【作者】 朱廷准；

【导师】 徐英辉；

【作者基本信息】 大连医科大学 ， 神经外科学， 2011， 博士

【摘要】 榄香烯(Elemene)是我国自主开发的、从中药莪术(温郁金)中提取的国家二类非细胞毒性抗肿瘤药,以β-、γ-和δ-榄香烯的油状混合物形式存在。作为其主要的活性成分,β-榄香烯在体内和体外可以明显抑制胶质瘤细胞的增殖。但由于抗胶质瘤作用的分子机制尚不明确,限制了其在临床上的应用。丝裂原活化蛋白激酶激酶-3(Mitogen-activated protein kinase kinase 3,MKK3)和-6(Mitogen-activated protein kinase kinase 6,MKK6)是丝裂原活化蛋白激酶p38(Mitogen-activated protein kinase p38,p38 MAPK)信号通路的两个上游激活因子。MKK3/6在多种肿瘤组织中被发现表达异常,同时MKK3/6也介导了许多药物的抗肿瘤作用。神经胶质成熟因子β(Glia Maturation Factor Beta,GMFβ)是一种17 kDa的小分子脑蛋白,在神经胶质细胞和神经元的生长发育、增殖、分化和凋亡过程中起着关键性的调节作用,这种作用常常通过调节MAPK信号通路的活性得以实现。我们前期的研究结果表明,p38 MAPK的激活以及细胞外信号调节蛋白激酶1/2(Extracellular Signal-regulated Protein Kinase1/2,ERK1/2)的失活在β-榄香烯抗胶质瘤细胞增殖中是不可或缺的。然而,β-榄香烯通过什么途径引起了胶质瘤细胞中上述信号通路的活性改变仍不得而知。目的:1、评价β-榄香烯对人U87及大鼠C6胶质瘤细胞的抗增殖及细胞周期阻滞作用。2、探讨MKK3和MKK6在β-榄香烯抗胶质瘤细胞增殖中的作用。3、明确GMFβ在β-榄香烯抗胶质瘤细胞增殖中的作用,及其与MKK3/6的关系。4、检测β-榄香烯对顺铂抗胶质瘤是否具有化疗增敏作用。方法:1、第一部分实验:分别以不同浓度(0、20、40、60和80μg/ml)的β-榄香烯作用于U87及C6细胞不同的时间(0、12、24、36和48小时),噻唑蓝(Methyl Thiazolyl Tetrazolium,MTT)法检测细胞增殖活性,流式细胞术检测细胞周期,并计算各组细胞周期中G0/G1期的百分比。2、第二部分实验:用β-榄香烯处理胶质瘤细胞后,提取各组细胞的总蛋白,用Western blot检测MKK3和MKK6的总蛋白及磷酸化蛋白含量,RT-PCR检测MKK3和MKK6的mRNA表达水平,应用Gel-Pro Analyzer 4.0软件半定量分析条带灰度,对结果进行统计学分析。用脂质体法将MKK3和MKK6的显性负突变(Dominant Negative,DN)质粒DN-MKK3和DN-MKK6转染进入胶质瘤细胞中,以抑制MKK3和MKK6的活性,免疫荧光法检测转染效率,同时行MTT法检测β-榄香烯对胶质瘤细胞活性的抑制能力。免疫组织化学方法检测人正常脑组织及胶质瘤组织中总的及磷酸化的MKK3/6的表达水平,探索MKK3/6与胶质瘤发病的关系。3、第三部分实验:免疫沉淀联合Western blot法检测经β-榄香烯处理的胶质瘤细胞中总的及磷酸化的GMFβ蛋白的表达水平。采用RNA干扰技术,使胶质瘤细胞中GMFβ的表达沉默,然后以MTT法检测β-榄香烯的抗胶质瘤增殖的能力。同时,用Western blot法检测β-榄香烯对MKK3和MKK6的激活作用是否因GMFβ沉默而受到影响。4、第四部分实验:用化疗药顺铂和β-榄香烯单独或联合作用于胶质瘤细胞,细胞计数法检测细胞数量,计算细胞生长抑制率,进而确定β-榄香烯对胶质瘤细胞的化疗增敏作用。结果:1、第一部分实验:β-榄香烯显著抑制了人U87及大鼠C6胶质瘤细胞系的增殖活性,并增加了细胞周期中G0/G1期细胞的比例。β-榄香烯对胶质瘤细胞的增殖抑制作用具有时间依赖性和浓度依赖性。2、第二部分实验:β-榄香烯使胶质瘤细胞中MKK3和MKK6的磷酸化水平上调。相反,通过转染显性负突变质粒抑制MKK3和MKK6的活性则可使β-榄香烯的抗胶质瘤增殖作用明显减弱。同时,在β-榄香烯的作用下,MKK3和MKK6其中一种的活性被抑制时,另一种的活性会代偿性地增强,MKK3和MKK6呈现出相互代偿性的激活。人胶质瘤组织中MKK3和MKK6的活性较正常脑组织显著增强。3、第三部分实验:β-榄香烯可使GMFβ的磷酸化水平增加。相反,通过RNA干扰技术抑制GMFβ的表达,可以使MKK3和MKK6的磷酸化水平下调,并降低β-榄香烯的抗胶质瘤细胞增殖的效果。4、第四部分实验:β-榄香烯与顺铂联合应用时,胶质瘤细胞的生长抑制率显著高于单独用药组,β-榄香烯对胶质瘤具有化疗增敏作用。结论:1、β-榄香烯可以通过将U87和C6细胞的细胞周期阻滞于G0/G1期,进而有效地抑制胶质瘤细胞的增殖。2、依赖于GMFβ磷酸化的MKK3和MKK6的相互代偿性的激活介导了β-榄香烯的抗胶质瘤增殖作用。同时,MKK3/6在人胶质瘤的发病中扮演了重要角色。3、在顺铂的抗胶质瘤增殖过程中,β-榄香烯表现出较强的化疗增敏作用。更多还原

