2,3-吲哚醌抑制人神经母瘤细胞侵袭和转移的分子机制研究

发布时间：2018-06-12 15:37

本文选题：2 + 3-吲哚醌　；参考：《青岛大学》2017年硕士论文

【摘要】：研究目的:神经母细胞瘤是小儿最常见的恶性肿瘤之一,恶性程度高,早期即发生转移,且转移是神经母细胞瘤患儿最主要的死亡原因。2,3-吲哚醌(2,3-dioxoindoline,ISA,Isatin)是存在于哺乳动物体液及组织中的一种天然物质,其化学结构明确,是我国I类抗癌新药靛玉红的单体结构,已发现Isatin可以抑制人神经母细胞瘤SH-SY5Y细胞的增殖,诱导其凋亡,并初步发现其有抑制侵袭转移的作用,因此本研究拟进一步明确Isatin对神经母细胞瘤侵袭转移的抑制作用,并对其机制进行探讨,为Isatin的临床应用提供理论依据,为最终将该药开发成一种新型的具有我国自主知识产权的天然抗癌药物奠定基础。研究方法:体外培养人神经母细胞瘤SH-SY5Y细胞,采用不同浓度(10、25、50、100、150、200、250、300、400、500、800μmol/L)的Isatin作用于体外培养的SH-SY5Y细胞,采用CCK-8检测细胞活力,并计算其IC50值;采用划痕实验检测不同浓度Isatin(0、50、100、200μmol/L)作用后SH-SY5Y细胞的迁移能力;采用transwell小室检测不同浓度Isatin(0、50、100、200μmol/L)作用后SHSY5Y细胞侵袭和运动能力;使用基因表达谱芯片技术检测Isatin对SH-SY5Y细胞基因表达的影响,并筛选出与侵袭转移相关的通路及基因,并采用RT-PCR验证其相关基因的相对表达;采用Western blotting检测与侵袭转移相关蛋白磷酸化水平或表达水平的变化。研究结果:Isatin对SH-SY5Y细胞作用的IC50值为279.7μmol/L,CCK-8结果显示Isatin能抑制SH-SY5Y细胞增殖且呈浓度依赖性,随着药物浓度的增大,细胞出现明显的皱缩、且细胞数目明显减少;与对照组相比,Isatin处理组细胞的迁移能力明显减弱(P0.05),并且具有浓度依赖性;与对照组相比,细胞侵袭运动能力显著下降(P0.01),且呈浓度依赖性;基因表达谱芯片结果发现,Isatin作用后,共有284个基因表达上调,145个基因表达下调,芯片提示mTOR信号通路可能是Isatin的效应靶点,其中DDIT4、RHEB、EIF4EBP1、RPS6KB1为mTOR信号通路的相关基因;RT-PCR结果与基因表达谱芯片预测的结果相一致,DDIT4和EIF4EBP1基因表达上调,RHEB和RPS6KB1基因表达下调;Western blotting检测与mTOR信号通路相关蛋白的表达,结果显示AMPK的磷酸化水平显著上调(P0.01),且呈浓度依赖性;mTOR的磷酸化水平下调(P0.01),呈浓度依赖性;HIF-1α和VEGF的蛋白相对表达量下调(P0.01),呈浓度依赖性;PI3K和Akt的磷酸化水平随药物浓度的增加而无明显变化。研究结论:Isatin对SH-SY5Y细胞的增殖和侵袭迁移具有抑制作用;基因表达谱芯片结果提示Isatin抑制SH-SY5Y细胞侵袭转移的作用可能是多靶点的,对mTOR通路相关蛋白的影响是Isatin抗SH-SY5Y细胞侵袭转移作用的可能机制之一,为该药用于临床提供了理论基础。
[Abstract]:Objective: neuroblastoma is one of the most common malignant tumors in children. Metastasis is the leading cause of death in children with neuroblastoma. Isatin 3-dioxoindoline isatin is a natural substance in mammalian body fluids and tissues. Its chemical structure is clear and it is the monomer structure of indirubin, a class I anticancer drug in China. It has been found that Isatin can inhibit the proliferation of human neuroblastoma SH-SY5Y cells, induce its apoptosis, and preliminarily find that Isatin can inhibit the invasion and metastasis of neuroblastoma. Therefore, this study intends to further clarify the inhibitory effect of Isatin on the invasion and metastasis of neuroblastoma. The mechanism of Isatin was discussed to provide a theoretical basis for the clinical application of Isatin, and to establish the foundation for the development of Isatin as a new type of natural anticancer drug with independent intellectual property rights in China. Methods: human neuroblastoma SH-SY5Y cells were cultured in vitro. Different concentrations of Isatin were used to treat SH-SY5Y cells in vitro. The activity of SH-SY5Y cells was measured by CCK-8 and its IC50 was calculated. The migration ability of SH-SY5Y cells exposed to different concentrations of Isatinine 050100,200 渭 mol / L, transwell chamber, invasion and motility of SH-SY5Y cells treated with different concentrations of Isatinine 050100,200 渭 mol / L, gene expression microarray technique, and the expression of SH-SY5Y cells were detected by scratch test. The pathway and gene associated with invasion and metastasis were screened, and the relative expression of the related genes was verified by RT-PCR, and the phosphorylation level or expression level of protein associated with invasion and metastasis was detected by Western blotting. Results the IC50 value of the effect of 1: Isatin on SH-SY5Y cells was 279.7 渭 mol / L CCK-8. The results showed that Isatin inhibited the proliferation of SH-SY5Y cells in a concentration-dependent manner. Compared with the control group, the migration ability of the cells treated with Isatin was significantly decreased in a concentration-dependent manner, and the cell invasion and motility decreased significantly in a concentration-dependent manner compared with the control group. A total of 284 genes were up-regulated and 145 genes were down-regulated, suggesting that the mTOR signaling pathway might be the target of Isatin. The results of RT-PCR showed that DDIT4 and EIF4EBP1RPS6KB1 were mTOR signaling pathway. The results of RT-PCR were consistent with the predicted results of gene expression microarray. The expression of DDIT4 and EIF4EBP1 up-regulated the expression of RHEB and RPS6KB1 genes. Western blotting detection and mTOR signaling pathway related protein expression were down-regulated. The results showed that the phosphorylation level of AMPK was significantly up-regulated, and the phosphorylation level of mTOR was down-regulated in a concentration-dependent manner. The relative expression of HIF-1 伪 and VEGF protein was down-regulated in a concentration-dependent manner. The phosphorylation levels of PI3K and Akt were in a concentration-dependent manner. The concentration of the substance increased without obvious change. Conclusion Isatin can inhibit the proliferation, invasion and migration of SH-SY5Y cells, and the results of gene expression microarray suggest that Isatin can inhibit the invasion and metastasis of SH-SY5Y cells. The effect of Isatin on mTOR transduction pathway is one of the possible mechanisms of Isatin in inhibiting the invasion and metastasis of SH-SY5Y cells, which provides a theoretical basis for its clinical application.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.4

【参考文献】