miR-125b抑制剂增敏脱甲氧基姜黄素和替莫唑胺对胶质瘤干细胞作用的机制研究

发布时间：2018-03-25 12:11

本文选题：miRNA　切入点：胶质瘤干细胞　出处：《南京医科大学》2016年博士论文

【摘要】：目的：恶性胶质瘤是人类颅内恶性程度最高的肿瘤之一,易复发,难治愈,胶质瘤干细胞的存在被认为是其主要原因之一。近年来越来越多的研究表明miRNAs及其相关信号传导通路和人类肿瘤干细胞的发生发展关系密切。其中特别是miR-125b与胶质瘤干细胞关系密切,其表达水平可调节胶质瘤干细胞增殖和凋亡的进程,但其作用的具体机制仍然不是十分明确。替莫唑胺(temozolomide,TMZ)是临床一线抗胶质瘤药物,而研究发现胶质瘤干细胞普遍对TMZ耐药。但抑制miR-125b后可有效提高胶质瘤干细胞对TMZ的敏感性。新近有研究表明胶质瘤干细胞对TMZ的耐受可能与胶质瘤干细胞中PI3K/Akt,Bcl-2/caspase和高表达ABCG2蛋白等相关,而与胶质瘤细胞表达的06-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)无关。PI3K/Akt通路被抑制后发现对胶质瘤干细胞(GSCs)生物学特性的影响大于非胶质瘤干细胞(GSCs).而直接抑制Bcl-2表达或Bcl-2/caspase信号通路可诱导胶质瘤干细胞发生凋亡。ABCG2转运蛋白是一种与肿瘤MDR有关的新的药物泵,可把具有不同化学结构和作用于细胞内不同位置靶点的化疗药物泵出胞外,从而导致细胞抗药性产生。脱甲氧基姜黄素(demethoxycurcumin, DMC)被发现是ABCG2基因的一种有效的天然抑制剂,可有效抑制细胞内ABCG2表达。本课题旨在通过DMC抑制胶质瘤干细胞内ABCG2表达提高胶质瘤干细胞内TMZ浓度,进一步结合抑制胶质瘤干细胞miR-125b表达水平来提高胶质瘤干细胞对TMZ的敏感性,达到更好的抗胶质瘤干细胞效果,并探讨其联合作用的机制。通过此项研究为增强胶质瘤干细胞化疗疗效提供一定的理论与实践基础。方法：收集临床不同等级的胶质瘤组织标本和脑外伤脑膨出需切除的正常脑组织,利用实时荧光定量PCR检测miR-125b在不同等级胶质瘤组织与正常脑组织中的表达差异。从病理诊断WHO-IV级临床胶质瘤母细胞瘤组织中分离培养原代胶质瘤细胞,然后利用免疫磁珠法分离出CD133阳性的胶质瘤干细胞,并通过Nestin和CD133免疫荧光染色验证,建立原代胶质瘤干细胞体外培养及体内种植的模型。评价不同药物浓度DMC对胶质瘤干细胞生长的抑制作用,研究其抗胶质瘤干细胞机理。进一步探讨DMC联合TMZ抗胶质瘤干细胞的作用及其机制。然后,合成上调和下调miR-125b的表达载体转染胶质瘤干细胞,实时荧光定量PCR验证转染效果,进一步分析改变胶质瘤干细胞内miR-125b表达水平后,DMC或TMZ对胶质瘤干细胞增殖、凋亡作用的影响,分析相关信号通路的变化,找出调节的靶信号通路。最后,评价miR-125b抑制剂、TMZ和DMC三者联合抗胶质瘤干细胞效果。结果：(1)miR-125b在正常脑组织中呈现过低表达,在WHO-Ⅱ、Ⅲ级胶质瘤组织中表达较正常脑组织仅轻度增高,但WHO-IV级胶质瘤组织中呈现明显高表达,为正常脑组织数十倍以上,并在胶质瘤干细胞中也呈现过表达状态。(2)我们在体外成功分离培养出原代胶质瘤干细胞,经免疫组化鉴定为CD133和Nestin双阳性。(3)DMC可有效抑制抑制胶质瘤干细胞增殖,并诱导其凋亡,作用效果明显优于TMZ,体外实验表明两者联合使用有明显的加强效应,但体内实验结果显示两者联合使用无明显的叠加效应,体内DMC的抑瘤效果仍然明显优于TMZ。其作用与抑制JAK/STAT3通过活化有关。(4) miR-125b抑制剂和TMZ单独并不能诱导胶质瘤干细胞凋亡,联合miR-125b抑制剂和TMZ干预结果表明抑制miR-125b表达可促进TMZ诱导胶质瘤干细胞增殖抑制,并诱导其凋亡,其作用机制与miR-125b靶向BAK1基因,PI3K/AKT信号通路抑制相关等有关。(5)miR-125b可调节DMC对胶质瘤干细胞的敏感性,过表达的miR-125b可抵制DMC诱导胶质瘤干细胞凋亡的作用,其机制可能与miR-125b靶向调控BAK1表达有关。(6)miR-125b抑制剂联合DMC联合TMZ抗胶质瘤干细胞作用明显优于两两联合组。结论：本研究结果证实抑制胶质瘤干细胞中miR-125b过表达可有效增敏TMZ和DMC的抗胶质瘤干细胞效果,并发现DMC抗胶质瘤干细胞效果无论体内外均明显优于TMZ。由此,我们提出通过靶向miRNA调控可能是增敏胶质瘤干细胞对化疗药物敏感性的一种新的途径,为将来治疗胶质瘤干细胞的提供了一个新的方向。
[Abstract]:Objective: malignant glioma is one of the most malignant tumors in human intracranial recurrence, difficult to cure, the presence of glioma stem cells is considered to be one of the main reasons. In recent years, more and more studies show that miRNAs and its related signal transduction pathway and human tumor stem cells had a close relationship. Especially miR-125b with glioma stem cells is closely related to its expression level can regulate glioma stem cell proliferation and apoptosis process, but the specific mechanism of the effect is still not very clear. Temozolomide (temozolomide, TMZ) is a clinical first-line anti glioma drugs, the study found that glioma stem cells are generally resistant to TMZ. But after the inhibition of miR-125b can effectively improve the sensitivity of glioma stem cells to TMZ. Recent studies have shown that glioma stem cells to TMZ tolerance and glioma stem cells in PI3K/Akt, Bcl-2/ caspase The high expression of ABCG2 and other related 06- methylguanine -DNA- expression and glioma cells and methyl transferase (MGMT) on glioma stem cells found by inhibiting irrelevant.PI3K/Akt pathway (GSCs) effect is greater than the non biological characteristics of glioma stem cells (GSCs). While the direct inhibition of the expression of Bcl-2 or Bcl-2/caspase signaling the cell apoptosis induced by.ABCG2 protein in glioma stem is a kind of new MDR associated with tumor drug pump, can have different chemical structure and function in cells in different position of target drug efflux pump outside cells, leading to cell resistance. Demethoxycurcumin (Demethoxycurcumin, DMC) is that is a kind of effective natural inhibitors of ABCG2 gene can effectively inhibit the expression of ABCG2 in cells. The purpose of this project is to inhibit the expression of ABCG2 in stem cells improve glioma stem cells in TMZ glioma by DMC With the further concentration, inhibition of