糖代謝在前列腺癌細胞干性調控作用中的研究
發(fā)布時間：2018-07-18 07:28
【摘要】：癌癥是全球范圍內非常重要的公共健康問題。長期以來,前列腺癌被認為是歐美國家男性中最常見的惡性腫瘤,然而,在我國,隨著人們平均壽命的延長、人口的老齡化的加劇,前列腺癌的發(fā)病率也逐漸增高,因此有效地防治前列腺癌是我國亟待解決的一個公共衛(wèi)生難題。在現階段,前列腺癌的治療方法主要有手術切除、雄激素去勢治療、放療、化療;但是,這些手段通常僅在起始階段有效,最終多數患者對這些傳統(tǒng)的治療都產生抵抗,發(fā)展為廣泛轉移。多年來,針對前列腺癌的基礎研究并沒有為治療和預后帶來突破性的進展,需要從新的角度尋找特異性的治療藥物和方法,這有賴于從不同的角度了解和掌握前列腺癌發(fā)生發(fā)展的機制。腫瘤的發(fā)生發(fā)展是一個動態(tài)而復雜的過程,目前研究者普遍認為:惡性腫瘤中含有的腫瘤干細胞樣細胞是維持腫瘤生長的主要因素。腫瘤干細胞是腫瘤組織內含有的一小部分腫瘤細胞,其特性與干細胞類似,表現出能夠自我復制和更新的能力,可以進行一定程度的分化。自1990s年加拿大的腫瘤研究者Dick在白血病中鑒定出腫瘤干細胞之后,不斷有研究者通過各種手段在包括腦、乳腺、結腸、前列腺、胰腺等不同來源的腫瘤中檢測出腫瘤干細胞的存在,日益增多的證據表明:腫瘤干細胞極有可能是癌癥復發(fā)、轉移的根源,如何有效地和特異性地殺滅腫瘤干細胞是當前腫瘤研究領域的一大挑戰(zhàn)。因而,充分了解腫瘤干細胞的性質、特征,從而通過靶向維持腫瘤干細胞的調控途徑,達到有效殺滅腫瘤干細胞最終根治癌癥的目的。近年的研究發(fā)現,正常干細胞特性的維持不但包括了表觀遺傳層次的修飾,同時也需要細胞內代謝模式轉化的協(xié)同參與。但是對于腫瘤干細胞代謝特征,現階段我們了解得遠遠不夠。對正常干細胞能量代謝模式中的研究可以為我們提供思路,研究發(fā)現胚胎干細胞,造血干細胞,間充質干細胞以及誘導的多能干細胞(i PS)在不同的生理狀態(tài)下,有不同的代謝模式,體現在:這些干細胞中的線粒體氧化磷酸化水平低下,細胞更多地依賴糖酵解途徑進行生存。這種代謝模式目前被認為是維持細胞干性特征的必要因素;相反,當這些干細胞的代謝模式從糖酵解為主轉變?yōu)橐匝趸姿峄癁橹鲿r,通常認為是干細胞起始分化的標志。這個理論目前可以很好地在各種干細胞培養(yǎng)和實驗中得到驗證,同時i PS的誘導過程可以為我們在腫瘤干細胞研究中帶來更深的啟發(fā)。我們知道,i PS細胞的本質是對已分化的成體細胞進行基因編輯,以期恢復細胞分化的全能性。研究發(fā)現,這些終末分化細胞的代謝方式主要是線粒體介導的氧化磷酸化;然而當在這些終末分化的細胞中導入干細胞四因子(Sox2、c-Myc、Oct3/4、Klf),細胞去分化,重新獲得分化潛能,同時其代謝方式也被同步轉換為糖酵解。由于這些細胞表現出在常氧條件下的嗜糖酵解特性,因此這種特性有時也被稱作做有氧糖酵解(Aerobic Glycolysis),而這些細胞內總體代謝框架的改變也被稱為代謝重編程(Metabolic reprogramming)�，F階段的研究發(fā)現,細胞干性重編程的過程偶聯(lián)了代謝重編程,盡管表觀遺傳修飾在這個過程中處于核心位置,但是大量的實驗室研究強烈提示:代謝的可編程性是細胞重獲多潛能的先決條件。同時研究人員已經發(fā)現,將已分化的細胞暴露于低氧或者抑制其氧化磷酸化將有助于提高干性的重編程的效率;相反,通過刺激干細胞中的線粒體功能發(fā)育或者抑制糖酵解將會顯著提高ATP的產量并促進干細胞分化。有趣的是,根據文獻記載,代謝重編程最早并非在干細胞中被發(fā)現,而是在高度惡性的腹水腫瘤細胞和肝癌組織中首次被記錄,早在1924年,一名叫Warburg的德國科學家發(fā)現,他所使用的小鼠腹水腫瘤細胞和大鼠肝癌組織即便在氧氣充足的情況下,仍然主要依靠糖酵解進行葡萄糖代謝提供能量,這些由葡萄糖分解產生的丙酮酸大部分經乳酸脫氫酶轉化為乳酸而排出細胞外。