糖代谢在前列腺癌细胞干性调控作用中的研究
发布时间：2018-07-18 07:28
【摘要】：癌症是全球范围内非常重要的公共健康问题。长期以来,前列腺癌被认为是欧美国家男性中最常见的恶性肿瘤,然而,在我国,随着人们平均寿命的延长、人口的老龄化的加剧,前列腺癌的发病率也逐渐增高,因此有效地防治前列腺癌是我国亟待解决的一个公共卫生难题。在现阶段,前列腺癌的治疗方法主要有手术切除、雄激素去势治疗、放疗、化疗;但是,这些手段通常仅在起始阶段有效,最终多数患者对这些传统的治疗都产生抵抗,发展为广泛转移。多年来,针对前列腺癌的基础研究并没有为治疗和预后带来突破性的进展,需要从新的角度寻找特异性的治疗药物和方法,这有赖于从不同的角度了解和掌握前列腺癌发生发展的机制。肿瘤的发生发展是一个动态而复杂的过程,目前研究者普遍认为:恶性肿瘤中含有的肿瘤干细胞样细胞是维持肿瘤生长的主要因素。肿瘤干细胞是肿瘤组织内含有的一小部分肿瘤细胞,其特性与干细胞类似,表现出能够自我复制和更新的能力,可以进行一定程度的分化。自1990s年加拿大的肿瘤研究者Dick在白血病中鉴定出肿瘤干细胞之后,不断有研究者通过各种手段在包括脑、乳腺、结肠、前列腺、胰腺等不同来源的肿瘤中检测出肿瘤干细胞的存在,日益增多的证据表明:肿瘤干细胞极有可能是癌症复发、转移的根源,如何有效地和特异性地杀灭肿瘤干细胞是当前肿瘤研究领域的一大挑战。因而,充分了解肿瘤干细胞的性质、特征,从而通过靶向维持肿瘤干细胞的调控途径,达到有效杀灭肿瘤干细胞最终根治癌症的目的。近年的研究发现,正常干细胞特性的维持不但包括了表观遗传层次的修饰,同时也需要细胞内代谢模式转化的协同参与。但是对于肿瘤干细胞代谢特征,现阶段我们了解得远远不够。对正常干细胞能量代谢模式中的研究可以为我们提供思路,研究发现胚胎干细胞,造血干细胞,间充质干细胞以及诱导的多能干细胞(i PS)在不同的生理状态下,有不同的代谢模式,体现在:这些干细胞中的线粒体氧化磷酸化水平低下,细胞更多地依赖糖酵解途径进行生存。这种代谢模式目前被认为是维持细胞干性特征的必要因素;相反,当这些干细胞的代谢模式从糖酵解为主转变为以氧化磷酸化为主时,通常认为是干细胞起始分化的标志。这个理论目前可以很好地在各种干细胞培养和实验中得到验证,同时i PS的诱导过程可以为我们在肿瘤干细胞研究中带来更深的启发。我们知道,i PS细胞的本质是对已分化的成体细胞进行基因编辑,以期恢复细胞分化的全能性。研究发现,这些终末分化细胞的代谢方式主要是线粒体介导的氧化磷酸化;然而当在这些终末分化的细胞中导入干细胞四因子(Sox2、c-Myc、Oct3/4、Klf),细胞去分化,重新获得分化潜能,同时其代谢方式也被同步转换为糖酵解。由于这些细胞表现出在常氧条件下的嗜糖酵解特性,因此这种特性有时也被称作做有氧糖酵解(Aerobic Glycolysis),而这些细胞内总体代谢框架的改变也被称为代谢重编程(Metabolic reprogramming)。现阶段的研究发现,细胞干性重编程的过程偶联了代谢重编程,尽管表观遗传修饰在这个过程中处于核心位置,但是大量的实验室研究强烈提示:代谢的可编程性是细胞重获多潜能的先决条件。同时研究人员已经发现,将已分化的细胞暴露于低氧或者抑制其氧化磷酸化将有助于提高干性的重编程的效率;相反,通过刺激干细胞中的线粒体功能发育或者抑制糖酵解将会显著提高ATP的产量并促进干细胞分化。有趣的是,根据文献记载,代谢重编程最早并非在干细胞中被发现,而是在高度恶性的腹水肿瘤细胞和肝癌组织中首次被记录,早在1924年,一名叫Warburg的德国科学家发现,他所使用的小鼠腹水肿瘤细胞和大鼠肝癌组织即便在氧气充足的情况下,仍然主要依靠糖酵解进行葡萄糖代谢提供能量,这些由葡萄糖分解产生的丙酮酸大部分经乳酸脱氢酶转化为乳酸而排出细胞外。后来的研究发Warburg在文献中描述的现象的本质即有氧糖酵解,为了纪念Warburg的杰出贡献,细胞进行有氧糖酵解的现象也被称作为Warburg效应(Warburg effect。近90余年来,Warburg效应反复地在多种肿瘤细胞中被证实,目前被认为是肿瘤的一个显著特征,然而Warburg效应的背后所隐含的机制和生物学意义仍不明确。由于这些研究将细胞的干性、代谢可塑性、Warburg效应以及肿瘤联系起来,因此,研究肿瘤的Warburg效应理论上是研究肿瘤干细胞的一个有效手段。基于这些背景知识和理论推导,我们集中研究了Warburg效应在前列腺癌发展过程中的地位,并探究了可能的机制。我们知道,线粒体氧化磷酸化和糖酵解是细胞中两大产能途径,其中丙酮酸是联系糖酵解和三羧酸循环的关键节点,干扰这一关键节点可能改变线粒体氧化磷酸化和糖酵解相对比率,在正常的已分化细胞中,由葡糖糖代谢生成的丙酮酸,首先由位于线粒体内膜上的丙酮酸转运载体(mitochondrial pyruvate carrier,MPC)自胞浆转运进入线粒体基质,随后在丙酮酸脱氢酶(pyruvate dehydrogenase complex,PDHc)的作用之下氧化脱羧,生成乙酰辅酶A,最终进入三羧酸循环进行氧化磷酸化;其中丙酮酸脱氢酶复合体的E1α亚单位(PDHA1)的磷酸化和去磷酸化,是PDHc失活和激活的关键调节方式,PDHA1蛋白的正常表达是线粒体中三羧酸循环和氧化磷酸化正常进行的前提条件。鉴于上述,我们首先以PDHA1为切入点,用免疫组化的方法检测前列腺癌组织中PDHA1蛋白的表达,分析其表达与临床病理学特征以及预后的关系,初步了解能量代谢中的关键酶在前列腺癌中的表达情况;随后在前列腺癌细胞系中敲除PDHA1,导致PDHc功能失活,分析PDHA1基因敲除后细胞中能量代谢的变化,同时研究了该细胞模型的细胞生物学与干细胞特征;此外,我们应用MPC特异性抑制剂UK5099处理前列腺癌细胞,分析处理前后细胞的能量代谢特点的变化,并分析处理前后细胞的生物学特性和干细胞特征,分析能量代谢变化与前列腺癌干细胞特性之间的联系,目的在于探讨能量代谢模式转换是否可调控前列腺癌细胞干性程度。第一部分:PDHA1蛋白在前列腺癌组织中的表达以及与预后的分析研究方法1.利用免疫组化的方法,检测88例前列腺癌组织中PDHA1蛋白的表达情况。并分析PDHA1蛋白表达与临床病理学特征、患者生存期之间的关系。2.应用SPSS13.0软件,采用单因素方差分析检验PDHA1蛋白表达与各临床病理学特征之间的关系;生存曲线采用Kaplan-Meier和log-rank分析检验。研究结果1.在88例前列腺癌中,34(38.64%)例阳性表达PDHA1蛋白,54(61.36%)例为阴性,PDHA1蛋白表达与前列腺癌Gleason分级相关,Gleason分级小于7的27例前列腺癌标本中,15例(55.6%)为阳性,在Gleason分级等于7的41例标本中14例(34.15.9%)为阳性,但在Gleason分级大于7的20例标本中仅有5例(25%)为阳性性(p0.05),PDHA1蛋白表达与其它临床病理学参数无相关性。2.88例患者中PDHA1蛋白表达阴性者的患者,总生存率显著低于PDHA1蛋白阳性患者(p0.05)。第二部分:PDHA1基因敲除对前列腺癌细胞代谢模式和干性程度的影响研究方法1.构建TALEN质粒,利用TALEN介导的基因编辑技术在前列腺癌细胞Ln Cap中进行了PDHA1基因的纯合性敲除,挑选单克隆,建立稳定细胞系。2.通过检测细胞内ATP、葡萄糖含量、以及应用Seahorse Extracellular Flux24F能量代谢分析设备分析细胞内葡萄糖代谢偶联的细胞氧耗(OCR)和胞外酸化速率(ECAR)的变化,分析敲除PDHA1基因后细胞代谢中糖酵解速率和线粒体氧化磷酸化程度的变化。3.PDHA1基因敲除后,用细胞计数法了解细胞增殖能力,Transwell assay了解迁移能力,Hoechst 33342染色及流式细胞仪分析侧群细胞(SP)比率,测试对化疗药物的敏感性,放疗后的克隆形成实验了解放疗敏感性,以及流式细胞仪和Western blot分析干细胞标记CD44、ABCG2、Oct3/4、Nanog表达的改变。研究结果1.成功建立了PDHA1基因敲除的稳定细胞系。2.PDHA1基因敲除后,线粒体氧化磷酸化程度被抑制,糖酵解速率提高,表现为基础OCR降低,基础ECAR升高,葡萄糖摄取能力升高,ATP产量降低。3.PDHA1基因敲除后,肿瘤细胞的增殖受到抑制,但是肿瘤细胞的体外移动能力增强,并且这些细胞呈现显著的化疗和放疗抵抗,SP细胞比率增加,干细胞标记CD44、ABCG2、Oct3/4、Nanog表达升高等,提示PDHA1基因敲除后的前列腺癌细胞更加具有肿瘤干细胞样细胞的特性。第三部分:MPC抑制剂UK5099对前列腺癌细胞代谢模式和干性程度的影响研究方法1.用合适浓度的MPC抑制剂处理Ln Cap细胞。2.以丙酮酸试剂盒、ATP试剂盒分析细胞胞浆中丙酮酸的浓度的变化、细胞ATP产量,以及使用线粒体膜电位JC-1探针和通过流式细胞仪检测细胞线粒体膜电位的变化,初步分析细胞代谢中线粒体氧化磷酸化程度和糖酵解速率的转变。2.用UK5099抑制丙酮酸转运进入线粒体后,观察Ln Cap细胞增殖的情况,流式细胞仪分析细胞周期、Hoechst 33342染色及流式细胞仪分析侧群细胞(SP)比率以及Western blot检测干细胞标记的表达水平的变化。研究结果1.应用MPC抑制剂UK5099处理细胞,证实了UK5099可以抑制丙酮酸转运进入线粒体基质。2.UK5099处理Ln Cap细胞后,ATP产量降低,乳酸产量增高,线粒体膜电势降低,提示线粒体氧化磷酸化程度收到抑制,糖酵解速率提高。3.UK5099处理Ln Cap细胞后,细胞增殖受到抑制,细胞G1/G0比例增加,SP细胞比率增加,干细胞标记Oct3/4和Nanog表达升高。研究结论1.我们检测PDHA1蛋白在前列腺癌组织中的表达情况,发现PDHA1蛋白阴性表达与较差的预后相关,提示糖酵解通路可能在前列腺癌发生发展中发挥了重要的作用。2.敲除PDHA1基因以造成丙酮酸氧化脱羧障碍,或外源性应用UK5099抑制丙酮酸转运进入线粒体,均可以抑制线粒体氧化磷酸化,促进糖酵解速率,可以作为研究Warburg效应和肿瘤干细胞的模型。3.糖酵解程度升高的代谢模式促进了前列腺癌细胞干性程度的提高,这可能是PDHA1阴性表达患者预后差的基础,本研究也间接提示了肿瘤干细胞样细胞的存在可能是Warburg现象产生的原因,我们的研究对于理解前列腺癌的发生发展机制以及探索新的治疗靶点提供新的视角。
