Glsl介导肝癌细胞耐酸并促进肝癌恶性进展的作用及机制

发布时间：2018-06-25 14:05

  本文选题：谷氨酰胺酶 + 肝癌　； 参考：《第四军医大学》2017年博士论文

【摘要】：【研究背景与目的】肝癌是高发和高致死的恶性肿瘤,约50%的肝癌发病及死亡都发生在中国。随着中国人口结构日趋老龄化,肝癌的发病率和死亡率将会进一步增长。肝细胞肝癌(Hepatocellular Carcinoma,HCC)是肝癌最主要的疾病类型,占全部肝癌的70%-90%,如何有效的治疗HCC,成为我们防治肝癌的首要任务。肝癌是一种典型的高代谢型癌症。自Otto Warburg于1924年发现了肿瘤代谢过程中特有的有氧糖酵解现象,从而奠定了肿瘤代谢理论的基础以来,癌症甚至被有些学者称为是一种代谢病。肿瘤在代谢重编程过程中,显著上调了糖酵解速率和谷氨酰胺的摄取,并产生大量中间产物用于生物合成,谷氨酰胺(Glutamine,Gln)及葡萄糖(Glucose,Glc)成为了肿瘤能量的最主要的来源。2011年,Hanahan和Weinberg在肿瘤原有六大特征的基础上增加了四个新的特征,而能量代谢异常是其中最为重要的一条,肿瘤代谢相关研究也成为了研究热点。内环境稳态是生理活动的基础,肿瘤的高糖酵解率使大量酸性产物堆积,导致肿瘤胞内pH(Intracellular pH,pHi)和胞外pH(Extracellular pH,pHe)发生显著下调,形成肿瘤局部酸性微环境(Acidic tumor microenvironment,A-TME)。一方面,降低的pHe可损伤正常细胞、降解基质、促进肿瘤侵袭转移,及获得免疫耐受、化疗抵抗;另一方面,肿瘤pHi的下降会自我损伤,需要极为高效、快速的抵抗酸胁迫(Acid resistance,AR)从而保证代谢、增殖需求。有观点认为囊泡型ATP酶(Vacuolar ATPase,V-ATPase)和Na+/H+交换体(Na+/H+Exchanger,NHE)起到主要作用,但不能很好解释肿瘤细胞对能量的迫切需求与持续供应离子泵用于更多消耗的矛盾;新的研究认为肾型谷氨酰胺酶(Kidney-type glutaminase,Gls1)能够促进宫颈癌及乳腺癌对抗酸胁迫,但仍缺乏足够的证据。结合谷氨酰胺对肿瘤的能量供应来看,显然这种又能提供氮源与碳源的能量物质如果能够进一步帮助肿瘤耐酸,就更为符合肿瘤乃至机体的生物特性。但是否肿瘤细胞确实如此,特别是肝癌这种具有显著代谢异质性的癌症如此,仍需大量研究加以阐述。本课题旨在通过Gls1介导肝癌耐酸相应的研究,试图寻找Gls1及Gln对肝癌代谢作用的新的认识。【研究方法】第一部分肝癌细胞的谷氨酰胺依赖1、不同条件下肝癌细胞的谷氨酰胺及葡萄糖缺失耐受检测选取5种肝癌细胞系,分别用不含谷氨酰胺与不含葡萄糖的培养基培养细胞3天,对比含有谷氨酰胺及葡萄糖培养条件下的细胞相对增殖;2、肝癌细胞系的Gln浓度依赖检测利用不同浓度的Gln培养肝癌细胞系4天,在不同时间点检测所选细胞系的增殖情况,从而了解Gln的浓度依赖情况;3、Gln对肝癌细胞侵袭和迁移能力的影响通过含有或不含有Gln的培养条件进行培养,分别利用利用Transwell实验和细胞迁移实验,检测并比对各个肝癌细胞系中,缺失Gln对肝癌细胞系侵袭能力和迁移能力的影响;第二部分Gls1及Gls2的组织分布1、Gls1及Gls2在不同肝脏疾病中的表达选取肝细胞肝癌、胆管细胞癌、结节性肝硬化及肝腺瘤病人的组织标本,通过免疫组织化学检测,对比病灶与病灶远端组织中Gls1及Gls2的表达情况;2、Gls1及Gls2的肝癌组织表达选取9例肝癌病人的癌、癌旁、远端组织,通过免疫组织化学检测,对比癌灶、癌旁及远端组织Gls1及Gls2的表达;通过选取6对肝癌的癌与癌旁组织,通过Western blotting检测Gls1及Gls2的蛋白表达情况;第三部分Gls1通过介导Gln促进肝癌细胞获得耐酸能力1、Glu及α-KG补救实验选取HepG2细胞及Hep3B细胞系,通过分别补充Gln的下游分解产物Glu及α-KG,检测对所选细胞的增殖恢复情况;2、Gln缺失的pHe检测通过含有或不含有Gln情况下,培养所选细胞5天,检测pHe随时间增加的变化情况,以了解胞外酸性环境的产生情况;3、缺失Gln、抑制Gls1对肝癌细胞系耐酸能力的影响检测分别通过含有或不含有Gln下,检测HepG2细胞及Hep3B细胞增殖变化,以反应细胞耐酸能力的变化;通过si-Gls1及小分子化合物抑制剂抑制Gls1活性的方法,对比不抑制时HepG2细胞及Hep3B细胞增殖变化,以反应细胞耐酸能力的变化;4、抑制剂补充实验及Gln补救实验通过抑制剂补充实验,检测HepG2细胞及Hep3B细胞在含有Gln,不同pH情况下培养时的细胞增殖变化;利用Gln的补救实验,观察上述细胞在抑制了Gls1后,不同pH下的细胞增殖变化;第四部分抑制Gls1可进一步抑制酸性微环境下肝癌细胞系恶性表型1、抑制Gls1对肝癌细胞系pHe的影响利用siRNA及小分子化合物抑制剂分别抑制HepG2细胞及Hep3B细胞的Gls1,培养5天,在不同时间点检测细胞pHe的变化情况;2、去除Gln和抑制Gls1对不同pH下肝癌细胞系侵袭能力影响分别通过含有或不含有Gln下培养细胞,利用Transwell实验检测HepG2细胞及Hep3B细胞在不同pH下侵袭能力变化;通过si-Gls1及小分子化合物抑制剂抑制Gls1活性的方法,利用Transwell实验对比不抑制时HepG2细胞及Hep3B细胞在不同pH条件下侵袭能力的变化;3、去除Gln和抑制Gls1对不同pH下肝癌细胞系迁移能力影响分别通过含有或不含有Gln下培养细胞,利用迁移实验检测HepG2细胞及Hep3B细胞在不同pH下迁移能力变化;通过si-Gls1及小分子化合物抑制剂抑制Gls1活性的方法,利用迁移实验对比不抑制时HepG2细胞及Hep3B细胞在不同pH条件下迁移能力的变化;第五部分Gls1小分子化合物抑制剂对小鼠肝癌的治疗作用1、联合化学法诱导肝癌小鼠模型的建立及Gls1抑制剂干预利用BALB/c小鼠,通过DEN+CCl4+Ethanol联合诱导方案构建肝癌模型;利用Gls1抑制剂C968及BPTES干预肝癌小鼠6周后,进行相关检测;2、观察应用Gls1抑制剂后的相关指标变化通过HE染色观察对比对照组与各处置组肝脏与肺的病理变化,通过Western blotting检测Gls1及Gls2在干预后的变化情况;通过测量一般指标及生化指标,观察并对比各组小鼠差异;第六部分肝癌中Gls1的DNA甲基化水平变化选取完全正常、肝癌与癌旁组织,利用MassARRAY定量分析Gls1的DNA甲基化水平差异。