本文關(guān)鍵詞： 草氨酸鈉 乳酸脫氫酶 鼻咽癌 周期阻滯 凋亡 放射敏感性　出處：《蘇州大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：目的研究草氨酸鈉作為乳酸脫氫酶（LDH）抑制劑對(duì)人鼻咽癌生長(zhǎng)及其放射敏感性的影響，探討抑制糖代謝對(duì)鼻咽癌的治療作用，，發(fā)現(xiàn)新的治療靶點(diǎn)。 方法選用鼻咽癌CNE-1、CNE-2細(xì)胞系作為研究對(duì)象，同時(shí)在Balb/c裸鼠進(jìn)行CNE-1移植瘤實(shí)驗(yàn)。 (1)采用四甲基偶氮唑鹽比色法（MTT）檢測(cè)草氨酸鈉對(duì)鼻咽癌細(xì)胞CNE-1、CNE-2生長(zhǎng)的影響。 (2)采用傷口愈合試驗(yàn)觀察草氨酸鈉抑制LDH對(duì)鼻咽癌細(xì)胞遷移能力的影響，transwell小室侵襲試驗(yàn)觀察草氨酸鈉對(duì)鼻咽癌侵襲能力的影響。并采用Western blot檢測(cè)遷移侵襲相關(guān)蛋白的表達(dá)變化。 (3)采用流式細(xì)胞術(shù)觀察草氨酸鈉對(duì)鼻咽癌細(xì)胞周期的影響，磷脂酰絲氨酸外翻法（AnnexinⅤ和PI雙染法）檢測(cè)草氨酸鈉對(duì)鼻咽癌細(xì)胞凋亡的影響。 (4)采用乳酸脫氫酶（LDH）試劑盒、活性氧（ROS）檢測(cè)試劑盒，輔酶ⅡNADP(H)含量測(cè)定試劑盒，ATP含量檢測(cè)試劑盒等，檢測(cè)草氨酸鈉對(duì)鼻咽癌細(xì)胞中LDH、ROS、NADP(H)及ATP等能量代謝指標(biāo)變化的影響。 (5)采用Western blot法檢測(cè)草氨酸鈉對(duì)鼻咽癌細(xì)胞中周期凋亡相關(guān)蛋白及其它蛋白表達(dá)的變化的影響。 (6)細(xì)胞克隆形成法檢測(cè)草氨酸鈉對(duì)鼻咽癌細(xì)胞放射敏感性的影響，并采用細(xì)胞流式術(shù)檢測(cè)草氨酸鈉聯(lián)合X射線對(duì)鼻咽癌細(xì)胞周期及細(xì)胞凋亡的影響。 (7)采用H2AX免疫熒光檢測(cè)照射后DNA損傷情況，Western blot用于進(jìn)一步檢測(cè)草氨酸鈉對(duì)照射后細(xì)胞內(nèi)DNA損傷修復(fù)相關(guān)蛋白的影響。 (8)在Balb/c裸鼠上進(jìn)行CNE-1移植成瘤試驗(yàn)，并觀察草氨酸鈉聯(lián)及放療單獨(dú)及聯(lián)合的作用，在體內(nèi)評(píng)價(jià)抑制LDH在鼻咽癌治療中的作用。 結(jié)果 (1)草氨酸鈉對(duì)鼻咽癌（NPC）具有明顯的增殖抑制作用，在CNE-1細(xì)胞中，在24h、48h、72h時(shí)，草氨酸鈉的50%抑制生長(zhǎng)濃度（IC50）分別是74.6mmol/L,32.4mmol/L和17.8mmol/L，而在CNE-2中，不同時(shí)間的IC50分別是62.3mmol/L,44.5mmol/L,31.6mmol/L。 (2)在鼻咽癌細(xì)胞中，當(dāng)LDH酶受到抑制后，糖酵解通路受到干擾,隨著草氨酸鈉濃度的增加，兩種細(xì)胞內(nèi)的LDH酶的活性均明顯下降，并呈現(xiàn)一定的劑量依賴性。 (3)抑制LDH酶后，鼻咽癌的侵襲能力同樣受到了明顯的抑制，抑制程度與劑量呈現(xiàn)良好的正相關(guān)。MMP-2、MMP9、VEGF下調(diào)，而E-cadherin、-cadherin增加。 (4)草氨酸鈉可引起NPC細(xì)胞G2/M期阻滯。誘導(dǎo)的G2/M期阻滯可能是通過(guò)調(diào)節(jié)cyclinB1和CDK1的表達(dá)實(shí)現(xiàn)的。 (5)草氨酸鈉抑制LDH酶后，鼻咽癌細(xì)胞確實(shí)發(fā)生了不同程度的凋亡，且與處理濃度及時(shí)間具有良好的相關(guān)性。草氨酸鈉處理后線粒體膜電位下降，線粒體凋亡途徑在這種凋亡過(guò)程中扮演著重要的角色。 (6)草氨酸鈉抑制LDH后可誘導(dǎo)鼻咽癌細(xì)胞中ROS的爆發(fā)。ROS是其抑制鼻咽癌細(xì)胞生長(zhǎng)的作用中扮演著重要的角色，且當(dāng)這種抑制作用可被ROS清除劑所緩解。 (7)草氨酸鈉對(duì)鼻咽癌CNE-1和CNE-2均具有明顯的放射增敏作用，增敏比分別為1.26和1.37，并且草氨酸鈉可增加照射后誘導(dǎo)兩種細(xì)胞凋亡的比例。(8)照射后，鼻咽癌細(xì)胞發(fā)生了顯著的G2/M期阻滯，也表明當(dāng)加入草氨酸鈉后，細(xì)胞的G2/M期阻滯更加嚴(yán)重，說(shuō)明草氨酸鈉與電離輻射對(duì)細(xì)胞周期阻滯具有一定的協(xié)同作用。 (9)加入草氨酸鈉后，照射后細(xì)胞中的H2AX位點(diǎn)明顯增多，意味著當(dāng)加入草氨酸鈉后，CNE-1細(xì)胞的DNA受到的損傷更為嚴(yán)重。 (10)當(dāng)加入LDH酶抑制劑草氨酸鈉后，電離輻射誘導(dǎo)的DNA修復(fù)蛋白DNA-PKcs、Ku70、Ku80和XRCC4等的表達(dá)受到了明顯的抑制。 (11)在動(dòng)物水平上進(jìn)一步證實(shí)了草氨酸鈉可抑制鼻咽癌生長(zhǎng)及提高其放射敏感性，更重要的是，抑制LDH-A后，在小鼠體內(nèi)可觀察到明顯的毒性。 結(jié)論 (1)草氨酸鈉可有效地抑制鼻咽癌細(xì)胞的生長(zhǎng)及能量代謝； (2)草氨酸鈉可明顯抑制鼻咽癌細(xì)胞的轉(zhuǎn)移及侵襲； (3)草氨酸鈉可通過(guò)降低cyclin B1/CDK1的表達(dá)顯著誘導(dǎo)鼻咽癌細(xì)胞G2/M期阻滯； (4)草氨酸鈉可誘導(dǎo)鼻咽癌細(xì)胞內(nèi)ROS爆發(fā)，并通過(guò)OXPHOS引起線粒體途徑凋亡； (5)草氨酸鈉可增加鼻咽癌的放射敏感性，其機(jī)制與ROS增加細(xì)胞凋亡并且抑制DNA修復(fù)有關(guān)； (6)草氨酸鈉在體內(nèi)動(dòng)物水平上，依然具有良好的抑制鼻咽癌生長(zhǎng)及放射增敏作用，且無(wú)明顯的毒性。
[Abstract]:Objective to study the effect of sodium alginate as lactate dehydrogenase (LDH) inhibitor on the growth and radiosensitivity of human nasopharyngeal carcinoma, and to explore the therapeutic effect of inhibiting glycometabolism on nasopharyngeal carcinoma and find a new therapeutic target.
Methods the CNE-1 and CNE-2 cell lines of nasopharyngeal carcinoma were selected as the research object, and the CNE-1 xenografts were carried out in Balb/c nude mice.
(1) the effect of sodium azotriate on the growth of CNE-1 and CNE-2 in nasopharyngeal carcinoma cells was detected with four methyl azazolus colorimetric assay (MTT).
(2) using wound healing test to observe the effect of sodium alginate inhibiting LDH on the migration of nasopharyngeal carcinoma cells. Transwell cell invasion test was used to observe the effect of sodium alginate on invasion of nasopharyngeal carcinoma. Western blot was used to detect the expression changes of migration and invasion related proteins.
(3) flow cytometry was used to observe the effect of sodium alginate on the cell cycle of nasopharyngeal carcinoma. The effect of sodium alginate on the apoptosis of nasopharyngeal carcinoma cells was detected by phosphatidylserine eversion (Annexin V and PI double staining).
(4) lactate dehydrogenase (LDH) kit, reactive oxygen species (ROS) kit, coenzyme II NADP (H) assay kit and ATP content detection kit were used to detect the effects of sodium oxalate on the changes of LDH, ROS, NADP (H) and ATP in nasopharyngeal carcinoma cells.
(5) the effect of sodium alanine on the changes of apoptosis related protein and other protein expression in nasopharyngeal carcinoma cells was detected by Western blot method.
