草氨酸钠抑制乳酸脱氢酶对鼻咽癌生长及放射敏感性的影响

发布时间：2018-03-01 03:17

本文关键词： 草氨酸钠乳酸脱氢酶鼻咽癌周期阻滞凋亡放射敏感性　出处：《苏州大学》2014年博士论文　论文类型：学位论文

【摘要】：目的研究草氨酸钠作为乳酸脱氢酶（LDH）抑制剂对人鼻咽癌生长及其放射敏感性的影响，探讨抑制糖代谢对鼻咽癌的治疗作用，，发现新的治疗靶点。方法选用鼻咽癌CNE-1、CNE-2细胞系作为研究对象，同时在Balb/c裸鼠进行CNE-1移植瘤实验。 (1)采用四甲基偶氮唑盐比色法（MTT）检测草氨酸钠对鼻咽癌细胞CNE-1、CNE-2生长的影响。 (2)采用伤口愈合试验观察草氨酸钠抑制LDH对鼻咽癌细胞迁移能力的影响，transwell小室侵袭试验观察草氨酸钠对鼻咽癌侵袭能力的影响。并采用Western blot检测迁移侵袭相关蛋白的表达变化。 (3)采用流式细胞术观察草氨酸钠对鼻咽癌细胞周期的影响，磷脂酰丝氨酸外翻法（AnnexinⅤ和PI双染法）检测草氨酸钠对鼻咽癌细胞凋亡的影响。 (4)采用乳酸脱氢酶（LDH）试剂盒、活性氧（ROS）检测试剂盒，辅酶ⅡNADP(H)含量测定试剂盒，ATP含量检测试剂盒等，检测草氨酸钠对鼻咽癌细胞中LDH、ROS、NADP(H)及ATP等能量代谢指标变化的影响。 (5)采用Western blot法检测草氨酸钠对鼻咽癌细胞中周期凋亡相关蛋白及其它蛋白表达的变化的影响。 (6)细胞克隆形成法检测草氨酸钠对鼻咽癌细胞放射敏感性的影响，并采用细胞流式术检测草氨酸钠联合X射线对鼻咽癌细胞周期及细胞凋亡的影响。 (7)采用H2AX免疫荧光检测照射后DNA损伤情况，Western blot用于进一步检测草氨酸钠对照射后细胞内DNA损伤修复相关蛋白的影响。 (8)在Balb/c裸鼠上进行CNE-1移植成瘤试验，并观察草氨酸钠联及放疗单独及联合的作用，在体内评价抑制LDH在鼻咽癌治疗中的作用。结果 (1)草氨酸钠对鼻咽癌（NPC）具有明显的增殖抑制作用，在CNE-1细胞中，在24h、48h、72h时，草氨酸钠的50%抑制生长浓度（IC50）分别是74.6mmol/L,32.4mmol/L和17.8mmol/L，而在CNE-2中，不同时间的IC50分别是62.3mmol/L,44.5mmol/L,31.6mmol/L。 (2)在鼻咽癌细胞中，当LDH酶受到抑制后，糖酵解通路受到干扰,随着草氨酸钠浓度的增加，两种细胞内的LDH酶的活性均明显下降，并呈现一定的剂量依赖性。 (3)抑制LDH酶后，鼻咽癌的侵袭能力同样受到了明显的抑制，抑制程度与剂量呈现良好的正相关。MMP-2、MMP9、VEGF下调，而E-cadherin、-cadherin增加。 (4)草氨酸钠可引起NPC细胞G2/M期阻滞。诱导的G2/M期阻滞可能是通过调节cyclinB1和CDK1的表达实现的。 (5)草氨酸钠抑制LDH酶后，鼻咽癌细胞确实发生了不同程度的凋亡，且与处理浓度及时间具有良好的相关性。草氨酸钠处理后线粒体膜电位下降，线粒体凋亡途径在这种凋亡过程中扮演着重要的角色。 (6)草氨酸钠抑制LDH后可诱导鼻咽癌细胞中ROS的爆发。ROS是其抑制鼻咽癌细胞生长的作用中扮演着重要的角色，且当这种抑制作用可被ROS清除剂所缓解。 (7)草氨酸钠对鼻咽癌CNE-1和CNE-2均具有明显的放射增敏作用，增敏比分别为1.26和1.37，并且草氨酸钠可增加照射后诱导两种细胞凋亡的比例。(8)照射后，鼻咽癌细胞发生了显著的G2/M期阻滞，也表明当加入草氨酸钠后，细胞的G2/M期阻滞更加严重，说明草氨酸钠与电离辐射对细胞周期阻滞具有一定的协同作用。 (9)加入草氨酸钠后，照射后细胞中的H2AX位点明显增多，意味着当加入草氨酸钠后，CNE-1细胞的DNA受到的损伤更为严重。 (10)当加入LDH酶抑制剂草氨酸钠后，电离辐射诱导的DNA修复蛋白DNA-PKcs、Ku70、Ku80和XRCC4等的表达受到了明显的抑制。 (11)在动物水平上进一步证实了草氨酸钠可抑制鼻咽癌生长及提高其放射敏感性，更重要的是，抑制LDH-A后，在小鼠体内可观察到明显的毒性。结论 (1)草氨酸钠可有效地抑制鼻咽癌细胞的生长及能量代谢； (2)草氨酸钠可明显抑制鼻咽癌细胞的转移及侵袭； (3)草氨酸钠可通过降低cyclin B1/CDK1的表达显著诱导鼻咽癌细胞G2/M期阻滞； (4)草氨酸钠可诱导鼻咽癌细胞内ROS爆发，并通过OXPHOS引起线粒体途径凋亡； (5)草氨酸钠可增加鼻咽癌的放射敏感性，其机制与ROS增加细胞凋亡并且抑制DNA修复有关； (6)草氨酸钠在体内动物水平上，依然具有良好的抑制鼻咽癌生长及放射增敏作用，且无明显的毒性。
[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.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.63

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