【Abstract】 Elemene, a novel self-developed anticancer medicine in China with low cytotoxic-ity, is extracted from Curcuma wenyujin and exists as an essential oil mixture ofβ-,γ- andδ-elemenes. As the major active component,β-elemene has strong anti-proliferative effects on glioblastoma cells in vitro and in vivo. However, lack of a defined molecular mechanism for the antitumor action ofβ-elemene hinders its application in clinical treatment of glioblastoma. Mitogen-activated protein kinase kinase-3 (MKK3) and -6 (MKK6) are two upstream kinases of Mitogen-activated protein kinase p38 (p38 MAPK). Altered activity of MKK3/6 has also been seen in various malignant tumors. Glia maturation factorβ(GMFβ) is a 17 kDa intracellular protein and is necessary for the growth, development, proliferation, differentiation and apoptosis of glial cells and neurons. GMFβusually acts through changing activity of MAPK signaling pathway. Our previous research found thatβ-elemene inhibited glioblastoma cell proliferation by activating p38 MAPK and decreasing the expression of p-ERK1/2, BCL-2 and BCL-X/L. However, the mechanism underlying the change of aforementioned mole-cules activity remains unclear.Objective:1. To evaluate the anti-glioblastoma and cell-cycle arrest effect of ?-elemene on human U87 and rat C6 cells.2. To investigate the role of MKK3 and MKK6 in the anti-glioblastoma prolifera-tion effect of ?-elemene.3. To confirm the role of GMFβin the antitumor effect ofβ-elemene on glioblas-toma and the relationship between GMFβand MKK3/6. 4. To confirm whether treatment withβ-elemene sensitizes glioblastoma cells to cisplatin.Methods:1. The first section research: human U87 and rat C6 glioblastoma cells were treated withβ-elemene at different drug doses (0, 20, 40, 60 and 80μg/ml) or for dif-ferent time amounts (0, 12, 24, 36 and 48 h). Cell viability was determined using a methyl thiazolyl tetrazolium (MTT) assay. Cell cycle progression was evaluated by flow cytometry, and the percentages of cells in G0/G1 phase were calculated.2. The second section research: glioblastoma cells were treated with different concentrations of ?-elemene and extracted with protein lysis buffer. p-MKK3, MKK3, p-MKK6 and MKK6 were measured by Western blot analysis. Total RNA was ex-tracted and RT-PCR was performed to examine the mRNA expression of MKK3 and MKK6. Band results were further semi-quantitatively estimated using Gel-Pro Analyzer 4.0 software. Data were analyzed by statistics. We transfected U87 glioblastoma cells with Dominant Negative-MKK3 (DN-MKK3), Dominant Negative-MKK6 (DN-MKK6) to inhibit the activity of MKK3 and MKK6, and then examined transfection efficiency by fluorescence microscopy and cell growth by MTT assay. To confirm the relationship between glioblastoma and MKK3/6, immunohistochemistry on human glioblastoma and normal brain tissues was performed using antibodies against MKK3, p-MKK3, MKK6 and p-MKK6 to evaluate the expression levels of MKK3/6 and p-MKK3/6.3. The third section research: to investigate the role of GMFβin the anti-glioblastoma effect ofβ-elemene, we examined the levels of total GMFβand p-GMFβinβ-elemene-treated U87 cells by immunoprecipitation and Western blot analysis. Upon GMFβsilencing using RNA interference, the antitumor action ofβ-elemene was evaluated in a MTT assay, and MKK3/6 and p-MKK3/6 expressions were examined by semiquantitative Western blot analysis.4. The fourth section research: after single or combined treatment with cisplatin andβ-elemene, glioblastoma cell numbers were measured by cell counting. The cell growth inhibitory rate (GIR) was calculated to examine the chemosensitization to cis-platin byβ-elemene.Results:1. The first section research:β-elemene inhibited the proliferation activity of U87 and C6 glioblastoma cells and arrested cell cycle in the G0/G1 phase. The anti-proliferation effect ofβ-elemene is dose- and time-dependent. 2. The second section research:β-elemene increased phosphorylation of both MKK3 and MKK6 in human glioblastoma cells. In contrast, inhibition of MKK3 and MKK6 with dominant-negative plasmids reversed the antitumor effect of ?-elemene. Furthermore, when either MKK3 or MKK6 was inhibited, the other was compensatorily activated in the presence ofβ-elemene. The expression of both total and phosphorylated forms of MKK3/6 were up-regulated in human glioblastoma.3. The third section research:β-elemene increased phosphorylation of GMFβin human glioblastoma cells. Conversely, silencing the expression of GMFβreversed the anti-glioblastoma effect ofβ-elemene and impaired the phosphorylation of MKK3/6.4. The fourth section research: the GIR in the combination (β-elemene + cisplatin) group was higher than that in both the single cisplatin andβ-elemene groups.β-elemene increased the sensitivity of U87 glioblastoma cells to cisplatin-induced cytotoxicity.Conclusion:1.β-elemene inhibited the proliferation of U87 and C6 glioblastoma cells through arresting them in G0/G1 phase.2. The activation of the GMFβ-MKK3/6-p38 signaling pathway underlay the anti–proliferative effect ofβ-elemene in glioblastoma. MKK3 and MKK6 were in-volved in the development of human glioblastoma.3.β-elemene and cisplatin had synergistic inhibitory effects on glioblastoma cell proliferation.更多还原