[Abstract]:Cancer is a very important public health problem around the world. Prostate cancer has long been considered the most common malignant tumor in men and women in Europe and America. However, in our country, the incidence of prostate cancer is increasing with the prolongation of the average life span, the aging of the population and the increase of the incidence of prostate cancer. Therefore, effective prevention and control of prostate cancer is mine. At the present stage, the main treatment of prostate cancer is surgical resection, androgen castration, radiotherapy, chemotherapy; however, these methods are usually only effective at the beginning stage, and most of the patients are eventually resistant to these traditional treatments and develop widely. For years, prostate cancer is targeted. The basic research does not bring about breakthrough in the treatment and prognosis. It is necessary to find specific therapeutic drugs and methods from a new perspective. This depends on understanding and mastering the mechanism of the development of prostate cancer from different angles. The development of the tumor is a dynamic and complex process. Tumor stem cell like cells are the main factors to maintain tumor growth. The tumor stem cells are a small part of tumor cells in the tumor tissue. Their characteristics are similar to those of the stem cells, showing the ability to self replicate and renew themselves. In 1990s, cancer researchers in Canada Dick are in white. After the identification of cancer stem cells in the blood disease, a variety of methods have been used to detect the existence of cancer stem cells in different sources including brain, breast, colon, prostate, and pancreas. The increasing evidence suggests that cancer stem cells are most likely to be cancer recurrence, the root of metastasis, and how effective and specific. Killing tumor stem cells is a major challenge in the field of cancer research. Therefore, we fully understand the properties and characteristics of cancer stem cells, so as to achieve the goal of effectively killing cancer stem cells by targeting the regulation of cancer stem cells. Recent studies have found that the maintenance of normal stem cell characteristics is not only included in the maintenance of cancer stem cells. The modification of epigenetic levels also requires synergistic participation in the transformation of intracellular metabolic patterns. However, we do not know much about the metabolic characteristics of cancer stem cells at the present stage. Cells and induced pluripotent stem cells (I PS) have different metabolic patterns in different physiological states, which are reflected in the low level of oxidative phosphorylation of mitochondria in these stem cells and more dependent on the glycolysis pathway to survive. This metabolic pattern is now considered as a necessary factor in maintaining the dry characteristics of the cells; on the contrary, this is the case. When the metabolic patterns of some stem cells change from glycolysis to oxidative phosphorylation, they are usually considered as a sign of the initiation and differentiation of stem cells. This theory can be well verified in various stem cell culture and experiments, and the induction of I PS can bring us deeper inspiration in the research of cancer stem cells. We know that the essence of I PS cells is to gene editors of differentiated adult cells in order to restore the omnipotent of cell differentiation. Studies have found that the metabolic modes of these terminal cells are mainly mitochondrial mediated oxidative phosphorylation; however, four