本文選題：谷氨酰胺酶 + 肝癌　； 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：【研究背景與目的】肝癌是高發(fā)和高致死的惡性腫瘤,約50%的肝癌發(fā)病及死亡都發(fā)生在中國。隨著中國人口結(jié)構(gòu)日趨老齡化,肝癌的發(fā)病率和死亡率將會進(jìn)一步增長。肝細(xì)胞肝癌(Hepatocellular Carcinoma,HCC)是肝癌最主要的疾病類型,占全部肝癌的70%-90%,如何有效的治療HCC,成為我們防治肝癌的首要任務(wù)。肝癌是一種典型的高代謝型癌癥。自O(shè)tto Warburg于1924年發(fā)現(xiàn)了腫瘤代謝過程中特有的有氧糖酵解現(xiàn)象,從而奠定了腫瘤代謝理論的基礎(chǔ)以來,癌癥甚至被有些學(xué)者稱為是一種代謝病。腫瘤在代謝重編程過程中,顯著上調(diào)了糖酵解速率和谷氨酰胺的攝取,并產(chǎn)生大量中間產(chǎn)物用于生物合成,谷氨酰胺(Glutamine,Gln)及葡萄糖(Glucose,Glc)成為了腫瘤能量的最主要的來源。2011年,Hanahan和Weinberg在腫瘤原有六大特征的基礎(chǔ)上增加了四個新的特征,而能量代謝異常是其中最為重要的一條,腫瘤代謝相關(guān)研究也成為了研究熱點。內(nèi)環(huán)境穩(wěn)態(tài)是生理活動的基礎(chǔ),腫瘤的高糖酵解率使大量酸性產(chǎn)物堆積,導(dǎo)致腫瘤胞內(nèi)pH(Intracellular pH,pHi)和胞外pH(Extracellular pH,pHe)發(fā)生顯著下調(diào),形成腫瘤局部酸性微環(huán)境(Acidic tumor microenvironment,A-TME)。一方面,降低的pHe可損傷正常細(xì)胞、降解基質(zhì)、促進(jìn)腫瘤侵襲轉(zhuǎn)移,及獲得免疫耐受、化療抵抗;另一方面,腫瘤pHi的下降會自我損傷,需要極為高效、快速的抵抗酸脅迫(Acid resistance,AR)從而保證代謝、增殖需求。有觀點認(rèn)為囊泡型ATP酶(Vacuolar ATPase,V-ATPase)和Na+/H+交換體(Na+/H+Exchanger,NHE)起到主要作用,但不能很好解釋腫瘤細(xì)胞對能量的迫切需求與持續(xù)供應(yīng)離子泵用于更多消耗的矛盾;新的研究認(rèn)為腎型谷氨酰胺酶(Kidney-type glutaminase,Gls1)能夠促進(jìn)宮頸癌及乳腺癌對抗酸脅迫,但仍缺乏足夠的證據(jù)。結(jié)合谷氨酰胺對腫瘤的能量供應(yīng)來看,顯然這種又能提供氮源與碳源的能量物質(zhì)如果能夠進(jìn)一步幫助腫瘤耐酸,就更為符合腫瘤乃至機(jī)體的生物特性。但是否腫瘤細(xì)胞確實如此,特別是肝癌這種具有顯著代謝異質(zhì)性的癌癥如此,仍需大量研究加以闡述。本課題旨在通過Gls1介導(dǎo)肝癌耐酸相應(yīng)的研究,試圖尋找Gls1及Gln對肝癌代謝作用的新的認(rèn)識�！狙芯糠椒ā康谝徊糠指伟┘�(xì)胞的谷氨酰胺依賴1、不同條件下肝癌細(xì)胞的谷氨酰胺及葡萄糖缺失耐受檢測選取5種肝癌細(xì)胞系,分別用不含谷氨酰胺與不含葡萄糖的培養(yǎng)基培養(yǎng)細(xì)胞3天,對比含有谷氨酰胺及葡萄糖培養(yǎng)條件下的細(xì)胞相對增殖;2、肝癌細(xì)胞系的Gln濃度依賴檢測利用不同濃度的Gln培養(yǎng)肝癌細(xì)胞系4天,在不同時間點檢測所選細(xì)胞系的增殖情況,從而了解Gln的濃度依賴情況;3、Gln對肝癌細(xì)胞侵襲和遷移能力的影