本文選題：膠質(zhì)瘤　切入點(diǎn)：氨基酸饑餓　出處：《吉林大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：膠質(zhì)瘤是一種神經(jīng)系統(tǒng)常見(jiàn)的惡性腫瘤,其以發(fā)病率低、致死率高為主要特點(diǎn)。目前,治療膠質(zhì)瘤的標(biāo)準(zhǔn)方法是手術(shù)切除輔助以化療和放療。但是,該方法不能徹底治愈膠質(zhì)瘤,經(jīng)過(guò)治療后患者預(yù)后差,復(fù)發(fā)率高,5年生存率不足10%。膠質(zhì)瘤細(xì)胞在體內(nèi)迅速增殖需要消耗大量營(yíng)養(yǎng)物質(zhì),超出了周圍環(huán)境的供給能力,導(dǎo)致微環(huán)境中營(yíng)養(yǎng)物質(zhì)供應(yīng)不足。但是,在營(yíng)養(yǎng)物質(zhì)供應(yīng)不充分的條件下,膠質(zhì)瘤細(xì)胞是如何保持著頑強(qiáng)的生存能力成為該領(lǐng)域研究的熱點(diǎn)問(wèn)題。環(huán)氧化酶-2(Cyclooxygenase-2,COX-2)作為前列腺素合成的限速酶,在維持細(xì)胞的正常生命活動(dòng)方面發(fā)揮著關(guān)鍵作用。近些年的研究發(fā)現(xiàn),COX-2在腫瘤細(xì)胞內(nèi)的表達(dá)能夠提高腫瘤細(xì)胞的增殖能力、遷移能力和血管化進(jìn)程,同時(shí)抑制腫瘤細(xì)胞的凋亡。COX-2在細(xì)胞內(nèi)的表達(dá)受活性氧、炎癥和代謝壓力等因素的調(diào)節(jié)。那么,營(yíng)養(yǎng)物質(zhì)供應(yīng)不足是否會(huì)對(duì)膠質(zhì)瘤內(nèi)COX-2的表達(dá)產(chǎn)生影響,如果產(chǎn)生影響,具體的機(jī)制是什么?對(duì)于這一問(wèn)題,在國(guó)內(nèi)外還未見(jiàn)到相關(guān)報(bào)道。因此,本研究通過(guò)細(xì)胞的氨基酸饑餓模型模擬體內(nèi)營(yíng)養(yǎng)物質(zhì)供應(yīng)不足的生存環(huán)境,探索營(yíng)養(yǎng)物質(zhì)供應(yīng)不足對(duì)膠質(zhì)瘤細(xì)胞內(nèi)COX-2表達(dá)的影響,并對(duì)相關(guān)的機(jī)制進(jìn)行進(jìn)一步的探索。本研究通過(guò)甲硫氨酸/半胱氨酸(Methionine/Cysteine,Met/Cys)缺失的培養(yǎng)基和谷氨酸鹽/精氨酸/賴氨酸(Glutamine/Arginine/Lysine,Gln/Arg/Lys)缺失的培養(yǎng)基培養(yǎng)U87細(xì)胞和SF767細(xì)胞,收集不同時(shí)間點(diǎn)的細(xì)胞樣品,通過(guò)熒光定量PCR和免疫印跡檢測(cè)法,從m RNA和蛋白質(zhì)水平對(duì)COX-2的表達(dá)進(jìn)行檢測(cè)。然后,我們利用p38-MAPK、MEK和JNK的特異性抑制劑SB202190(SB)、U0126(U)和SP600125(SP)阻斷這些信號(hào)通路的信號(hào)傳遞功能,通過(guò)熒光定量PCR和免疫印跡檢測(cè)法,從m RNA和蛋白質(zhì)水平對(duì)COX-2的表達(dá)進(jìn)行檢測(cè)。為了更加準(zhǔn)確的闡明p38-MAPK信號(hào)通路對(duì)COX-2表達(dá)的影響,我們通過(guò)脂質(zhì)體轉(zhuǎn)染法將p38-MAPK特異性的si RNA導(dǎo)入到膠質(zhì)瘤細(xì)胞內(nèi),通過(guò)熒光定量PCR和免疫印跡檢測(cè)法,從m RNA和蛋白質(zhì)水平對(duì)p38-MAPK的基因敲除效果和COX-2的表達(dá)進(jìn)行檢測(cè)。為了進(jìn)一步探索p38-MAPK下游基因SP1對(duì)COX-2表達(dá)的影響,我們通過(guò)脂質(zhì)體轉(zhuǎn)染法將COX-2-luciferase報(bào)告基因?qū)氲侥z質(zhì)瘤細(xì)胞內(nèi),再利用SP1特異性的si RNA和抑制劑mithramycin探索SP1對(duì)COX-2表達(dá)的影響,通過(guò)檢測(cè)熒光的強(qiáng)度確定COX-2表達(dá)量的高低。最后,我們利用COX-2特異性的si RNA和抑制劑celecoxib抑制COX-2的功能,研究COX-2對(duì)VEGF表達(dá)和自噬發(fā)生的影響。通過(guò)熒光定量PCR確定COX-2對(duì)VEGF表達(dá)的影響;通過(guò)免疫印跡的方法檢測(cè)LC3-II的表達(dá)水平,進(jìn)而評(píng)價(jià)細(xì)胞自噬的強(qiáng)度。實(shí)驗(yàn)結(jié)果顯示:Met/Cys缺失的培養(yǎng)基和Gln/Arg/Lys缺失的培養(yǎng)基均可以誘導(dǎo)COX-2在U87和SF767細(xì)胞中的表達(dá),在誘導(dǎo)2h時(shí),細(xì)胞內(nèi)開(kāi)始表達(dá)COX-2,4-6h趨于穩(wěn)定。但是,相比之下,Met/Cys缺失能夠更快速的誘導(dǎo)COX-2的表達(dá)。因此,在后續(xù)的實(shí)驗(yàn)研究中,我們選擇通過(guò)Met/Cys缺失建立細(xì)胞饑餓模型。經(jīng)過(guò)p38-MAPK、MEK和JNK的特異性抑制劑SB202190(SB)、U0126(U)、SP600125(SP)以及si RNA處理后,我們發(fā)現(xiàn)p38-MAPK抑制劑SB對(duì)氨基酸饑餓誘導(dǎo)的COX-2表達(dá)的抑制效果最明顯,而JNK特異性抑制劑SP能夠顯著抑制U87細(xì)胞中COX-2的表達(dá),卻對(duì)SF767細(xì)胞中COX-2的表達(dá)幾乎沒(méi)有影響,U0126的作用效果剛好與SP相反。同樣,我們發(fā)現(xiàn)氨基酸饑餓能夠同時(shí)在U87細(xì)胞和SF767細(xì)胞誘導(dǎo)p38-MAPK的磷酸化,而JNK的磷酸化僅發(fā)生在U87細(xì)胞中。進(jìn)一步實(shí)驗(yàn)發(fā)現(xiàn)p38-MAPK是通過(guò)SP1調(diào)節(jié)COX-2的表達(dá),SP1基因的敲除和抑制劑的應(yīng)用能夠顯著降低氨基酸饑餓誘導(dǎo)的COX-2的表達(dá)。對(duì)COX-2下游的作用途徑進(jìn)行檢測(cè)分析發(fā)現(xiàn),COX-2的表達(dá)能夠促進(jìn)VEGF的表達(dá)及誘導(dǎo)自噬的發(fā)生。綜上所述,本研究以氨基酸饑餓模型模擬膠質(zhì)瘤組織內(nèi)營(yíng)養(yǎng)物質(zhì)供應(yīng)不足的生存環(huán)境,探索在該種條件下誘發(fā)COX-2表達(dá)的相關(guān)機(jī)制。研究結(jié)果闡明氨基酸饑餓誘導(dǎo)的COX-2表達(dá)主要受上游p38-MAPK/SP1信號(hào)通路的調(diào)控;COX-2能夠提高膠質(zhì)瘤細(xì)胞內(nèi)VEGF的表達(dá),為腫瘤組織內(nèi)的血管化奠定基礎(chǔ);COX-2通過(guò)激發(fā)膠質(zhì)瘤細(xì)胞的自噬作用,為細(xì)胞的生存提供所需能量。