factors (Sox2, c-Myc, Oct3/4, K) are introduced into these terminal cells. LF) cells dedifferentiated and regained their differentiation potential, and their metabolic patterns were synchronously converted to glycolysis. Because these cells exhibit glycolytic properties under the condition of normal oxygen, this characteristic is sometimes called Aerobic Glycolysis, and the changes in the overall metabolic framework in these cells are also called generations. Metabolic reprogramming. The present stage studies have found that the process of cell dry reprogramming coupled with thanks reprogramming, although epigenetic modification is at the core of this process, but a large number of laboratory studies strongly suggest that metabolic programmable is a prerequisite for cell reprocessing of multipotential. It has been found that exposing differentiated cells to hypoxia or inhibiting their oxidative phosphorylation will help to improve the efficiency of dry reprogramming; on the contrary, the development of mitochondrial function in stem cells or inhibition of glycolysis will significantly increase the yield of ATP and promote stem cell differentiation. Interestingly, metabolism is recorded in the literature. Reprogramming was first not found in stem cells, but was first recorded in highly malignant ascites tumor cells and liver cancer tissues. As early as 1924, a German scientist named Warburg found that the mouse ascites tumor cells and rat hepatoma tissues were still mainly dependent on glycolysis even if oxygen was sufficient. Glucose metabolism provides energy, and most of the pyruvic acid produced by glucose is discharged from the cells through the conversion of lactate dehydrogenase into lactic acid. Later, the essence of the phenomenon described in the Warburg literature is aerobic glycolysis. In memory of the outstanding contribution of Warburg, the phenomenon of aerobic glycolysis in cells is also known as the phenomenon. As the Warburg effect (Warburg effect. over the last 90 years, the Warburg effect has been repeatedly confirmed in a variety of tumor cells and is currently considered a significant feature of the tumor. However, the underlying mechanisms and biological implications behind the Warburg effect are still unclear. These studies have resulted in the stem, metabolic plasticity and Warburg effect of the cells. " It is associated with the tumor, so the study of the Warburg effect of the tumor is an effective method for the study of cancer stem cells. Based on these background knowledge and theoretical derivation, we focus on the status of the Warburg effect in the development of prostate cancer and explore possible mechanisms. We know that oxidative phosphorylation and sugar of mitochondria are used. Glycolysis is the two major productivity pathway in the cell, in which pyruvate is the key node associated with glycolysis and the cycle of three carboxylic acids. The interference of this key node may change the relative ratio of oxidative phosphorylation and glycolysis, in normal differentiated cells, pyruvic acid produced by glucose metabolism, first of the acetone located on the membrane of the mitochondria. The acid transport carrier (mitochondrial pyruvate carrier, MPC) transshipped from cytoplasm to the mitochondrial matrix and then oxidized decarboxylation under the action of pyruvate dehydrogenase (pyruvate dehydrogenase complex, PDHc) to produce acetyl coenzyme A and eventually entered the three carboxylic acid cycle for oxygenated phosphorylation, in which the E1 alpha subunit of the pyruvate dehydrogenase complex (a subunit of the pyruvate dehydrogenase complex). Phosphorylation and dephosphorylation of PDHA1) is the key regulation of PDHc inactivation and activation. The normal expression of PDHA1 protein is the prerequisite for the normal operation of the three carboxylic acid cycle and oxidative phosphorylation in mitochondria. In view of the above, we first use PDHA1 as the breakthrough point to detect the expression of PDHA1 protein in the