【作者】 王建民；

【导师】 刘佳；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 目的：在髓母细胞瘤细胞系UW228-3细胞和胶质母细胞瘤细胞系LN-18细胞中研究白藜芦醇(Resveratrol, Res)对STAT3及其相关因子LIF、c-Myc、Survivin、Cox-2、CyclinD1、Bcl-2表达情况的影响,以及STAT3和细胞的生长、分化、凋亡与耐药之间的关系,探讨白藜芦醇在抗髓母细胞瘤和胶质母细胞瘤作用中的分子机制,为改善髓母细胞瘤和胶质母细胞瘤的临床化学治疗现状提供新的实验室指标和理论根据。方法：人髓母细胞瘤细胞系UW228-3细胞和胶质母细胞瘤细胞系LN-18细胞分别培养于含10%胎牛血清的DMEM (Dulbecco’s Modified Eagle’s Medium)培养液中,以5×104/ml的初始细胞密度接种于Φ60mm和Φ100mm的培养皿中,24小时后用100μM白藜芦醇进行处理。将白藜芦醇溶解于二甲基亚砜(DMSO,demethylsulfoxide)溶液中,以5mM的浓度储存,使用前稀释到100μM。用100μM白藜芦醇(resveratrol, Res),处理细胞48小时,观察其对细胞的作用效果。为便于观察细胞形态学特征和进行免疫细胞化学(ICC)染色,将无菌盖玻片预先置于培养皿底部制备细胞爬片,待取出时用100%冷丙酮进行固定。同时收集细胞悬液并离心沉淀以用于RNA和蛋白质的提取。采用免疫细胞化学、RT-PCR和Western-blotting等方法系统检测髓母细胞瘤细胞系UW228-3细胞和胶质母细胞瘤细胞系LN-18细胞中STAT3及其相关因子LIF、c-Myc、Survivin、Cox-2、CyclinD1和Bcl-2在100μM白藜芦醇处理前后表达水平的改变、细胞内的分布状况及其与分化和凋亡的关系。结果：1)用100μM白藜芦醇处理细胞48h时后,对白藜芦醇敏感的髓母细胞瘤细胞系UW228-3细胞形态发生明显改变,而白藜芦醇并不能引起胶质母细胞瘤细胞系LN-18细胞形态的变化。2)在髓母细胞瘤细胞系UW228-3细胞中,白藜芦醇可以下调STAT3的表达水平并且使其核易位程度明显减少,而在胶质母细胞瘤细胞系LN-18细胞中,白藜芦醇可使STAT3发生核易位但对STAT3表达水平的影响很小。3)白藜芦醇可以明显上调髓母细胞瘤细胞系UW228-3细胞中LIF的表达水平,而对胶质母细胞瘤细胞系LN-18细胞中LIF表达水平的影响甚小。4)在髓母细胞瘤细胞系UW228-3细胞中,100μM白藜芦醇可不同程度地下调c-Myc、Survivin、Cox-2和CyclinD1的表达水平,上调Bcl-2的表达水平,而在胶质母细胞瘤细胞系LN-18细胞中100μM白藜芦醇对c-Myc、Survivin、Cox-2、CyclinD1和Bcl-2表达水平皆无明显影响。结论：1)本研究证实100μtM白藜芦醇在诱导髓母细胞瘤细胞系UW228-3细胞发生分化和调亡的同时,抑制了STAT3通路的活化,而在胶质母细胞瘤细胞系LN-18细胞中白藜芦醇未呈现其抗肿瘤作用但是STAT3发生核易位。2)髓母细胞瘤细胞系UW228-3细胞对白藜芦醇的作用较敏感,而胶质母细胞瘤细胞系LN-18细胞对白藜芦醇的作用则具有耐药性。3)本研究进一步证实了对STAT3信号转导通路的抑制是白藜芦醇抗原发性脑肿瘤作用的重要分子机制。更多还原

【Abstract】 Objective:To study the effect of STAT3 and its related factors LIF, c-Myc, Survivin, Cox-2, CyclinD1, Bcl-2 in resveratrol anti-medullo-blastoma cell line UW228-3 cells and anti-glioblastoma cell line LN-18 cells respectively, analyzing the relationship of STAT3 on cell growth, differentiation, apoptosis and drug resistance, investigating the molecular mechanism of resveratrol anti-medulloblastoma and anti-glioblastoma, so as to provide a new laboratory evidence and theoretic basis for improving the current clinical chemotherapy status of medulloblastoma and glioblastoma.Methods:Human medulloblastoma cell line UW228-3 cells and glioblastoma cell line LN-18 cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) culture medium containing 10% Fetal Bovine Serum (FBS). The cells (5×104/ml) were plated toΦ60mm andΦ100mm dishes and incubated for 24 hours before further experiments. Resveratrol was dissolved in dimethylsulfoxide to a stock concentration of 5mM and diluted with culture medium to an optimal working concentration of 100μM just before use. The cells were treated with resveratrol (100μM) for 48 hours, to observe the effect on the cells. Put sterilized cover slips in the dishes before cell seeding for cell morphology observation and immunocytochemistry (ICC) staining were performed. After above treatments, cover slips were collected and fixed with 100% cold acetone. Meanwhile, Cell suspension was collected and centrifuged to extract RNA and protein.Immunocyrochemistry (ICC) staining, RT-PCR Western blotting and other methods were used to analyze the expression levels and their intracellular distribution of STAT3 and its related factors LIF, c-Myc, Survivin, Cox-2, CyclinD1 and Bcl-2 in medulloblastoma cell line UW228-3 cells and glioblastoma cell line LN-18 cells with and without resveratrol treatment, in order to explore the expression levels, the distribution in intracellular and the correlation between STAT3 and its related factors LIF,c-Myc, Survivin, Cox-2, CyclinD1, Bcl-2 and differentiation, as well as apoptosis.Results:1) On resveratrol-sensitive medulloblastoma line UW228-3 cells, the cell morphology changes apparent with resveratrol(100μM) treatment for 48 hours, but no obvious morphological changes were observed on glioblastoma cell line LN-18 cells with resveratrol (100μM) treatment for 48 hours.2) In the medulloblastoma cell line UW228-3 cells, resveratrol reduced the expression of STAT3 and its nuclear translocation levels decreased, whereas in glioblastoma cell line LN-18 cells, resveratrol can induce STAT3 nuclear translocation, but not much change on STAT3 expression levels.3) The expression of LIF significantly up-regulated in medulloblastoma cell line UW228-3 cells with resveratrol(100μM) treatment, whereas in glioblastoma cell line LN-18 cells, the expression of LIF did not change much.4) The expression of c-Myc, Survivin, Cox-2, CyclinD1 was down-regulated, Bcl-2 expression levels were significantly up-regulated in medulloblastoma cell line UW228-3 cells with resveratrol (100μM) treatment; c-Myc, Survivin, Cox-2, CyclinD1, Bcl-2 were no significant changes in expression levels in glioblastoma cell line LN-18 cells with and without resveratrol(100μM) treatment.Conclusions:1) This study demonstrated that resveratrol(100μM) induced medulloblastoma cell line UW228-3 cells differentiation and apoptosis, while inhibited the STAT3 pathway activation; whereas in glioblastoma cell line LN-18 cells, resveratrol did not show its anti-tumor effect but the STAT3 nuclear translocation occured.2) The medulloblastoma cell line UW228-3 cells were sensitive to resveratrol, while the glioblastoma cell line LN-18 cells was resistant to resveratrol.3) The study further confirmed that the inhibition of STAT3 signaling pathway is an important anti-primary brain tumor molecular mechanism of resveratrol.更多还原