【作者】 平轶芳；

【导师】 卞修武；

【作者基本信息】 第三军医大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 恶性胶质瘤是中枢神经系统最常见的原发肿瘤,其高度侵袭性导致治疗十分困难、复发率高。丰富的新生血管是此肿瘤最为突出的间质特点,这些新生血管是瘤细胞快速增殖、高度侵袭和复发的重要结构基础。因此,深入研究血管生成机制、并有效阻抑血管新生对于恶性胶质瘤的治疗非常关键。肿瘤血管生成由瘤细胞及间质细胞分泌的促血管生成因子和抑血管生成因子共同调控,其中血管内皮生长因子(vascular endothelial growth factor, VEGF)和白细胞介素-8 (interleukin-8, IL-8)是两种重要的促血管生成因子,它们可以促进肿瘤相关内皮细胞增殖、迁移及形成血管腔。近年研究表明,趋化因子受体CXCR4在肿瘤血管生成和侵袭中发挥重要作用。本课题组前期发现,CXCR4表达与胶质瘤微血管密度及恶性程度呈正相关,提示此受体具有促胶质瘤血管生成的作用。但是,CXCR4促胶质瘤血管生成作用的机制和治疗学意义尚需深入探讨。最近,肿瘤干细胞(cancer stem cells, CSCs)或称肿瘤始动细胞(tumor initiating cells)因其独特的生物学特性及在肿瘤中的重要作用已越来越受到重视。CSCs在肿瘤中含量极微,却独具自我更新、无限增殖、多向分化和重建肿瘤的能力,被认为可能是肿瘤发生、侵袭转移、治疗抵抗以及复发的根源。然而,这群CSCs在血管生成中所扮演的角色尚不清楚:它们是否始动或参与了肿瘤血管生成过程?其分子机制如何?我们前期结果和国外的报道均提示胶质瘤中的CSCs,即胶质瘤干细胞(glioma stem cells, GSCs)高分泌VEGF,IL-8等血管生成因子,其上游调控机制不明。最近,我们发现GSCs高表达CXCR4。基于此,我们推测,GSCs高表达的CXCR4可能通过促进VEGF和IL-8产生而发挥促血管生成作用。本研究中,我们首先观察了人恶性胶质瘤细胞系U87细胞中CXCR4的表达及活化后促侵袭和血管生成因子产生的作用,并观测我室自行合成的化合物诺帝对CXCR4介导的上述效应的抑制作用;然后,我们检测了GSCs标志物CD133和nestin在原代胶质瘤、胶质瘤细胞系及其移植瘤中的表达,并从中分离、鉴定出GSCs,研究了CXCR4在GSCs中的表达及其活化后促血管生成作用机制;最后,我们探讨了拮抗CXCR4活化对GSCs移植瘤的治疗作用。主要结果和结论如下:1.CXCR4活化促进U87细胞迁移及产生VEGF和IL-8。①CXCR4在U87细胞之间表达强弱呈现非均一性。CXCR4配体CXCL12 (25~100 ng/ml)可引起细胞内钙流增加,效应在50 ng/ml时达到最强。用CXCR4特异性拮抗剂AMD3100 (1~10μM)预处理细胞,可抑制CXCL12诱发的胞内钙流。②CXCR4活化促进U87细胞迁移。对照组U87细胞跨膜迁移细胞数为11.68±3.78个/3个高倍视野(3HPF),而采用CXCL12 (25~100 ng/ml)活化CXCR4后,跨膜迁移的细胞数明显增加,其中以50 ng/ml剂量组效应最强,迁移细胞数为156.67±13.2个/3HPF。1和10μM AMD3100均可抑制CXCR4活化诱导的细胞迁移。CXCR4促迁移效应可能与其活化后诱导F-actin增加有关。③10~100 ng/ml CXCL12处理36 h均能促进U87细胞分泌VEGF和IL-8,浓度为50 ng/ml时作用最强,50 ng/ml CXCL12处理12 h可促进IL-8分泌,对VEGF分泌无影响,处理24~48 h可同时促进IL-8和VEGF蛋白分泌和mRNA表达。1~10μM AMD3100预处理能抑制CXCR4活化引起的VEGF和IL-8增加。2.诺帝抑制U87细胞CXCR4表达及活化效应。①25~100μM诺帝处理12 h能使CXCR4表达减弱,以100μM作用最强。