后來的研究發(fā)Warburg在文獻中描述的現象的本質即有氧糖酵解,為了紀念Warburg的杰出貢獻,細胞進行有氧糖酵解的現象也被稱作為Warburg效應(Warburg effect。近90余年來,Warburg效應反復地在多種腫瘤細胞中被證實,目前被認為是腫瘤的一個顯著特征,然而Warburg效應的背后所隱含的機制和生物學意義仍不明確。由于這些研究將細胞的干性、代謝可塑性、Warburg效應以及腫瘤聯(lián)系起來,因此,研究腫瘤的Warburg效應理論上是研究腫瘤干細胞的一個有效手段�；谶@些背景知識和理論推導,我們集中研究了Warburg效應在前列腺癌發(fā)展過程中的地位,并探究了可能的機制。我們知道,線粒體氧化磷酸化和糖酵解是細胞中兩大產能途徑,其中丙酮酸是聯(lián)系糖酵解和三羧酸循環(huán)的關鍵節(jié)點,干擾這一關鍵節(jié)點可能改變線粒體氧化磷酸化和糖酵解相對比率,在正常的已分化細胞中,由葡糖糖代謝生成的丙酮酸,首先由位于線粒體內膜上的丙酮酸轉運載體(mitochondrial pyruvate carrier,MPC)自胞漿轉運進入線粒體基質,隨后在丙酮酸脫氫酶(pyruvate dehydrogenase complex,PDHc)的作用之下氧化脫羧,生成乙酰輔酶A,最終進入三羧酸循環(huán)進行氧化磷酸化;其中丙酮酸脫氫酶復合體的E1α亞單位(PDHA1)的磷酸化和去磷酸化,是PDHc失活和激活的關鍵調節(jié)方式,PDHA1蛋白的正常表達是線粒體中三羧酸循環(huán)和氧化磷酸化正常進行的前提條件。鑒于上述,我們首先以PDHA1為切入點,用免疫組化的方法檢測前列腺癌組織中PDHA1蛋白的表達,分析其表達與臨床病理學特征以及預后的關系,初步了解能量代謝中的關鍵酶在前列腺癌中的表達情況;隨后在前列腺癌細胞系中敲除PDHA1,導致PDHc功能失活,分析PDHA1基因敲除后細胞中能量代謝的變化,同時研究了該細胞模型的細胞生物學與干細胞特征;此外,我們應用MPC特異性抑制劑UK5099處理前列腺癌細胞,分析處理前后細胞的能量代謝特點的變化,并分析處理前后細胞的生物學特性和干細胞特征,分析能量代謝變化與前列腺癌干細胞特性之間的聯(lián)系,目的在于探討能量代謝模式轉換是否可調控前列腺癌細胞干性程度。第一部分:PDHA1蛋白在前列腺癌組織中的表達以及與預后的分析研究方法1.利用免疫組化的方法,檢測88例前列腺癌組織中PDHA1蛋白的表達情況。并分析PDHA1蛋白表達與臨床病理學特征、患者生存期之間的關系。2.應用SPSS13.0軟件,采用單因素方差分析檢驗PDHA1蛋白表達與各臨床病理學特征之間的關系;生存曲線采用Kaplan-Meier和log-rank分析檢驗。研究結果1.在88例前列腺癌中,34(38.64%)例陽性表達PDHA1蛋白,54(61.36%)例為陰性,PDHA1蛋白表達與前列腺癌Gleason分級相關,Gleason分級小于7的27例前列腺癌標本中,15例(55.6%)為陽性,在Gleason分級等于7的41例標本中14例(34.15.9%)為陽性,但在Gleason分級大于7的20例標本中僅有5例(25%)為陽性性(p0.05),PDHA1蛋白表達與其它臨床病理學參數無相關性。2.88例患者中PDHA1蛋白表達陰性者的患者,總生存率顯著低于PDHA1蛋白陽性患者(p0.05)。第二部分:PDHA1基因敲除對前列腺癌細胞代謝模式和干性程度的影響研究方法1.構建TALEN質粒,利用TALEN介導的基因編輯技術在前列腺癌細胞Ln Cap中進行了PDHA1基因的純合性敲除,挑選單克隆,建立穩(wěn)定細胞系。