prostate cancer tissue by immunohistochemical method. The relationship between the expression of the expression and the clinicopathological features and the prognosis was analyzed. The expression of the key enzymes in the prostate cancer was preliminarily understood. Then PDHA1 was knocked out in the prostate cancer cell line, resulting in the deactivation of the PDHc function and the analysis of the changes in the energy metabolism in the cells after the PDHA1 knockout, and the cells of the cell model were studied. Biological and stem cell characteristics; in addition, we used the MPC specific inhibitor UK5099 to treat the prostate cancer cells, analyzed the changes in the energy metabolism of the cells before and after treatment, and analyzed the biological and stem cell characteristics of the cells before and after treatment, and analyzed the relationship between the energy metabolism and the characteristics of the prostate cancer stem cells. To investigate whether the transformation of energy metabolic pattern can regulate the degree of prostate cancer cell stem. Part 1: the expression of PDHA1 protein in the prostate cancer tissue and the analysis of the prognosis. 1. the expression of PDHA1 protein in the prostate cancer tissues was detected by immunohistochemical method, and the expression of PDHA1 protein and clinical disease were analyzed. The relationship between the patient's survival time and the relationship between the patients' survival time.2. applied SPSS13.0 software to test the relationship between the expression of PDHA1 protein and the clinicopathological features by single factor analysis of variance; the survival curve was examined by Kaplan-Meier and log-rank analysis. Results 1. in 88 cases of prostate cancer, 34 (38.64%) positive expression of PDHA1 protein, 54 (61.36%). The expression of PDHA1 protein was associated with Gleason classification of prostate cancer. In 27 cases of prostate cancer with Gleason classification less than 7, 15 cases (55.6%) were positive. 14 cases (34.15.9%) were positive in 41 cases of Gleason grading equal to 7, but only 5 cases (25%) were positive (P0.05) and PDHA1 protein expression in 20 cases with Gleason classification greater than 7. The total survival rate of the patients with negative PDHA1 protein expression in.2.88 patients with no correlation with other clinicopathological parameters was significantly lower than that of PDHA1 protein positive patients (P0.05). Second part: the effect of PDHA1 gene knockout on the metabolic pattern and dry degree of prostate cancer cell 1. construction of TALEN plasmids and TALEN mediated gene coding. The collection of homozygous knockout of PDHA1 gene in prostate cancer cell Ln Cap, select a monoclonal and establish a stable cell line.2. by detecting intracellular ATP, glucose content, and using Seahorse Extracellular Flux24F energy metabolism analysis equipment to analyze the cell oxygen consumption (OCR) and exoacidification speed of intracellular glucose metabolism coupling in cells. The change of rate (ECAR), analysis of the change of glycolysis rate and the degree of mitochondrial oxidative phosphorylation after PDHA1 gene knockout.3.PDHA1 gene knockout, the cell count method was used to understand the cell proliferation ability, Transwell assay was used to understand the migration ability, Hoechst 33342 staining and flow cytometry were used to analyze the ratio of side group cells (SP) and to test the chemotherapy. The sensitivity of the drug, the clone formation after radiotherapy, and the sensitivity of the radiotherapy, and the flow cytometer and Western blot analysis of the changes in the expression of CD44, ABCG2, Oct3/4, Nanog in the stem cells. Results 1. the degree of oxidative phosphorylation of mitochondria was suppressed after the knockout of the stable cell line.2.PDHA1 gene of the PDHA1 knockout. The rate of glycolysis was improved, which showed that the base OCR decreased, the base ECAR increased, the ability of glucose uptake increased, and the proliferation