【作者】 张煜；

【导师】 崔尧元； 吴伟忠； 刘银坤；

【作者基本信息】 复旦大学 ， 神经外科学， 2006， 博士

【摘要】 胶质瘤是最常见的颅内肿瘤，严重危害人类健康。由于胶质瘤侵袭性生长，极易术后复发，综合治疗效果不佳。目前研究发现，胶质瘤在多个基因的不同环节上发生异常，胶质瘤细胞在增殖、分化和凋亡等重要生物学过程中缺乏正常的基因调控而出现细胞的恶性增殖和侵袭生长。新的抗肿瘤治疗策略倾向于寻找能够针对肿瘤机制中不同环节同时作用的治疗方法，以获得更好的疗效。信号转导和转录激活因子3（Signal Transducer and Activator of Transcription 3，STAT3）的发现为这种设想供了理论依据。STAT3介导各种细胞因子和生长因子所引起的广泛的细胞反应，作用涉及细胞的增殖、分化和免疫调节等许多方面，研究在多种人类恶性肿瘤中发现了STAT3信号通路异常激活的现象，STAT3的异常激活能够通过生长因子信号系统与凋亡相关因子、促血管新生因子等相互作用而导致肿瘤生成并促进肿瘤的发展。以STAT3作为抗肿瘤治疗靶点的重要价值已逐步呈现出来。人类中枢神经系统恶性肿瘤由于生长部位和组织来源的特殊，具有不同的生物学特点。基因治疗的重要价值尤为明显。虽然有资料表明人脑胶质细胞瘤中有STAT3的异常表达，但对STAT3信号通路在胶质瘤发生发展中的作用以及阻断STAT3信号通路对胶质瘤的影响的研究尚未深入开展，相关机制不明。研究目的和思路：本项目拟观察STAT3在胶质瘤手术切除标本及体外培养胶质瘤细胞株中的表达，并应用酪氨酸激酶抑制剂AG490阻断STAT3信号通路，观察肿瘤细胞增殖、凋亡、侵袭等重要生物学过程发生的变化，以了解STAT3信号通路在人脑胶质瘤发生发展过程中所起的作用，并为这一通路能否成为胶质瘤治疗的有效靶点作一些前期的评估。方法和结果：一STAT3在星形细胞瘤手术标本中的表达特点星形细胞瘤手术标本30例行HE染色和STAT3免疫组化染色。对照HE染色切片情况，将STAT3免疫组化的切片根据其中阳性细胞的表达强度和分布范围分别进行评分量化。数据行等级资料非参数检验（Nonparametric Test）。结果发现STAT3蛋白在所有标本中的表达率为100％。STAT3抗原通常定位在细胞的胞浆部分。STAT3免疫组化染色切片与相应的HE染色切片对比，发现STAT3在瘤周组织正常组织中的表达通常较低或不表达。分化较好的标本中，STAT3蛋白在瘤内组织中的表达比较均匀。分化较差的标本中，STAT3阳性细胞分布和表达强度上差异很大。部分瘤巨细胞和分裂相的肿瘤细胞胞浆中见到STAT3的表达。较为恶性的胶质瘤内常有丰富的新生血管，STAT3蛋白在新生血管的内皮细胞中表达不仅非常明确，而且十分普遍。统计结果显示STAT3的表达在性别、年龄、病理级别的不同分组间P值大于0.05，无显著性差异。STAT3在瘤内与瘤周组织的表达P＜0.01，差异显著。因此，STAT3蛋白在胶质瘤标本中有广泛的表达，但瘤周组织表达很低。STAT3在分裂相细胞和肿瘤多核巨细胞及血管内皮细胞中的高表达提示STAT3参与了胶质瘤细胞分裂、增殖和血管新生关系密切。二STAT3、p-STAT3在人脑胶质瘤细胞株U251和U87中的表达体外培养人胶质瘤细胞，将人胶质瘤细胞株U251、U87细胞种植于25cm²细胞培养瓶，加含10％胎牛血清的DMEM培养液，在5％CO₂37℃恒温的条件下培养。用免疫细胞化学染色方法以及相差荧光显微镜观察检测STAT3和p-STAT3在人脑胶质瘤细胞株U251和U87中的表达。结果显示：体外培养的人胶质瘤细胞株U251和U87中，STAT3蛋白和STAT3的激活状态p-STAT3都有表达；STAT3蛋白在肿瘤细胞的胞浆表达非常丰富；p-STAT3蛋白仅表达于肿瘤细胞核；提示STAT3存在于胞浆，磷酸化激活后转入细胞核内实施转录。三AG490对STAT3信号通路的阻断作用以酪氨酸激酶抑制剂AG490阻断STAT3信号通路，胶质瘤细胞U251、U87接种于6孔板，加入AG490使最终浓度分别达到12.5uM，25uM，50uM，以不加药为对照。加药后肿瘤细胞与对照在条件完全相同的情况下培养72小时。提取细胞总蛋白，通过蛋白定量检测蛋白浓度，Western Blot半定量的方法检测STAT3和p-STAT3的表达情况。结果显示：STAT3和p-STAT3在两种胶质瘤细胞株中都有表达，U251细胞中的表达比U87细胞稍高。STAT3蛋白的表达不受AG490作用的影响，而p-STAT3蛋白的表达在两种细胞中都随AG490浓度的升高而逐步降低。U87细胞中STAT3和p-STAT3的表达较低，AG490作用后，p-STAT3的下降更为彻底，甚至完全不表达。所以，AG490能够阻断STAT3信号通路，其机制是通过抑制STAT3蛋白激活形成p-STAT3的过程来阻断这一信号转导通路。AG490对胶质瘤细胞株STAT3信号通路阻断的效果呈现浓度依赖的特点，并受到肿瘤细胞中STAT3和p-STAT3基础表达量的影响。四AG490阻断STAT3信号通路对胶质瘤细胞增殖和存活率的影响将胶质瘤细胞接种于96孔板，加入AG490，使终浓度分别为12.5uM，25uM，50uM，将不加药组设为对照。各组细胞在相同条件下培养，分别在加药前后24小时、48小时和72小时用SRB法染色，通过酶标仪570nm比色，对STAT3信号通路阻断的细胞模型进行细胞增殖检测，并以Excel和SPSS软件计算和统计分析。结果发现：阻断STAT3信号通路可明显抑制胶质瘤细胞株U251和U87的细胞增殖，并使肿瘤细胞的存活率降低。多因素方差分析的结果显示AG490对胶质瘤细胞增殖和存活率的抑制作用具有时间效应和浓度效应。作用时间和作用浓度两个影响因素的P值均小于0.01，具有统计学意义。析因分析显示，作用时间和作用浓度两者之间存在交换作用，两因素同时作用可以使效果因叠加而增强（P＜0.01）。因而，AG490能够通过阻断STAT3信号通路而抑制胶质瘤细胞株增殖并使肿瘤细胞存活率降低。五AG490阻断STAT3信号通路对胶质瘤细胞株凋亡的诱导作用用6孔板培养U251和U87细胞，加入AG490，使终浓度分别为12．5uM，2SuM，50uM，设不加药组对照。相同条件下继续培养，分别在加药后48小时和72小时收获细胞。按Annexin V—FITC凋亡检测试剂盒说明进行染色，以流式细胞仪进行细胞凋亡检测。发现：U251、U87细胞在不同浓度AG490作用后，部分肿瘤细胞发生形态改变。显微镜下表现为胞体缩小，呈圆形突起，细胞周边在显微镜下可见折光。部分凋亡细胞外形膨大，细胞浆呈泡沫样改变，甚至出现细胞破碎崩解。无论AG490作用48小时还是72小时，U25l细胞组和U87细胞组的凋亡比例都高于对照组。在AG490作用的两种细胞中，肿瘤细胞的凋亡比例均随AG490浓度的提高而升古同0因此，AG490阻断STAT3信号通路能够诱导胶质瘤细胞株发生凋亡。而大量肿瘤细胞凋亡的发生可以解释肿瘤细胞存活率下降的现象。六AG490阻断STAT3信号通路对胶质瘤细胞株细胞周期的阻滞作用在AG490阻断STAT3信号通路的细胞模型上，用低渗缓冲液对待测细胞行PI染色，用流式细胞仪检测肿瘤细胞的DNA细胞周期。检测发现，两种胶质瘤细胞的G1-S期比例增大，而细胞周期其他构成部分的比例均下降，提示STATB信号通路阻断后胶质瘤细胞的分裂过程受到阻滞而停留于分裂前的合成期。