[Abstract]:Glioma is the most common malignant tumor of a nervous system, with its low incidence, high death rate as the main characteristics. At present, the standard method for the treatment of glioma is surgical resection assisted by chemotherapy and radiotherapy. However, this method cannot be completely cured after treatment in patients with glioma, pre post difference, high recurrence rate. The 5 year survival rate of less than 10%. glioma cells need to consume a large amount of nutrients in the body rapidly proliferating, beyond the supply capacity of the surrounding environment, resulting in insufficient supply of nutrients to the micro environment. However, the supply of nutrients is not sufficient under the condition of curing glioma is how to maintain a strong survival ability has become a hot issue in the the field of research. Cyclooxygenase -2 (Cyclooxygenase-2, COX-2) is the rate limiting enzyme of prostaglandin synthesis, play a key role in maintaining the normal life activities of cells. Recent studies have found that in COX-2 The expression of tumor cells can enhance tumor cell proliferation, migration and vascularization, expression and inhibit the apoptosis of.COX-2 tumor cells in cells by regulating inflammation and metabolism of active oxygen, pressure and other factors. So, whether the nutrient supply will have an impact on the expression of COX-2 in glioma, if have an impact, what is the specific mechanism? For this problem, at home and abroad has not been reported. Therefore, this research through the model simulation of amino acid starvation cell nutrient in a living environment in short supply, insufficient supply of nutrients to explore effect on the expression of COX-2 in glioma cells, and further explore the related mechanism. This study by methionine / cysteine (Methionine/Cysteine, Met/Cys) medium and glutamate / lack of arginine / lysine (Glutamine/Argi Nine/Lysine, Gln/Arg/Lys) medium lack of cultured U87 cells and SF767 cells were collected at different time points of cell samples was detected by fluorescence quantitative PCR and Western blot, were detected from the m level of RNA and protein expression of COX-2. Then, we use p38-MAPK, a specific inhibitor of SB202190 MEK and JNK (SB). U0126 (U) and SP600125 (SP) signal pathway blocking the transfer function was detected by fluorescence quantitative PCR and Western blot, were detected from the m level of RNA and protein expression of COX-2. In order to accurately elucidate the p38-MAPK signaling pathway on the expression of