【主要研究结果】第一部分肝癌细胞的恶性生物学行为高度依赖谷氨酰胺肝癌细胞对Gln及Glc均具有显著依赖;在4mM以上Gln进行培养则对细胞增殖再无明显差异;而缺失Gln培养则能抑制肝癌细胞的侵袭及迁移能力;第二部分Gls1及Gls2的组织分布规律对比病灶于病灶旁组织,Gls1在肝癌与胆管癌中表达上调而在结节性肝硬化及肝腺瘤中无明显变化;并且,Gls在肝癌中特异性的表达上调,而Gls2则无明显变化;第三部分Gls1通过介导谷氨酰胺分解代谢促进肝癌细胞获取耐酸能力通过补充Gln分解的下游产物Glu和α-KG不能完全恢复肝癌细胞增殖能力;而含有Gln 4mM培养时,能导致细胞外酸性环境形成;分别通过pH 6.0及pH 7.0培养HepG2及Hep3B细胞系,缺失Gln或抑制Gls1能够显著降低酸性环境下细胞增殖能力;pH 6.0培养细胞时,含有Gln的情况下抑制Gls1能抑制细胞增殖,而抑制Gls1情况下补谷氨酰胺则不能恢复细胞增殖;第四部分抑制Gls1可降低酸性微环境下肝癌细胞恶性行为通过抑制Gls1能够阻止肝癌细胞的胞外酸性微环境形成;去除Gln或抑制Gls1,能够进一步降低pH 6.0下细胞的侵袭和迁移能力;第五部分Gls1小分子抑制剂对联合化学诱导小鼠原位肝癌的治疗作用Gls1干预肝癌小鼠6周后,可见干预组能有效降低肝癌病理分级,生存率显著高于未处理组,能显著缩小瘤体直径,抑制肺部转移,并部分改善肝功能。第六部分临床患者肝癌组织中Gls1的DNA甲基化水平变化规律肝癌癌灶组织内的Gls1甲基化水平较癌旁与完全正常肝组织显著降低,而癌旁与正常组织两者间无显著性差异。【结论】肝癌在代谢过程中产生酸性微环境,肝癌特异性上调表达的Gls1通过分解Gln产生Glu和NH3,一方面为肝癌增殖提供能量和生物合成原料,一方面促进介导肝癌细胞获得耐酸能力;抑制Gls1能够有效抑制酸性环境下肝癌细胞的增殖、侵袭和迁移,是一种很有前景的治疗方向,而肝癌组织发生特异性的去甲基化,是Gls1在肝癌上调的机制之一。
[Abstract]:[background and objective] liver cancer is a high and fatal malignant tumor, and about 50% of the incidence and death of liver cancer occur in China. With the aging of the population structure in China, the incidence and mortality of liver cancer will be further increased. Hepatoma (Hepatocellular Carcinoma, HCC) is the main disease type of liver cancer, which accounts for the total number of liver cancer. The 70%-90% of liver cancer, how to effectively treat HCC, is the primary task of preventing and controlling liver cancer. Liver cancer is a typical high metabolic cancer. Since Otto Warburg discovered the unique aerobic glycolysis in the process of tumor metabolism in 1924, it has laid the foundation of the theory of tumor metabolism, and cancer has even been called by some scholars. Glutamine, Gln and Glc (Glucose, Glc) have become the most important source of tumor energy in.2011 years, and Hanahan and Weinberg are the six major characteristics of the tumor. On the basis of four new features, energy metabolism abnormality is the most important one, and the research on tumor metabolism is also the focus of research. Internal environment homeostasis is the basis of physiological activity. The high glycolysis rate of the tumor accumulates a large number of acid products, resulting in the intracellular pH (Intracellular pH, pHi) and extracellular pH (Extracellular P). On the one hand, the reduced pHe can damage normal cells, degrade the matrix, promote tumor invasion and metastasis, and obtain immune tolerance, and chemotherapeutic resistance. On the other hand, the decrease of pHi in swelling tumor will be self damaging, which requires highly efficient and rapid resistance to acid. On the other hand, the decrease of pHe can damage normal cells (Acidic tumor microenvironment, A-TME). Acid resistance (AR) ensures metabolism and proliferation demand. There is a view that vesicular ATP (Vacuolar ATPase, V-ATPase) and Na+/H+ exchange (Na+/H+Exchanger, NHE) play a major role, but the contradiction between the urgent demand for energy and the use of the continuous supply ion pump for more consumption is not well explained; new studies believe that Kidney-type glutaminase (Gls1) can promote cervical cancer and breast cancer against acid stress, but there is still lack of sufficient evidence. The biological characteristics of the body. But