of the tumor cells was inhibited after the ATP production reduced.3.PDHA1 knockout, but the ability of tumor cells to move in vitro was enhanced, and these cells showed significant chemotherapy and radiation resistance, the ratio of SP cells increased, and the stem cell markers were increased. CD44, ABCG2, Oct3/4, Nanog expression is higher, suggesting that PDHA1 gene knockout prostate cancer cells have the characteristics of tumor stem cell like cells. Third part: the study of the effect of MPC inhibitor UK5099 on the metabolic pattern and dry degree of prostate cancer cells; 1. with the appropriate concentration of MPC inhibitor to treat Ln Cap cells.2. with pyruvate. The kit and ATP kit analysis the changes in the concentration of pyruvic acid in cytoplasm, the yield of cell ATP, and the changes in the mitochondrial membrane potential using the mitochondrial membrane potential JC-1 probe and the flow cytometry. The transformation of mitochondrial oxidation phosphorylation and glycolysis rate in cell metabolism is a preliminary analysis of.2. with UK5099 inhibition of acetone. After acid transport entered mitochondria, the proliferation of Ln Cap cells was observed, cell cycle was analyzed by flow cytometry, Hoechst 33342 staining and flow cytometry analysis of side group cells (SP) ratio and changes in expression level of Western blot detection of stem cell markers. Results 1. application of MPC inhibitor UK5099 processing cells proved that UK5099 could be used. After the inhibition of pyruvic acid transport into the mitochondrial matrix.2.UK5099 treatment Ln Cap cells, the output of ATP decreased, the production of lactic acid increased and the mitochondrial membrane potential decreased, suggesting that the degree of mitochondrial oxidative phosphorylation was suppressed. After the glycolysis rate increased.3.UK5099 treatment Ln Cap cells, the cell proliferation was inhibited, the proportion of cell G1/G0 increased, and the SP cell ratio was increased. Conclusion: 1.. We detected the expression of PDHA1 protein in prostate cancer tissues, and the expression of Oct3/4 and Nanog was increased.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25
本文编号：2131188
[Abstract]:Cancer is a very important public health problem around the world. Prostate cancer has long been considered the most common malignant tumor in men and women in Europe and America. However, in our country, the incidence of prostate cancer is increasing with the prolongation of the average life span, the aging of the population and the increase of the incidence of prostate cancer. Therefore, effective prevention and control of prostate cancer is mine. At the present stage, the main treatment of prostate cancer is surgical resection, androgen castration, radiotherapy, chemotherapy; however, these methods are usually only effective at the beginning stage, and most of the patients are eventually resistant to these traditional treatments and develop widely. For years, prostate cancer is targeted. The basic research does not bring about breakthrough in the treatment and prognosis. It is necessary to find specific therapeutic drugs and methods from a new perspective. This depends on understanding and mastering the mechanism of the development of prostate cancer from different angles. The development of the tumor is a dynamic and complex process. Tumor stem cell like cells are the main factors to maintain tumor growth. The tumor stem cells are a small part of tumor cells in the tumor tissue. Their characteristics are similar to those of the stem cells, showing the ability to self replicate and renew themselves. In 1990s, cancer researchers in Canada Dick are in white. After the identification of cancer stem cells in the blood disease, a variety of methods have been used to detect the existence of cancer stem cells in different sources including brain, breast, colon, prostate, and pancreas. The increasing evidence suggests that cancer stem cells are most likely to be cancer recurrence, the root of