【作者】 王爱东；

【导师】 黄强；

【作者基本信息】 苏州大学 ， 神经外科学， 2008， 博士

【摘要】 第一部分逆分化细胞是胶质瘤发生、发展、治疗的靶细胞【目的】探讨BTSCs体外分化过程中的回逆现象,为研究其分化抑制机制奠定基础。【方法】利用CD133免疫磁珠筛选系统,从肿瘤组织中分离获得CD133+细胞(BTSCs),分成4组进行培养:(1)含10%胎牛血清(FCS);(2)10%FCS+VPA;(3)无FCS+生长因子;(4)无FCS+生长因子+VPA。取不同时间点上的细胞,相差显微镜观察其形态变化;流式细胞术检测与分化相关的标志物、细胞周期和DNA倍体变化;免疫激光共聚焦分析与分化相关标志物的共表达情况。【结果】无FCS条件下培养的BTSCs呈悬浮球状生长,高表达CD133和Nestin,不表达GFAP和β-TubulinⅢ,G0/G1期细胞占大多数,G2/M期细胞接近0%,DNA都是异倍体,对VPA反应不敏感。含FCS培养原本悬浮的细胞约4 h开始贴壁,均呈圆形。此后逐渐向多形性分化,至7 d时分化的细胞部分又返回至圆形,至10 d~21 d时,有的还能重新恢复球形并呈悬浮生长。培养3 d、7 d、10 d和21 d时,CD133、Nestin阳性细胞数先降后升,GFAP+和β-TubulinⅢ+细胞数始终处于较低水平。含FCS培养液中加入VPA,细胞形态上未见上述的回逆现象,CD133和Nestin表达的先降后升现象消失,GFAP和β-TubulinⅢ在第7 d以后表达明显升高,但极大部分细胞共表达Nestin。而神经干细胞(NSCs)在含FCS培养至10 d时即以GFAP和β-TubulinⅢ表达为主,未见CD133+细胞。此外,含血清培养时BTSCs仍以异倍体为主,含少量的G2/M期细胞,加VPA诱导后细胞周期和DNA倍体变化不明显。【结论】BTSCs在含血清条件下培养出现的多向分化表型不稳定,时有去分化所导致的回逆。加入诱导分化剂VPA培养,虽然能阻止回逆现象出现,并有代表星形胶质细胞和神经元标志物表达上升,但因其共表达Nestin而仍属于未完全分化细胞,表明BTSCs分化始终处于受抑状态。第二部分p18INK4c基因失活参与胶质瘤的发生发展【目的】探讨p18INK4c基因在胶质瘤发生发展中的作用。【方法】分别用RT-PCR和免疫组化检测人脑胶质瘤细胞和组织中p18INK4c基因的表达变化;MS-PCR分析胶质瘤细胞中p18INK4c基因启动子甲基化状态;通过测序分析p18INK4c基因的序列变化;构建真核表达载体pcDNA-p18INK4c,脂质体Lipofectamine 2000介导转染人脑胶质瘤细胞SHG44,RT-PCR检测基因表达,流式细胞仪分析细胞增殖周期变化。【结果】8株人脑胶质瘤细胞系中5株细胞不表达p18INK4c基因,其中2株中基因启动子呈甲基化状态;3株细胞表达p18INK4c基因,基因启动子区都呈去甲基化。68例人脑胶质瘤组织中22例不表达、46例表达p18蛋白。BTSCs中p18INK4c表达缺失。转染p18INK4c基因后,SHG44细胞中出现明显p18INK4c表达,细胞周期G0/G1期细胞增加,S期细胞减少。【结论】部分胶质瘤细胞和组织中p18INK4c基因表达缺失,启动子甲基化是导致基因表达沉默的原因之一。p18INK4c基因失活参与了胶质瘤的发生发展过程。第三部分诱导分化治疗人脑胶质瘤的靶细胞与靶分子【目的】探讨丙戊酸钠诱导人脑胶质瘤细胞分化时作用的靶细胞和靶分子。【方法】丙戊酸钠治疗位于裸小鼠皮下的可移植性人脑胶质瘤,半定量RT-PCR检测移植瘤中HDAC1和Tob的表达;CD133免疫磁珠分离胶质瘤组织中的CD133+细胞,在分别含血清或不含血清加生长因子的条件下培养,加丙戊酸钠作用21d,流式细胞仪检测和共聚焦显微镜观察分化标志物表达。【结果】丙戊酸钠能明显抑制位于裸小鼠皮下移植瘤的增殖,使HDAC1表达下降,Tob表达升高。流式细胞仪检测丙戊酸钠作用21d的细胞,在含血清条件下,GFAP或β-tubulinⅢ阳性细胞数显著增加,而无血清加生长因子条件下两标志物表达均无显著差异;共聚焦显微镜观察显示上述的GFAP+或β-tubulinⅢ+细胞共表达Nestin。【结论】VPA逆转人脑胶质瘤细胞分化抑制的靶分子有HDAC1和Tob,靶细胞为处于分化过程中的脑肿瘤干细胞。第四部分ABCG2和MGMT基因启动子甲基化异常与胶质瘤细胞耐药【目的】分析胶质瘤细胞中耐药基因ABCG2和MGMT启动子甲基化与基因表达之间的关系。【方法】RT-PCR检测胶质瘤细胞中ABCG2和MGMT的表达,MS-PCR分析基因启动子甲基化状态,用去甲基化试剂5-氮-脱氧胞苷处理胶质瘤细胞U251后分析基因表达变化。【结果】8株人脑胶质瘤细胞中有6株细胞表达ABCG2,其中5株细胞中ABCG2基因呈去甲基化,1株甲基化与去甲基化共存;2株细胞不表达ABCG2,其中1株甲基化与去甲基化共存,1株没有检测出甲基化和去甲基化产物。5株细胞表达MGMT,3株细胞不表达,其中5株细胞中MGMT为去甲基化,4株为高甲基化。【结论】胶质瘤细胞中部分细胞表达ABCG2和/或MGMT,基因启动子区呈去甲基化状态,这些细胞可能对相关药物耐药;而启动子高甲基化是胶质瘤细胞中ABCG2和MGMT基因失活的重要因素。更多还原

【Abstract】 Part I Dedifferentiated cells: the targets of gliomagenesis, malignant progression, and therapy【Objective】To investigate in vitro differentiation reversion of brain tumor stem cells (BTSCs) for further study the inhibitory mechanisms of BTSCs differentiation.【Methods】CD133+ cells (BTSCs) were isolated from glioma specimens using the immunomagnetic bead separation system, and cultured in DMEM/F-12 medium supplemented with: (1) 10% fetal calf serum (FCS), (2) 10% FCS + sodium valproate (VPA) injection, (3) no FCS + growth factors, and (4) no FCS + growth factors + VPA injection, respectively. The morphological changes of cells were observed under phase contrast microscope at different time points; the cell surface markers relating with cell differentiation, cell cycles and changes in DNA ploids were detected with flow cytometry (FCM); co-expressions of cell surface markers relating with differentiation were analyzed with immunofluorescence staining and laser scanning confocal microscopy (LSCM).