whether it is true that the tumor cells are so, especially the cancer of the liver, which has significant metabolic heterogeneity, still needs a lot of research. This topic aims to find a new study on the acid tolerance of liver cancer mediated by Gls1 and try to find a new understanding of the metabolism of liver cancer by Gls1 and Gln. [method] the first part The glutamine of hepatoma cells depends on 1. Under different conditions, the glutamine and glucose tolerance test of hepatoma cells selected 5 kinds of hepatoma cell lines, and cultured cells without glutamine and glucose free medium for 3 days, compared with the relative proliferation of the cells containing glutamine and glucose culture. 2, liver cancer cell lines. Gln concentration depended on the detection of liver cancer cell lines with different concentrations of Gln for 4 days. The proliferation of selected cell lines was detected at different time points, so as to understand the concentration dependence of Gln; 3, the influence of Gln on the invasion and migration of hepatoma cells was cultured with or without Gln culture conditions, using Transwell respectively. Experiments and cell migration tests were used to detect and compare the effects of Gln deletion on the invasion and migration of hepatocellular carcinoma cell lines. Second the tissue distribution of Gls1 and Gls2 was 1, Gls1 and Gls2 were expressed in different liver diseases, and the tissues of hepatocellular carcinoma, cholangiocarcinoma, nodular cirrhosis and liver adenoma were selected. The expression of Gls1 and Gls2 in the lesion and the distal tissue of the lesion was compared by immunohistochemistry. 2, Gls1 and Gls2 were expressed in 9 cases of liver cancer, adjacent to cancer and distal tissue. The expression of Gls1 and Gls2 were compared by immunohistochemistry, cancer and distal tissue, and 6 of cancer were selected. The protein expression of Gls1 and Gls2 was detected by Western blotting with the para cancerous tissue, and the third part Gls1 promoted the acid resistance of hepatoma cells by mediating Gln, and Glu and alpha -KG remedial experiment selected HepG2 cell and Hep3B cell lines. The proliferation recovery of selected cells was detected by supplementation of Gln downstream decomposition products Glu and alpha replication. 2, the pHe detection of Gln deletion was used to cultivate the selected cells for 5 days by containing or without Gln to detect the increase of pHe with time to understand the occurrence of the extracellular acid environment; 3, the deletion of Gln, and the inhibition of Gls1 on the acid resistance of the hepatocellular carcinoma cell lines, detection of HepG2 cells and Hep3B, respectively, under or without Gln. The cell proliferation changes in response to the changes in the acid resistance of the cells. Through the methods of inhibiting the activity of Gls1 by si-Gls1 and the inhibitors of small molecular compounds, the proliferation of HepG2 cells and Hep3B cells in non inhibition is compared to respond to the changes in the acid resistance of the cells. 