響通過含有或不含有Gln的培養(yǎng)條件進(jìn)行培養(yǎng),分別利用利用Transwell實驗和細(xì)胞遷移實驗,檢測并比對各個肝癌細(xì)胞系中,缺失Gln對肝癌細(xì)胞系侵襲能力和遷移能力的影響;第二部分Gls1及Gls2的組織分布1、Gls1及Gls2在不同肝臟疾病中的表達(dá)選取肝細(xì)胞肝癌、膽管細(xì)胞癌、結(jié)節(jié)性肝硬化及肝腺瘤病人的組織標(biāo)本,通過免疫組織化學(xué)檢測,對比病灶與病灶遠(yuǎn)端組織中Gls1及Gls2的表達(dá)情況;2、Gls1及Gls2的肝癌組織表達(dá)選取9例肝癌病人的癌、癌旁、遠(yuǎn)端組織,通過免疫組織化學(xué)檢測,對比癌灶、癌旁及遠(yuǎn)端組織Gls1及Gls2的表達(dá);通過選取6對肝癌的癌與癌旁組織,通過Western blotting檢測Gls1及Gls2的蛋白表達(dá)情況;第三部分Gls1通過介導(dǎo)Gln促進(jìn)肝癌細(xì)胞獲得耐酸能力1、Glu及α-KG補(bǔ)救實驗選取HepG2細(xì)胞及Hep3B細(xì)胞系,通過分別補(bǔ)充Gln的下游分解產(chǎn)物Glu及α-KG,檢測對所選細(xì)胞的增殖恢復(fù)情況;2、Gln缺失的pHe檢測通過含有或不含有Gln情況下,培養(yǎng)所選細(xì)胞5天,檢測pHe隨時間增加的變化情況,以了解胞外酸性環(huán)境的產(chǎn)生情況;3、缺失Gln、抑制Gls1對肝癌細(xì)胞系耐酸能力的影響檢測分別通過含有或不含有Gln下,檢測HepG2細(xì)胞及Hep3B細(xì)胞增殖變化,以反應(yīng)細(xì)胞耐酸能力的變化;通過si-Gls1及小分子化合物抑制劑抑制Gls1活性的方法,對比不抑制時HepG2細(xì)胞及Hep3B細(xì)胞增殖變化,以反應(yīng)細(xì)胞耐酸能力的變化;4、抑制劑補(bǔ)充實驗及Gln補(bǔ)救實驗通過抑制劑補(bǔ)充實驗,檢測HepG2細(xì)胞及Hep3B細(xì)胞在含有Gln,不同pH情況下培養(yǎng)時的細(xì)胞增殖變化;利用Gln的補(bǔ)救實驗,觀察上述細(xì)胞在抑制了Gls1后,不同pH下的細(xì)胞增殖變化;第四部分抑制Gls1可進(jìn)一步抑制酸性微環(huán)境下肝癌細(xì)胞系惡性表型1、抑制Gls1對肝癌細(xì)胞系pHe的影響利用siRNA及小分子化合物抑制劑分別抑制HepG2細(xì)胞及Hep3B細(xì)胞的Gls1,培養(yǎng)5天,在不同時間點檢測細(xì)胞pHe的變化情況;2、去除Gln和抑制Gls1對不同pH下肝癌細(xì)胞系侵襲能力影響分別通過含有或不含有Gln下培養(yǎng)細(xì)胞,利用Transwell實驗檢測HepG2細(xì)胞及Hep3B細(xì)胞在不同pH下侵襲能力變化;通過si-Gls1及小分子化合物抑制劑抑制Gls1活性的方法,利用Transwell實驗對比不抑制時HepG2細(xì)胞及Hep3B細(xì)胞在不同pH條件下侵襲能力的變化;3、去除Gln和抑制Gls1對不同pH下肝癌細(xì)胞系遷移能力影響分別通過含有或不含有Gln下培養(yǎng)細(xì)胞,利用遷移實驗檢測HepG2細(xì)胞及Hep3B細(xì)胞在不同pH下遷移能力變化;通過si-Gls1及小分子化合物抑制劑抑制Gls1活性的方法,利用遷移實驗對比不抑制時HepG2細(xì)胞及Hep3B細(xì)胞在不同pH條件下遷移能力的變化;第五部分Gls1小分子化合物抑制劑對小鼠肝癌的治療作用1、聯(lián)合化學(xué)法誘導(dǎo)肝癌小鼠模型的建立及Gls1抑制劑干預(yù)利用BALB/c小鼠,通過DEN+CCl4+Ethanol聯(lián)合誘導(dǎo)方案構(gòu)建肝癌模型;利用Gls1抑制劑C968及BPTES干預(yù)肝癌小鼠6周后,進(jìn)行相關(guān)檢測;2、觀察應(yīng)用Gls1抑制劑后的相關(guān)指標(biāo)變化通過HE染色觀察對比對照組與各處置組肝臟與肺的病理變化,通過Western