【Results】BTSCs cultured grew with a spherical shape in a suspended way in FCS-free medium and the expression levels of CD133 and nestin were high, but expressions of glial fibrillary acidic protein (GFAP) andβ-TubulinⅢwere negative. Cells in G0/G1 phase were the majority among these cells and in G2/M phase, nearly 0%. Besides, chromosomes of these cells were all heteroploids and the cells were insensitive to VPA. Having been cultured in FCS medium for 4 h, the suspended BTSCs spheres became adherent and turned to the round shape. Later on, the cells differentiated gradually into various shapes, until 7 d after in vitro culturing, the differentiated cells partially returned to the round shape and during the 10-21 d, some even regained the spherical shape and again grew in the suspended way. In addition, the results of FCM showed that the number of CD133 or Nestin positive cells decreased first and then increased after having been cultured in FCS medium for 3, 7, 10 and 21 days, but the number of GFAP+ or β-TubulinⅢ+ cells were at low levels. On the contrary, when VPA was added to the FCS medium, the phenomenon of reversion in cell morphology and changes of CD133 and nestin expression levels disappeared. The GFAP+ andβ-TubulinⅢ+ cells increased significantly after treatment with VPA for 7 d, but nestin was co-expressed with most of the GFAP+ orβ-TubulinⅢ+ cells. After cultured in FCS medium for 10 d, the majority of neural stem cells (NSCs) were GFAP+ andβ-TubulinⅢ+ cells with no CD133+ cells. Besides, the BTSCs cultured in FCS medium were heteroploids primarily including a few G2/M phase cells. After treatment with VPA, there were no significant differences in cell cycle and DNA ploids.【Conclusions】The phenotype of multi-directional differentiation of BTSCs in FCS medium was instable. The differentiated cells reversed frequently due to dedifferentiation. The differentiation inducer VPA inhibited differentiation reversion of BTSCs and facilitated the expressions of cell surface markers representing astrocytes and neurons, but the differentiation of BTSCs was incomplete for their co-expression with nestin, suggesting that the differentiation of BTSCs was still consistently inhibited.Part II Inactivation of p18INK4c gene was involved in gliomagenesis and malignant progression【Objective】To investigate the role of p18INK4c gene in gliomagenesis and malignant progression.【Methods】The p18INK4c expression in glioma cell lines and tissues was detected uing RT-PCR and immunohistochemistry. The methylation status of the p18INK4c promoter was analyzed by methylation-specific PCR. The p18INK4c mRNA was amplified and sequenced from 4 cell lines with p18INK4c expression, and then ligated into pcDNA3.1+ vector and transfected into glioma cell line SHG44 with Lipofectamine 2000. After transfection, the p18INK4c expression and cell cycle of SHG44 cell line were detected with RT-PCR and FCS, respectively.