metastasis, and how effective and specific. Killing tumor stem cells is a major challenge in the field of cancer research. Therefore, we fully understand the properties and characteristics of cancer stem cells, so as to achieve the goal of effectively killing cancer stem cells by targeting the regulation of cancer stem cells. Recent studies have found that the maintenance of normal stem cell characteristics is not only included in the maintenance of cancer stem cells. The modification of epigenetic levels also requires synergistic participation in the transformation of intracellular metabolic patterns. However, we do not know much about the metabolic characteristics of cancer stem cells at the present stage. Cells and induced pluripotent stem cells (I PS) have different metabolic patterns in different physiological states, which are reflected in the low level of oxidative phosphorylation of mitochondria in these stem cells and more dependent on the glycolysis pathway to survive. This metabolic pattern is now considered as a necessary factor in maintaining the dry characteristics of the cells; on the contrary, this is the case. When the metabolic patterns of some stem cells change from glycolysis to oxidative phosphorylation, they are usually considered as a sign of the initiation and differentiation of stem cells. This theory can be well verified in various stem cell culture and experiments, and the induction of I PS can bring us deeper inspiration in the research of cancer stem cells. We know that the essence of I PS cells is to gene editors of differentiated adult cells in order to restore the omnipotent of cell differentiation. Studies have found that the metabolic modes of these terminal cells are mainly mitochondrial mediated oxidative phosphorylation; however, four factors (Sox2, c-Myc, Oct3/4, K) are introduced into these terminal cells. LF) cells dedifferentiated and regained their differentiation potential, and their metabolic patterns were synchronously converted to glycolysis. Because these cells exhibit glycolytic properties under the condition of normal oxygen, this characteristic is sometimes called Aerobic Glycolysis, and the changes in the overall metabolic framework in these cells are also called generations. Metabolic reprogramming. The present stage studies have found that the process of cell dry reprogramming coupled with thanks reprogramming, although epigenetic modification is at the core of this process, but a large number of laboratory studies strongly suggest that metabolic programmable is a prerequisite for cell reprocessing of multipotential. It has been found that exposing differentiated cells to hypoxia or inhibiting their oxidative phosphorylation will help to improve the efficiency of dry reprogramming; on the contrary, the development of mitochondrial function in stem cells or inhibition of glycolysis will significantly increase the yield of ATP and promote stem cell differentiation. Interestingly, metabolism is recorded in the literature. Reprogramming was first not found in stem cells, but was first recorded in highly malignant ascites tumor cells and liver cancer tissues. As early as 1924, a German scientist named Warburg found that the mouse ascites tumor cells and rat hepatoma tissues were still mainly dependent on glycolysis even if oxygen was sufficient. Glucose metabolism provides energy, and most of the pyruvic acid produced by glucose is discharged from the cells through the conversion of lactate dehydrogenase into lactic acid. Later, the essence of the phenomenon described in the Warburg literature is aerobic glycolysis. In memory of the outstanding contribution of Warburg, the phenomenon of aerobic glycolysis in cells is also known as the phenomenon. As the Warburg effect (Warburg effect. over the last 90 years, the Warburg effect has been repeatedly confirmed in a variety of tumor cells and is currently considered a significant feature of the tumor. However, the underlying mechanisms and biological implications behind the Warburg