dependent. In the high dose group, the 5-FU group and the water soluble total Huang Tonggao dose +5-FU group had the best effect. Compared with the model group, the drug group and the model group could significantly reduce the AFP. and the results of the tumor / body weight ratio of each group. The basic situation of the high dose of water soluble total flavonoids in the high dose +5-FU group was obviously improved. The combination of water soluble total flavonoids and 5-FU not only inhibited the growth of the tumor, but also overcame the side effects of the chemicals, and improved the quality of life. Histopathological examination found that the tumor cells in the tumor bodies of the total water soluble total flavonoids were apoptotic and had moderate necrosis, which showed that the water-soluble total flavonoids could promote apoptosis and inhibit the division of the cancer cells. No effusion was found in the breast and abdominal cavity of nude mice. No metastasis nodules of cancer cells and other abnormal.Tunel staining were found in the heart, liver, kidney, spleen, lung and brain. The number of apoptotic cells increased with the increase of concentration and showed a certain dose effect relationship. Five, the water soluble total flavonoids of Creek grasses inhibit the transplantation tumor of human liver cancer in nude mice. The production mechanism study electron microscope to observe the water soluble total flavonoids inhibit the formation of autophago in hepatoma cells, which is in accordance with the conclusion in the cell experiment. The ultrastructure of the cell also has obvious morphological changes and a large number of apoptotic bodies.RT-PCR assay showed that the water soluble total flavonoids were down regulated by the anti apoptotic protein gene Bcl-2 mRNA and survivi The expression of nmRNA and Bax mRNA and Caspase-3 mRNA induced the apoptosis of HepG2 cells in transplanted tumor. Water soluble total flavonoids can inhibit the expression of autophagic gene Beclin1mRNA, Atg5mRNA, and inhibit the expression of CyclinD1 mRNA, MMP-9mRNA gene, and the concentration dependence of concentration is higher and more obvious. On the contrary, water soluble total flavonoids can be found. The higher the expression of nm23-H1 gene, the higher the concentration of the.Western blot method, the results also further confirmed that the water soluble total flavonoids can induce the apoptosis of the transplanted tumor HepG2 cells by down regulation of the anti apoptotic protein gene Bcl-2 and Survivin and up regulation of the expression level of the apoptotic protein Bax and Caspase-3. The water soluble total flavonoids can also inhibit Beclin1, Atg5, Cyc. The expression of linD1, MMP-9 and other proteins, in contrast, water soluble total flavonoids can promote the expression of nm23-H1 protein. Conclusion: in this study, a variety of methods were used to study the inhibitory activity and mechanism of HepG2 cells in vitro and in vitro. In vitro, the water soluble total flavonoids effectively block the HepG2 cells in the G0/G1 phase. Interfering with the mitochondrial membrane potential and increasing the intracellular ROS level and inducing apoptosis of HepG2 cells, the mechanism may be to induce the release of cytochrome C into the cytoplasm by down regulation of the anti apoptotic protein (Bcl-2 and Mcl-1) and Survivin and up regulation of apoptotic protein (Bax), inducing the apoptosis of HepG2 cells and inhibiting the proliferation of cells. In vivo, water solubility Total flavonoids can inhibit the growth of xenografts in nude mice. Its possible mechanism, by inhibiting autophagy, prevents tumor cells from obtaining energy and nutrients from the surrounding matrix cells, thus inhibiting the growth of tumor cells. On the other hand, down regulation of anti apoptotic protein gene Bcl-2 and Survivin and up regulation of apoptotic protein Bax and Caspase-3 Levels of HepG2 cells can induce apoptosis of transplanted tumor cells and inhibit the growth of tumor by effectively inhibiting the invasion and metastasis of HepG2 cells. These studies provide a reliable theoretical basis for the clinical application and further development and application of Creek grasses.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5

【参考文献】