可见，AG490阻断STAT3信号通路对胶质瘤细胞株的细胞周期有阻滞作用。细胞周期的抑制是造成肿瘤细胞增殖抑制的一个重要原因。七阻断STAT3信号通路对胶质瘤细胞株侵袭性特征的影响通过Transwell细胞侵袭试验检查阻断STAT3信号通路对胶质瘤细胞株侵袭性特征的影响。以24孔板为基座，采用直径6.5mm的Transwell小室，聚碳酸酯微孔膜孔径8um。使用前铺Matrigel Matrix及湿化小室。5×10⁴个胶质瘤细胞加入上室，条件培养液及AG490加入下室。以不加药为对照。继续培养10小时后将穿过微孔膜的细胞用Giemsa溶液染色。显微镜下细胞计数，并进行统计分析。结果发现AG490作用后，能够穿过微孔膜小孔的胶质瘤细胞数量比对照组明显减少，P＜0.01，有显著性差异。说明肿瘤细胞中STAT3通路的阻断导致细胞侵袭能力下降。明胶酶谱检测MMP-2和MMP-9的表达。用6孔板培养U251和U87细胞，分别加入AG490使浓度达到25uM和50uM，设不加药组对照，72小时后取细胞培养液，通过电泳、复性、显影、染色、脱色的过程在凝胶上显示MMP-2和MMP-9的表达。凝胶用扫描器获取条带图像进行形态定量。结果显示AG490作用后MMP-2和MMP-9的表达与对照组相比均有明显下降，且下降程度随AG490浓度的提高而加重。因此，AG490阻断STAT3信号通路对胶质瘤细胞株体外侵袭力的抑制作用可能是通过下调MMP-2、MMP-9来实现的。八AG490阻断胶质瘤细胞STAT3信号通路对部分基因mRNA表达的影响通过RT-PCR观察AG490阻断胶质瘤细胞STAT3信号通路对部分基因mRNA表达的影响。用6孔板培养胶质瘤细胞。分别加入AG490达到25uM和50uM的最终浓度，以不加药组对照，相同条件下继续培养72小时。用TRIZOL提取细胞总RNA，经浓度检测后用RT方法将RNA逆转录为cDNA，再用MasterMix在PCR扩增仪上扩增。琼脂糖电泳显示结果。结果发现，抗凋亡因子Mcl-1、Bci-XL、Survivin的mRNA在AG490作用后表达受到抑制。促进肿瘤血管发生和侵袭的VEGF表达下降。细胞周期相关的Cyclin D1有下降趋势而p21没有明显变化。STAT3 mRNA的表达不受AG490影响。这样，AG490阻断STAT3信号通路对胶质瘤细胞株增殖、凋亡、细胞周期和侵袭能力的影响可能是通过对这些基因的调控来实现的。结论：STAT3在人脑胶质瘤中有广泛的表达，STAT3信号通路与胶质瘤细胞株的增殖、凋亡、细胞周期、肿瘤侵袭以及血管新生等诸多方面均有密切联系。因此，STAT3信号通路可能成为抑制胶质瘤侵袭生长的新靶点。更多还原

【Abstract】 The critical reason for glioma recurrence following the resection was the invasiveness of progression. It has been discovered that there was accumulation of genetic lesions in astrocytoma. Without regulation in proliferation, differentiation and apoptosis, cells of tissue tended to proliferate without limitation and progress invasively. Thus, many researches focused on the molecular mechanism of glioma for the final destination of control the disease effectively. STAT3 (Signal Transducer and Activator of Transcription 3) regulated gene expression in response to the signaling from cytokine and growth factor receptors, which correlated with the biological process of cell proliferation, differentiation and immunoloregulation. Constitutive activation of STAT3 protein has been detected in a variety of malignant tumors of human beings. It has also been reported that constitutive activation of STAT3 contributed to oncogenesis or tumor promotion through its intimate connection to the signaling relating to growth, apoptosis or angiogenesis. Thus, STAT3 as a novel target for therapeutic intervention of human cancers has been shown to be valuable.Though the constitutive activation of STAT3 has been found in astrocytoma, study on mechanisms of STAT3 signaling around the development of glioma did not actually expend. And the reports on STAT3 as a new therapeutic target for tumor of central nervous system were quite few. Therefore, we studied the function of STAT3 signaling pathway in astrocytoma in genesis and progress. We observed the expression of STAT3 in astrocytoma resection specimens. By inhibiting the STAT3 signaling pathway in U251 and U87 cell lines, we manage to reveal the effects of STAT3 on cell proliferation, cell cycle, apoptosis and invasiveness of astrocytoma in vitro. Our study tried to provid a basis for further evaluation of STAT3 as a novel target of glioma therapy and lead to a new path to understand the mechanism of astrocytoma invasiveness and progress as well as