2.通過檢測細胞內ATP、葡萄糖含量、以及應用Seahorse Extracellular Flux24F能量代謝分析設備分析細胞內葡萄糖代謝偶聯(lián)的細胞氧耗(OCR)和胞外酸化速率(ECAR)的變化,分析敲除PDHA1基因后細胞代謝中糖酵解速率和線粒體氧化磷酸化程度的變化。3.PDHA1基因敲除后,用細胞計數法了解細胞增殖能力,Transwell assay了解遷移能力,Hoechst 33342染色及流式細胞儀分析側群細胞(SP)比率,測試對化療藥物的敏感性,放療后的克隆形成實驗了解放療敏感性,以及流式細胞儀和Western blot分析干細胞標記CD44、ABCG2、Oct3/4、Nanog表達的改變。研究結果1.成功建立了PDHA1基因敲除的穩(wěn)定細胞系。2.PDHA1基因敲除后,線粒體氧化磷酸化程度被抑制,糖酵解速率提高,表現為基礎OCR降低,基礎ECAR升高,葡萄糖攝取能力升高,ATP產量降低。3.PDHA1基因敲除后,腫瘤細胞的增殖受到抑制,但是腫瘤細胞的體外移動能力增強,并且這些細胞呈現顯著的化療和放療抵抗,SP細胞比率增加,干細胞標記CD44、ABCG2、Oct3/4、Nanog表達升高等,提示PDHA1基因敲除后的前列腺癌細胞更加具有腫瘤干細胞樣細胞的特性。第三部分:MPC抑制劑UK5099對前列腺癌細胞代謝模式和干性程度的影響研究方法1.用合適濃度的MPC抑制劑處理Ln Cap細胞。2.以丙酮酸試劑盒、ATP試劑盒分析細胞胞漿中丙酮酸的濃度的變化、細胞ATP產量,以及使用線粒體膜電位JC-1探針和通過流式細胞儀檢測細胞線粒體膜電位的變化,初步分析細胞代謝中線粒體氧化磷酸化程度和糖酵解速率的轉變。2.用UK5099抑制丙酮酸轉運進入線粒體后,觀察Ln Cap細胞增殖的情況,流式細胞儀分析細胞周期、Hoechst 33342染色及流式細胞儀分析側群細胞(SP)比率以及Western blot檢測干細胞標記的表達水平的變化。研究結果1.應用MPC抑制劑UK5099處理細胞,證實了UK5099可以抑制丙酮酸轉運進入線粒體基質。2.UK5099處理Ln Cap細胞后,ATP產量降低,乳酸產量增高,線粒體膜電勢降低,提示線粒體氧化磷酸化程度收到抑制,糖酵解速率提高。3.UK5099處理Ln Cap細胞后,細胞增殖受到抑制,細胞G1/G0比例增加,SP細胞比率增加,干細胞標記Oct3/4和Nanog表達升高。研究結論1.我們檢測PDHA1蛋白在前列腺癌組織中的表達情況,發(fā)現PDHA1蛋白陰性表達與較差的預后相關,提示糖酵解通路可能在前列腺癌發(fā)生發(fā)展中發(fā)揮了重要的作用。2.敲除PDHA1基因以造成丙酮酸氧化脫羧障礙,或外源性應用UK5099抑制丙酮酸轉運進入線粒體,均可以抑制線粒體氧化磷酸化,促進糖酵解速率,可以作為研究Warburg效應和腫瘤干細胞的模型。3.糖酵解程度升高的代謝模式促進了前列腺癌細胞干性程度的提高,這可能是PDHA1陰性表達患者預后差的基礎,本研究也間接提示了腫瘤干細胞樣細胞的存在可能是Warburg現象產生的原因,我們的研究對于理解前列腺癌的發(fā)生發(fā)展機制以及探索新的治療靶點提供新的視角。
[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.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.25
本文編號：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.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.25
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