【Results】Five of 8 human glioma cell lines did not expressed p18INK4c, with promoter methylation in two. Three of 8 cell lines expressed p18INK4c, with all promoter hypomethylation. Twenty two of 68 glioma specimens did not express P18 protein, 46 of the specimens expressed. P18 mRNA and protein expression were negative in BTSCs. After transfected with pcDNA-p18INK4c, human glioma cell line SHG44 expressed p18INK4c significantly, the ratio of G0/G1 phase cells increased, S phase cells decreased compared with negative control.【Conclusions】Part of glioma cells and specimens did not express p18INK4c gene. Promoter methylation might contribute to p18INK4c silencing in some glioma cells. The p18INK4c gene inactivation might play a role in gliomagenesis and malignant progression.Part III Target cells being induced to differentiate by VPA in glioma【Objective】To investigate the target cells and molecules with sodium valproate induced differentiation of human glioma cells.【Methods】Nude mice bearing human glioma xenogenic graft subcutaneously were treated with VPA. The expressions of HDAC1 and Tob genes of xenografts were analyzed with semiquantitative RT-PCR. The CD133+ cells (BTSCs) were isolated from glioma specimens by immunomagnetic sorting, and cultured in the medium containing FCS or in the serum-free medium supplemented with growth factors, respectively, followed by treatment with VPA in vitro for 21days. The cell surface markers were detected with flow cytometry and confocal microscopy.【Results】VPA inhibited the growth of subcutaneous xenografs bearing on nude mice, and up-regulated the HDAC1 expression, down-regulated the Tob expression. The cell surface markers of BTSCs were detected by flow cytometry after VPA treatment for 21 days. In the FCS group, the GFAP orβ-tublinⅢpositive cells increased significantly, but in the growth factor group, no statistical differences were observed in the GFAP orβ-tublinⅢexpression. The results of confocal microscopy indicated that the GFAP+ orβ-tublinⅢ+ cells coexpressed with Nestin.【Conclusions】HDAC1 and Tob genes were the potential target molecules in reversion of the differential inhibition of human glioma cells with VPA. The BTSCs undergoing the processes of differentiation were the target cells for VPA. Part IV Promoter methylation of ABCG2 and MGMT gene in glioma cells【Objective】To analyze the correlations of ABCG2 and MGMT gene expression with the promoter methylation status in glioma cell lines.【Methods】The expressions of ABCG2 and MGMT in glioma cell lines were detected with RT-PCR. The promoter methylation status of ABCG2 and MGMT were analyzed by methylation-specific PCR. The expressions of ABCG2 and MGMT in U251 glioma cell line were analyzed after 5-Aza-dC treatment.【Results】Six of 8 glioma cell lines expressed ABCG2, with amplification only in U(unmethylated) reaction in five, and amplification in both U and M(methylated) reaction in one. Two of 8 cell lines did not expressed ABCG2, one displayed amplification bands in both the M and U reaction, and the other one failed to amplify either M or U bands. Five of 8 cell lines expressed MGMT, hypomethylation was detected in five, and hypermethylation in four.【Conclusions】Part of glioma cell lines expressed ABCG2 and/or MGMT, with promoter hypomethylation. These cell lines might be drug resistance. Promoter methyltion might contribute to inactivation of ABCG2 and MGMT in glioma cell lines.更多还原