4, the inhibitor supplementation experiment and the Gln remedial experiment are used to detect HepG2 by the inhibitor supplementation experiment. Cells and Hep3B cells were cultured in Gln and different pH conditions, and Gln remedial experiments were used to observe the cell proliferation changes under the inhibition of Gls1 and different pH, and the fourth inhibition of Gls1 could further inhibit the malignant phenotype of hepatoma cell line 1 in the acid microenvironment, and inhibit Gls1 to the liver cancer cell line pHe. The influence of siRNA and small molecular compound inhibitors on the inhibition of Gls1 of HepG2 cells and Hep3B cells for 5 days was detected at different time points. 2, the effects of removing Gln and inhibiting Gls1 on the invasion ability of hepatocellular carcinoma cell lines under different pH were detected by containing or not containing Gln, and using Transwell test. The invasiveness of HepG2 cells and Hep3B cells at different pH; by means of si-Gls1 and inhibition of Gls1 activity by inhibitors of small molecular compounds, Transwell experiments were used to compare the invasiveness of HepG2 cells and Hep3B cells under different pH conditions without inhibition; 3, removal of Gln and inhibitory Gls1 on the migration of hepatocellular carcinoma cell lines under different pH. The migration ability of HepG2 cells and Hep3B cells under different pH was detected by migration experiments by the culture cells containing or without Gln, and the migration ability of HepG2 cells and Hep3B cells under different pH conditions was compared by means of si-Gls1 and the inhibition of Gls1 activity by the inhibitors of si-Gls1 and small molecular compounds. The changes in the fifth part of Gls1 small molecule compound inhibitor on mice liver cancer 1, combined with chemical induced liver cancer mice model and Gls1 inhibitor intervention using BALB/c mice, through the DEN+CCl4+Ethanol joint induction scheme for the construction of liver cancer model, Gls1 inhibitor C968 and BPTES intervention for 6 weeks after liver cancer mice, the phase 2, observe the changes of the related indexes after the application of Gls1 inhibitor, observe the pathological changes of liver and lung in comparison control group and each treatment group by HE staining, and detect the changes of Gls1 and Gls2 in the dry prognosis through Western blotting, and observe and compare the difference of the mice in each group by measuring the general index and biochemical index, and the sixth part of liver cancer. The changes in the level of DNA methylation of Gls1 were completely normal, liver cancer and para cancer tissue, and the difference of DNA methylation level of Gls1 was quantitatively analyzed by MassARRAY. [main results] the malignant biological behavior of liver cancer cells in part 1 was highly dependent on the Gln and Glc of HCC cells, and Gln carried out above 4mM. There was no significant difference in cell proliferation, but the absence of Gln culture