stem cells to improve the expression level of miR-125b glioma stem cell sensitivity to TMZ glioma, better anti glioma stem cells, and to explore the mechanism of the combined effect. Through this research provided to enhance the theory and practice basis of glioma stem cells. Methods: the curative effect of chemotherapy collect the clinical grades of gliomas and brain injury encephalocele to normal brain tissue resection, differential expression using real-time fluorescence quantitative PCR detection of miR-125b in different grades of glioma and normal brain tissues. The cultured glioma cells from the pathological diagnosis of WHO-IV clinical glioma glioblastoma tissues. Then using immunomagnetic beads CD133 positive glioma stem cell separation method, and verified by Nestin staining and CD133 immunofluorescence, establish primary glioma stem cells in vitro Keep and in vivo model. Evaluation of different concentration DMC to inhibit the growth of glioma stem cells, to study the anti glioma stem cells. To further explore the mechanism of DMC and TMZ on anti glioma stem cells. Then the effect and mechanism of synthesis, up-regulated and down regulated miR-125b expression vector was transfected into glioma stem cells, real-time quantitative verification of PCR transfection effect, further analysis of changes in the expression level of miR-125b cells of glioma stem, DMC or TMZ on the proliferation of glioma stem cells, apoptotic effect, analysis of the changes of related signaling pathways, identify the target signal pathway. Finally, the evaluation of miR-125b inhibitors, TMZ and DMC three combined anti glioma stem the effect of cell. Results: (1) miR-125b showed low expression in normal brain, WHO- II, III grade glioma than normal brain tissues was slightly increased, but WHO-IV grade of glioma Obviously showed high expression in normal brain tissue as dozens of times, and in glioma stem cells also showed overexpression of state. (2) we successfully isolated in vitro cultured glioma stem cells, identified by immunohistochemistry for CD133 and Nestin double positive. (3) DMC can effectively the inhibition of the proliferation of glioma stem cells, and induce apoptosis, the effect is better than that of TMZ, in vitro experiments show that the combination use of strengthening effect is obvious, but the in vivo experiment results show that the combination use of superposition effect is obvious, the anti-tumor effect of DMC in vivo is still significantly better than TMZ. and its role in the inhibition of JAK/STAT3 activation by. (4) miR-125b and TMZ inhibitors alone did not induce apoptosis of glioma stem cells, combined with miR-125b inhibitor TMZ intervention and the results show that inhibition of the expression of miR-125b may promote TMZ induced glioma stem cell proliferation inhibition and induction The cell apoptosis and its mechanism of miR-125b targeting BAK1 gene, PI3K/AKT signaling pathway inhibition and so on. (5) miR-125b can regulate DMC on glioma stem cells sensitivity, overexpression of miR-125b can resist DMC induced glioma stem cells apoptosis, and its mechanism may be related to the regulation of BAK1 expression of miR-125b target. (6) miR-125b DMC combined with TMZ inhibitor combined with anti glioma stem cells was superior to that of 22 combined group. Conclusion: the results of this study confirmed that inhibition of stem cells in the expression of miR-125b TMZ and DMC can effectively sensitized the anti glioma effect of glioma stem cells, and found that DMC anti glioma stem cells both in vitro and in vivo this is significantly better than that of TMZ., we may put forward by targeting miRNA regulation is sensitizing glioma stem a new pathway of cell sensitivity to chemotherapy, for future treatment of glioma stem cells provides a new The direction.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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