本文選題：2 + 3-吲哚醌��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：研究目的:神經(jīng)母細(xì)胞瘤是小兒最常見(jiàn)的惡性腫瘤之一,惡性程度高,早期即發(fā)生轉(zhuǎn)移,且轉(zhuǎn)移是神經(jīng)母細(xì)胞瘤患兒最主要的死亡原因。2,3-吲哚醌(2,3-dioxoindoline,ISA,Isatin)是存在于哺乳動(dòng)物體液及組織中的一種天然物質(zhì),其化學(xué)結(jié)構(gòu)明確,是我國(guó)I類(lèi)抗癌新藥靛玉紅的單體結(jié)構(gòu),已發(fā)現(xiàn)Isatin可以抑制人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞的增殖,誘導(dǎo)其凋亡,并初步發(fā)現(xiàn)其有抑制侵襲轉(zhuǎn)移的作用,因此本研究擬進(jìn)一步明確Isatin對(duì)神經(jīng)母細(xì)胞瘤侵襲轉(zhuǎn)移的抑制作用,并對(duì)其機(jī)制進(jìn)行探討,為Isatin的臨床應(yīng)用提供理論依據(jù),為最終將該藥開(kāi)發(fā)成一種新型的具有我國(guó)自主知識(shí)產(chǎn)權(quán)的天然抗癌藥物奠定基礎(chǔ)。研究方法:體外培養(yǎng)人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞,采用不同濃度(10、25、50、100、150、200、250、300、400、500、800μmol/L)的Isatin作用于體外培養(yǎng)的SH-SY5Y細(xì)胞,采用CCK-8檢測(cè)細(xì)胞活力,并計(jì)算其IC50值;采用劃痕實(shí)驗(yàn)檢測(cè)不同濃度Isatin(0、50、100、200μmol/L)作用后SH-SY5Y細(xì)胞的遷移能力;采用transwell小室檢測(cè)不同濃度Isatin(0、50、100、200μmol/L)作用后SHSY5Y細(xì)胞侵襲和運(yùn)動(dòng)能力;使用基因表達(dá)譜芯片技術(shù)檢測(cè)Isatin對(duì)SH-SY5Y細(xì)胞基因表達(dá)的影響,并篩選出與侵襲轉(zhuǎn)移相關(guān)的通路及基因,并采用RT-PCR驗(yàn)證其相關(guān)基因的相對(duì)表達(dá);采用Western blotting檢測(cè)與侵襲轉(zhuǎn)移相關(guān)蛋白磷酸化水平或表達(dá)水平的變化。研究結(jié)果:Isatin對(duì)SH-SY5Y細(xì)胞作用的IC50值為279.7μmol/L,CCK-8結(jié)果顯示Isatin能抑制SH-SY5Y細(xì)胞增殖且呈濃度依賴(lài)性,隨著藥物濃度的增大,細(xì)胞出現(xiàn)明顯的皺縮、且細(xì)胞數(shù)目明顯減少;與對(duì)照組相比,Isatin處理組細(xì)胞的遷移能力明顯減弱(P0.05),并且具有濃度依賴(lài)性;與對(duì)照組相比,細(xì)胞侵襲運(yùn)動(dòng)能力顯著下降(P0.01),且呈濃度依賴(lài)性;基因表達(dá)譜芯片結(jié)果發(fā)現(xiàn),Isatin作用后,共有284個(gè)基因表達(dá)上調(diào),145個(gè)基因表達(dá)下調(diào),芯片提示mTOR信號(hào)通路可能是Isatin的效應(yīng)靶點(diǎn),其中DDIT4、RHEB、EIF4EBP1、RPS6KB1為mTOR信號(hào)通路的相關(guān)基因;RT-PCR結(jié)果與基因表達(dá)譜芯片預(yù)測(cè)的結(jié)果相一致,DDIT4和EIF4EBP1基因表達(dá)上調(diào),RHEB和RPS6KB1基因表達(dá)下調(diào);Western blotting檢測(cè)與mTOR信號(hào)通路相關(guān)蛋白的表達(dá),結(jié)果顯示AMPK的磷酸化水平顯著上調(diào)(P0.01),且呈濃度依賴(lài)性;mTOR的磷酸化水平下調(diào)(P0.01),呈濃度依賴(lài)性;HIF-1α和VEGF的蛋白相對(duì)表達(dá)量下調(diào)(P0.01),呈濃度依賴(lài)性;PI3K和Akt的磷酸化水平隨藥物濃度的增加而無(wú)明顯變化。研究結(jié)論:Isatin對(duì)SH-SY5Y細(xì)胞的增殖和侵襲遷移具有抑制作用;基因表達(dá)譜芯片結(jié)果提示Isatin抑制SH-SY5Y細(xì)胞侵襲轉(zhuǎn)移的作用可能是多靶點(diǎn)的,對(duì)mTOR通路相關(guān)蛋白的影響是Isatin抗SH-SY5Y細(xì)胞侵襲轉(zhuǎn)移作用的可能機(jī)制之一,為該藥用于臨床提供了理論基礎(chǔ)。
[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 偽 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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R739.4

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