(6) cell clone formation assay was used to detect the effect of sodium alginate on radiosensitivity of nasopharyngeal carcinoma cells. Cell flow cytometry was used to detect the effect of sodium alginate combined with X ray on cell cycle and apoptosis of nasopharyngeal carcinoma.
(7) H2AX immunofluorescence was used to detect DNA damage after irradiation. Western blot was used to further detect the effect of sodium alginate on the DNA damage and repair related proteins after irradiation.
(8) CNE-1 transplantation and tumorigenicity test on Balb/c nude mice. We observed the effect of sodium alginate combined with radiotherapy alone and combined, and evaluated the inhibitory effect of LDH on nasopharyngeal carcinoma in vivo.
Result
(1) oxamate on nasopharyngeal carcinoma (NPC) significantly inhibited the proliferation in CNE-1 cells, in 24h, 48h, 72h, oxamate, 50% growth inhibition concentration (IC50) were 74.6mmol/L, 32.4mmol/L and 17.8mmol/L, and in CNE- 2, different time respectively is IC50 62.3mmol/L, 44.5mmol/L, 31.6mmol/L.
(2) in nasopharyngeal carcinoma cells, when the LDH enzyme was inhibited, the glycolysis pathway was disturbed. With the increase of sodium alginate concentration, the activity of LDH enzyme in two kinds of cells decreased significantly, and showed a dose-dependent manner.
(3) after inhibiting LDH enzyme, the invasive ability of nasopharyngeal carcinoma was also significantly inhibited. The degree of inhibition was positively correlated with the dose..MMP-2, MMP9 and VEGF were down regulated, while E-cadherin and -cadherin increased.
(4) sodium alanine can induce G2/M phase block in NPC cells. The induced G2/M phase block may be achieved by regulating the expression of cyclinB1 and CDK1.
(5) inhibition of oxamate LDH enzyme after nasopharyngeal carcinoma cells did occur apoptosis in different degree, and has a good correlation with the concentration and time. A decrease in mitochondrial membrane potential after treatment with oxamate, mitochondrial apoptosis pathway plays an important role in the apoptosis process.
(6) sodium alginate inhibits the ROS burst in nasopharyngeal carcinoma cells after inhibiting LDH..ROS plays an important role in inhibiting the growth of nasopharyngeal carcinoma cells, and this inhibition can be alleviated by ROS scavenger.
(7) oxaminic acid sodium has obvious radiosensitizing effect on CNE-1 and CNE-2 in nasopharyngeal carcinoma, the sensitization enhancement ratio were 1.26 and 1.37, and oxamate two induced apoptosis increased after irradiation. The proportion (8) after irradiation, there is a significant G2/M phase arrest of nasopharyngeal carcinoma cells, but also when adding oxamate, cell G2/M arrest is more serious, and that the ionization radiation oxaminic acid sodium has a synergistic effect on cell cycle arrest.
(9) after the addition of sodium alanine, the H2AX site in the cells increased significantly after irradiation, which means that when sodium alanine was added, the damage to the DNA of CNE-1 cells was more serious.
(10) when sodium alanine was added to the LDH enzyme inhibitor, the expression of DNA repair protein DNA-PKcs, Ku70, Ku80 and XRCC4 induced by ionizing radiation was significantly inhibited.
(11) at the animal level, it is further confirmed that sodium alginate inhibits the growth and radiosensitivity of nasopharyngeal carcinoma, and more importantly, it can observe obvious toxicity in mice after inhibiting LDH-A.
conclusion
(1) sodium alanine can effectively inhibit the growth and energy metabolism of nasopharyngeal carcinoma cells.
(2) sodium alanine can obviously inhibit the metastasis and invasion of nasopharyngeal carcinoma cells.
(3) sodium alanine can significantly induce G2/M phase block in nasopharyngeal carcinoma cells by reducing the expression of cyclin B1/CDK1.
(4) sodium alanine can induce ROS outbreak in nasopharyngeal carcinoma cells and induce mitochondrial pathway apoptosis through OXPHOS.
(5) sodium alanine can increase the radiosensitivity of nasopharyngeal carcinoma, and its mechanism is related to the increase of ROS apoptosis and the inhibition of DNA repair.
(6) sodium alanine still has a good inhibitory effect on the growth and radiosensitization of nasopharyngeal carcinoma and has no obvious toxicity in the animal level in vivo.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.63

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 馮青青;劉紅梅;彭秋平;柯傳慶;;HK-Ⅱ、TS和Ki-67在青年人結(jié)腸癌組織中的表達(dá)及其意義[J];腫瘤防治研究;2011年06期

本文編號(hào)：1550168