other biological features.1, The expression of STAT3 in astrocytoma specimen30 cases of astrocytoma resection specimens were selected and sliced. Immunohistochemistry staining was applied to observe the STAT3 expression in astrocytoma in tissue section. We found that every astrocytoma case we checked expressed STAT3. In most cases, STAT3 stain located in the plasma of the tumor cells. The level of STAT3 expression was significantly higher in the tumor area than in the peritumoral regions (P < 0.01). However, there was no significant difference of STAT3 expression in groups of histolpathalogical grade, sex and age (P > 0.05). The STAT3 positive cells seemed more even in group of low pathological grade than that of high grade, not only in distribution but also stain intensity. More, we found that the cytoplasm of the mitotic cells as well as multinucleated giant cells in astrocytoma appeared to be STAT3 positive. Moreover, the endothelial cells of neoplastic capillaries commonly expressed STAT3 protein in evidence.2, The expression of STAT3 in glioma cell lines U251 and U87We checked STAT3 and p-STAT3 expression in glioma cell lines U251 and U87 by way of immunocytochemistry staining and found that both U251 and U87 intensely expressed STAT3 as well p-STAT3 protein. The STAT3 protein was located in cytoplasm of tumor cells while p-STAT3 in cell nucleus. There seemed to be more STAT3 positive cells than p-STAT3 positive ones.3, The STAT3 signaling pathway being blocked by AG490 in glioma cells in vitroBoth U251 and U87 were treated with AG490 at concentrations of 12.5uM, 25uM and 50uM. The groups of no AG490 treating were set as control. At the time point of 72 hours, cells of all groups were harvested and both cytoplasmic and nuclear protein was obtained by way of whole cell extract. Western Blot was applied to detect and evaluate the expression of STAT3 and p-STAT3 in the cell extract, by which we found that both U251 and U87 expressed STAT3 as well as p-STAT3. The level of the two antigens was a little bit higher in U251 than in U87. Also in both of the glioma cell lines, the expression of STAT3 appeared be not influenced by AG490 but the level of p-STAT3 declined progressively till none in accompany with elevating of AG490 concentration. Thus, we proved that AG490 prevented the STAT3 protein from activating so that the STAT3 signaling pathway in glioma cells was blocked.4, The proliferation and survival rate of U251 and U87 cells were depressed after STAT3 signaling pathway inhibitionU251 and U87 were treated with AG490 respectively at the concentrations of 12.5uM, 25uM and 50uM in 96-well plates and groups of no AG490 treating were set as control. At the time point of 24 hours, 48 hours and 72 hours, cells were fixed and stained by method of SRB (Sulforhodamine B Protein Stain). The proliferation and survival rate of the two glioma cell lines were evaluated according to the OD values checked thereafter. The data were analyzed in SPSS 11.0 by way of Univariate Analysis of Variance. The result showed that both the proliferation and survival rate of U251 and U87 cells were significantly depressed by AG490 in