blotting檢測Gls1及Gls2在干預(yù)后的變化情況;通過測量一般指標(biāo)及生化指標(biāo),觀察并對比各組小鼠差異;第六部分肝癌中Gls1的DNA甲基化水平變化選取完全正常、肝癌與癌旁組織,利用MassARRAY定量分析Gls1的DNA甲基化水平差異�！局饕芯拷Y(jié)果】第一部分肝癌細(xì)胞的惡性生物學(xué)行為高度依賴谷氨酰胺肝癌細(xì)胞對Gln及Glc均具有顯著依賴;在4mM以上Gln進(jìn)行培養(yǎng)則對細(xì)胞增殖再無明顯差異;而缺失Gln培養(yǎng)則能抑制肝癌細(xì)胞的侵襲及遷移能力;第二部分Gls1及Gls2的組織分布規(guī)律對比病灶于病灶旁組織,Gls1在肝癌與膽管癌中表達(dá)上調(diào)而在結(jié)節(jié)性肝硬化及肝腺瘤中無明顯變化;并且,Gls在肝癌中特異性的表達(dá)上調(diào),而Gls2則無明顯變化;第三部分Gls1通過介導(dǎo)谷氨酰胺分解代謝促進(jìn)肝癌細(xì)胞獲取耐酸能力通過補(bǔ)充Gln分解的下游產(chǎn)物Glu和α-KG不能完全恢復(fù)肝癌細(xì)胞增殖能力;而含有Gln 4mM培養(yǎng)時,能導(dǎo)致細(xì)胞外酸性環(huán)境形成;分別通過pH 6.0及pH 7.0培養(yǎng)HepG2及Hep3B細(xì)胞系,缺失Gln或抑制Gls1能夠顯著降低酸性環(huán)境下細(xì)胞增殖能力;pH 6.0培養(yǎng)細(xì)胞時,含有Gln的情況下抑制Gls1能抑制細(xì)胞增殖,而抑制Gls1情況下補(bǔ)谷氨酰胺則不能恢復(fù)細(xì)胞增殖;第四部分抑制Gls1可降低酸性微環(huán)境下肝癌細(xì)胞惡性行為通過抑制Gls1能夠阻止肝癌細(xì)胞的胞外酸性微環(huán)境形成;去除Gln或抑制Gls1,能夠進(jìn)一步降低pH 6.0下細(xì)胞的侵襲和遷移能力;第五部分Gls1小分子抑制劑對聯(lián)合化學(xué)誘導(dǎo)小鼠原位肝癌的治療作用Gls1干預(yù)肝癌小鼠6周后,可見干預(yù)組能有效降低肝癌病理分級,生存率顯著高于未處理組,能顯著縮小瘤體直徑,抑制肺部轉(zhuǎn)移,并部分改善肝功能。第六部分臨床患者肝癌組織中Gls1的DNA甲基化水平變化規(guī)律肝癌癌灶組織內(nèi)的Gls1甲基化水平較癌旁與完全正常肝組織顯著降低,而癌旁與正常組織兩者間無顯著性差異。【結(jié)論】肝癌在代謝過程中產(chǎn)生酸性微環(huán)境,肝癌特異性上調(diào)表達(dá)的Gls1通過分解Gln產(chǎn)生Glu和NH3,一方面為肝癌增殖提供能量和生物合成原料,一方面促進(jìn)介導(dǎo)肝癌細(xì)胞獲得耐酸能力;抑制Gls1能夠有效抑制酸性環(huán)境下肝癌細(xì)胞的增殖、侵襲和遷移,是一種很有前景的治療方向,而肝癌組織發(fā)生特異性的去甲基化,是Gls1在肝癌上調(diào)的機(jī)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.7
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本文編號：2066253