effect are still unclear. These studies have resulted in the stem, metabolic plasticity and Warburg effect of the cells. " It is associated with the tumor, so the study of the Warburg effect of the tumor is an effective method for the study of cancer stem cells. Based on these background knowledge and theoretical derivation, we focus on the status of the Warburg effect in the development of prostate cancer and explore possible mechanisms. We know that oxidative phosphorylation and sugar of mitochondria are used. Glycolysis is the two major productivity pathway in the cell, in which pyruvate is the key node associated with glycolysis and the cycle of three carboxylic acids. The interference of this key node may change the relative ratio of oxidative phosphorylation and glycolysis, in normal differentiated cells, pyruvic acid produced by glucose metabolism, first of the acetone located on the membrane of the mitochondria. The acid transport carrier (mitochondrial pyruvate carrier, MPC) transshipped from cytoplasm to the mitochondrial matrix and then oxidized decarboxylation under the action of pyruvate dehydrogenase (pyruvate dehydrogenase complex, PDHc) to produce acetyl coenzyme A and eventually entered the three carboxylic acid cycle for oxygenated phosphorylation, in which the E1 alpha subunit of the pyruvate dehydrogenase complex (a subunit of the pyruvate dehydrogenase complex). Phosphorylation and dephosphorylation of PDHA1) is the key regulation of PDHc inactivation and activation. The normal expression of PDHA1 protein is the prerequisite for the normal operation of the three carboxylic acid cycle and oxidative phosphorylation in mitochondria. In view of the above, we first use PDHA1 as the breakthrough point to detect the expression of PDHA1 protein in the prostate cancer tissue by immunohistochemical method. The relationship between the expression of the expression and the clinicopathological features and the prognosis was analyzed. The expression of the key enzymes in the prostate cancer was preliminarily understood. Then PDHA1 was knocked out in the prostate cancer cell line, resulting in the deactivation of the PDHc function and the analysis of the changes in the energy metabolism in the cells after the PDHA1 knockout, and the cells of the cell model were studied. Biological and stem cell characteristics; in addition, we used the MPC specific inhibitor UK5099 to treat the prostate cancer cells, analyzed the changes in the energy metabolism of the cells before and after treatment, and analyzed the biological and stem cell characteristics of the cells before and after treatment, and analyzed the relationship between the energy metabolism and the characteristics of the prostate cancer stem cells. To investigate whether the transformation of energy metabolic pattern can regulate the degree of prostate cancer cell stem. Part 1: the expression of PDHA1 protein in the prostate cancer tissue and the analysis of the prognosis. 1. the expression of PDHA1 protein in the prostate cancer tissues was detected by immunohistochemical method, and the expression of PDHA1 protein and clinical disease were analyzed. The relationship between the patient's survival time and the relationship between the patients' survival time.2. applied SPSS13.0 software to test the relationship between the expression of PDHA1 protein and the clinicopathological features by single factor analysis of variance; the survival curve was examined by Kaplan-Meier and log-rank analysis. Results 1. in 88 cases of prostate cancer, 34 (38.64%) positive expression of PDHA1 protein, 54 (61.36%). The expression of PDHA1 protein was associated with Gleason classification of prostate cancer. In 27 cases of prostate cancer with Gleason classification less than 7, 15 cases (55.6%) were positive. 