COX-2, we transfected into Si RNA p38-MAPK specific to glioma cells was detected by fluorescent quantitative PCR and Western blotting, from m RNA and protein levels of p38-MAPK gene knockdown the expression and effect of COX-2 were detected. Further explore the effect of p38-MAPK on the expression of COX-2 downstream gene SP1, we transfected COX-2-luciferase gene into glioma cells, then using SP1 specific Si RNA and explore the effect of SP1 inhibitor mithramycin on the expression of COX-2, COX-2 expression level was determined by measuring the fluorescence intensity. Finally, we use the COX-2 specific Si and RNA Inhibitor Celecoxib can inhibit the function of COX-2, the effects of COX-2 on the expression of VEGF and autophagy. Effect of COX-2 on the expression of VEGF was determined by fluorescence quantitative PCR; expression by Western blot analysis to detect LC3-II, and evaluate the autophagy intensity. Experimental results show that the medium of Met/Cys the lack of culture medium and deficiency in Gln/Arg/Lys could induce the expression of COX-2 in U87 and SF767 cells, induced by 2H, the cells began to express COX-2,4-6h Stable. But, in contrast, more rapid loss of Met/Cys can induce the expression of COX-2. Therefore, in the following experiments, we chose to establish a cell model by Met/Cys. The lack of hunger by p38-MAPK, a specific inhibitor of SB202190 MEK and JNK (SB), U0126 (U), SP600125 (SP) and Si after RNA treatment, we found that the inhibitory effect of p38-MAPK inhibitor SB on the expression of amino acid starvation induced COX-2, JNK specific inhibitor SP can inhibit the expression of COX-2 in U87 cells, but the expression of COX-2 in SF767 cells does not influence the effect of U0126 is just the opposite of SP. Also, we found that the amino acid at the same time to starvation induced p38-MAPK in U87 cells and SF767 phosphorylation, JNK phosphorylation occurs only in U87 cells. Further experiments found that p38-MAPK regulates COX-2 expression through SP1, SP1 gene The application of knockout and inhibitors can significantly reduce the expression of amino acid starvation induced COX-2. The effect of COX-2 downstream of the detection analysis showed that the expression of COX-2 can promote and induce autophagy of VEGF expression. In summary, based on the amino acid starvation model to simulate glioma tissue nutrient supply environment. To explore the related mechanisms of COX-2 expression induced by this kind of conditions. The results illustrate the amino acid starvation induced COX-2 expression is regulated by upstream of the p38-MAPK/SP1 signaling pathway; COX-2 can enhance the expression of glioma cells VEGF, lay the foundation for tumor angiogenesis; COX-2 through autophagy stimulate glioma cells, provide as the energy required for cell survival.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41

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本文編號(hào)：1560804