could inhibit the invasion and migration of hepatoma cells. The tissue distribution of the second part of Gls1 and Gls2 was compared with the lesion in the paratiasal tissue. The expression of Gls1 in HCC and cholangiocarcinoma was up and there was no significant change in nodular cirrhosis and liver adenoma; and Gls was in the liver cancer. The expression of medium specificity is up, but Gls2 does not change obviously. Third part of Gls1 can not completely restore the proliferation ability of hepatoma cells by supplementing the metabolism of glutamine catabolism and promoting the ability of liver cancer cells to gain acid resistance by supplementing the downstream products of Gln decomposition, Glu and alpha -KG, while Gln 4mM culture can lead to the formation of extracellular acidic environment. Don't cultivate HepG2 and Hep3B cell lines through pH 6 and pH 7, the absence of Gln or inhibition of Gls1 can significantly reduce the proliferation of cells in the acidic environment; pH 6 inhibits the proliferation of cells in the case of Gln in the case of Gln, while the inhibition of Gls1 is not able to restore the proliferation of cells; the fourth part inhibits Gls1 can be reduced. The malignant behavior of hepatoma cells in acid microenvironment can inhibit the formation of extracellular acidic microenvironment of hepatoma cells by inhibiting Gls1; removal of Gln or inhibition of Gls1 can further reduce the invasion and migration of pH 6 cells; the fifth part of Gls1 small molecule inhibitors can intervene in the treatment of liver cancer in mice induced by chemical induced mice by Gls1 intervention in liver 6 weeks after cancer mice, it can be seen that the intervention group can effectively reduce the pathological grade of liver cancer, the survival rate is significantly higher than that of the untreated group, which can significantly reduce the diameter of the tumor body, inhibit the pulmonary metastasis, and partly improve the liver function. The level of DNA methylation in the Gls1 in the liver cancer tissues of the sixth clinical patients is more than that of the cancer in the carcinoma of the liver cancer. There is no significant difference between the para and normal liver tissues, but there is no significant difference between the paracancerous and normal tissues. [Conclusion] the liver cancer produces an acidic microenvironment during the metabolic process. The specific up Gls1 expression of HCC produces Glu and NH3 by decomposing Gln, on the one hand, it provides energy and biosynthesis materials for the proliferation of liver cancer. Cancer cells obtain acid resistance, and inhibition of Gls1 can effectively inhibit the proliferation, invasion and migration of hepatoma cells in acidic environment, and it is a promising treatment direction. The specific demethylation of liver cancer tissue is one of the mechanisms of Gls1 in the up regulation of liver cancer.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7
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本文编号：2066253