a time- and concentration- dependent way (P < 0.01). The tests of between-subjects effects further revealed that both facts, time and concentration were responsible for the drop of cell proliferation and survival rate (P < 0.01) and the effect enhanced when both of them functioned simultaneously (P < 0.01).5, That AG490 inhibiting STAT3 induced apoptosis in glioma cell lines U251 and U87U251 and U87 cells were pretreated with AG490 for 48 and 72 hours respectively. All groups of tumor cells including control were harvested and stained according to the protocol of Annexin V-FITC apoptosis detection kit. The stained cells were then measured by flow cytometry. We found that the apoptosis rates of tumor cells were markedly higher in groups of AG490 treatment than those of control. More, the higher AG490 concentration applied, the more apoptosis tumor cells detected in the group.6, Cell cycle of U251 and U87 were arrested after AG490 applicationU251 and U87 cells were pretreated with AG490 of 50uM for 48 hours and harvested with groups of control. Then we stained the cells by method of PI staining and measured them with flow cytometry to see the change of DNA cell cycle of the glioma cell lines. It was observed that the cell cycle of both U251 and U87 showed arrested in G1-S phase after AG490 treatment.7, Inhibition of invasiveness of glioma Cell lines by blocking the STAT3 signaling pathway in vitroCell invasion was assayed in a 24-well Transwell cell culture chambers. Tumor cells were suspended into the upper chamber with AG490, while the controls without. After 10 hours of incubation, the invading tumor cells on the lower surface of the chamber were stained with Giemsa. The number of invading cells were counted and analyzed by student’s t test. Furthermore, we analyzed the secretion of MMP-2 and MMP-9 via a zymography assay. The result showed that AG490, which blocked the STAT3 signaling pathwa, significantly decreased the number of invading tumor cells (P < 0.01) and down regulated the secretion of both MMP-2 and MMP-9. Therefore, AG490 inhibited the invasiveness of glioma cell lines cells in vitro.8, Change of mRNA expression of some related genes in U251 cell in case of STAT3 pathway being blockedU251 cells were pretreated with AG490 for 72 hours. The whole cell RNA of GBM cell was extracted. mRNA expression of several related genes was initially assessed by RT-PCR using corresponding specific primers. The PCR result indicated that mRNA expression of apoptosis inhibitors genes Mcl-1, Bcl-XL and Survivin were down-regulated in respond to AG490 treatment. Similarly, angiogenesis gene VEGF decreased too. While cell cycle relevant gene Cyclin D1 also came down to some extent with p21 kept unchanged. The STAT3 mRNA expression did not affected by AG490.Conclusion:The findings that STAT3 widely expressed in astrocytoma and involved in the cell proliferation, survival, cell cycle, apoptosis, invasiveness and angiogenesis of glioma cell lines contributed to the conception that STAT3 signaling pathway is important in the pathogenesis of glioma and, therefore, possible to be a promising target in glioma therapy.更多还原