14 cases (34.15.9%) were positive in 41 cases of Gleason grading equal to 7, but only 5 cases (25%) were positive (P0.05) and PDHA1 protein expression in 20 cases with Gleason classification greater than 7. The total survival rate of the patients with negative PDHA1 protein expression in.2.88 patients with no correlation with other clinicopathological parameters was significantly lower than that of PDHA1 protein positive patients (P0.05). Second part: the effect of PDHA1 gene knockout on the metabolic pattern and dry degree of prostate cancer cell 1. construction of TALEN plasmids and TALEN mediated gene coding. The collection of homozygous knockout of PDHA1 gene in prostate cancer cell Ln Cap, select a monoclonal and establish a stable cell line.2. by detecting intracellular ATP, glucose content, and using Seahorse Extracellular Flux24F energy metabolism analysis equipment to analyze the cell oxygen consumption (OCR) and exoacidification speed of intracellular glucose metabolism coupling in cells. The change of rate (ECAR), analysis of the change of glycolysis rate and the degree of mitochondrial oxidative phosphorylation after PDHA1 gene knockout.3.PDHA1 gene knockout, the cell count method was used to understand the cell proliferation ability, Transwell assay was used to understand the migration ability, Hoechst 33342 staining and flow cytometry were used to analyze the ratio of side group cells (SP) and to test the chemotherapy. The sensitivity of the drug, the clone formation after radiotherapy, and the sensitivity of the radiotherapy, and the flow cytometer and Western blot analysis of the changes in the expression of CD44, ABCG2, Oct3/4, Nanog in the stem cells. Results 1. the degree of oxidative phosphorylation of mitochondria was suppressed after the knockout of the stable cell line.2.PDHA1 gene of the PDHA1 knockout. The rate of glycolysis was improved, which showed that the base OCR decreased, the base ECAR increased, the ability of glucose uptake increased, and the proliferation of the tumor cells was inhibited after the ATP production reduced.3.PDHA1 knockout, but the ability of tumor cells to move in vitro was enhanced, and these cells showed significant chemotherapy and radiation resistance, the ratio of SP cells increased, and the stem cell markers were increased. CD44, ABCG2, Oct3/4, Nanog expression is higher, suggesting that PDHA1 gene knockout prostate cancer cells have the characteristics of tumor stem cell like cells. Third part: the study of the effect of MPC inhibitor UK5099 on the metabolic pattern and dry degree of prostate cancer cells; 1. with the appropriate concentration of MPC inhibitor to treat Ln Cap cells.2. with pyruvate. The kit and ATP kit analysis the changes in the concentration of pyruvic acid in cytoplasm, the yield of cell ATP, and the changes in the mitochondrial membrane potential using the mitochondrial membrane potential JC-1 probe and the flow cytometry. The transformation of mitochondrial oxidation phosphorylation and glycolysis rate in cell metabolism is a preliminary analysis of.2. with UK5099 inhibition of acetone. After acid transport entered mitochondria, the proliferation of Ln Cap cells was observed, cell cycle was analyzed by flow cytometry, Hoechst 33342 staining and flow cytometry analysis of side group cells (SP) ratio and changes in expression level of Western blot detection of stem cell markers. Results 1. application of MPC inhibitor UK5099 processing cells proved that UK5099 could be used. After the inhibition of pyruvic acid transport into the mitochondrial matrix.2.UK5099 treatment Ln Cap cells, the output of ATP decreased, the production of lactic acid increased and the mitochondrial membrane potential decreased, suggesting that the degree of mitochondrial oxidative phosphorylation was suppressed. After the glycolysis rate increased.3.UK5099 treatment Ln Cap cells, the cell proliferation was inhibited, the proportion of cell G1/G0 increased, and the SP cell ratio was increased. Conclusion: 1.. We detected the expression of PDHA1 protein in prostate cancer tissues, and the expression of Oct3/4 and Nanog was increased.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25
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