100μM诺帝处理12、24、36、48 h,平均荧光强度分别为24.13±8.53、24.52±4.41、17.36±3.91和13.45±4.23,与对照组(38.54±11.42)相比CXCR4表达显著降低(P<0.05)。27 mg/kg诺帝体内治疗能够降低U87移植瘤CXCR4蛋白的表达。但是,诺帝处理并不影响CXCR4 mRNA表达。②25~100μM诺帝预处理能够抑制CXCR4活化所引起的细胞迁移活性增加,穿膜细胞数分别为99.67±9.45、82.67±7.02和54.33±8.62个/3HPF,与对照组(156.67±13.2个/ 3HPF)相比有显著统计学差异(P<0.05)。此效应与诺帝抑制CXCR4活化后诱导的F-actin增多有关。③诺帝对U87细胞CXCR4活化后的促VEGF和IL-8 mRNA转录及蛋白分泌效应均具有抑制作用。3.人原代胶质瘤组织、胶质瘤细胞系及其移植瘤中均含有nestin~+/CD133~+ GSCs。①人原代星形胶质细胞瘤,胶质瘤细胞系U87、SHG-44、CHG-5及其相应移植瘤均表达GSCs标志物nestin和CD133。其中nestin的表达与肿瘤级别呈正相关,高级别胶质瘤(III、IV级)显著高于低级别胶质瘤(I、II级),而CD133表达与肿瘤级别无关。大部分CD133阳性细胞同时表达nestin。②nestin~+/CD133~+细胞具有GSCs特性。CD133~+细胞在神经干细胞培养基中成球生长;所形成的细胞球表达nestin和CD133,经诱导后能分化为表达GFAP、MBP和β-tubulin III的细胞,且能在体内连续成瘤。③创建了4种简便、易行的获取肿瘤干细胞的方法,并应用于GSCs的筛选。这些方法包括克隆分型筛选法、基于侵袭异质性的筛选法、耐药细胞筛选法和逐步添加条件培养基筛选法。4.GSCs优势性高表达CXCR4,此受体活化后介导GSCs诱导的胶质瘤血管生成。①胶质瘤细胞系(U87、SHG-44、CHG-5)、U87移植瘤和人胶质母细胞瘤来源的CD133~+细胞均比相应的CD133-群体高表达CXCR4。在CD133~+细胞形成GSCs球的过程中,CXCR4保持这种高表达状态。②10~100 ng/ml CXCL12刺激GSCs球均能引起胞内钙流增加,以50 ng/ml剂量组作用最强,此效应可被CXCR4特异性拮抗剂AMD3100抑制。③CXCR4活化能够增强GSCs的软琼脂克隆形成能力。对照组细胞软琼脂克隆形成率(CFE)为8.2±1.3%,而CXCL12刺激组增加至17.4±4.8%,AMD3100预处理能够抑制CXCR4的效应,CFE为8±1.15%。此外,AMD3100单独处理也能够抑制GSCs的CFE。④CXCR4活化后,从转录和翻译两个水平促进GSCs产生VEGF和IL-8。25 ~100 ng/ml CXCL12均能促进GSCs产生VEGF和IL-8,50 ng/ml剂量组效应最强。1~10μM AMD3100能抑制CXCR4活化引起的VEGF和IL-8产生增加。⑤CD133~+细胞培养上清对脐静脉内皮细胞增殖的诱导能力强于CD133-细胞。CD133~+细胞培养上清刺激组2、3、4、5、6和7 d时的OD值均较CD133-细胞培养上清刺激组显著增高。⑥无论在U87细胞移植瘤还是在人原代胶质母细胞瘤中,肿瘤微血管周围可见更多的CD133~+、nestin~+的GSCs。⑦GSCs高表达CXCR4的机制可能与其独特的miRNA表达谱有关。CD133~+细胞和CD133-细胞的miRNA表达谱不同,大部分miRNA在CD133~+细胞高表达。以CXCR4为靶点的几种miRNA表达趋势如下:miRNA-9在从U87细胞和人原代胶质母细胞瘤中分离得到的CD133~+细胞中表达上调,上调倍数分别为3.96和3.93倍;miRNA-93上调倍数分别为6.63和2.06倍;miRNA-372上调倍数分别为10.79和20.24倍;miRNA-373上调最为显著,倍数分别为77.86和12.95倍。miRNA-302在U87细胞来源的CD133~+细胞中表达上调8.68倍,而在人原代胶质母细胞瘤来源的CD133~+细胞中表达下调1.51倍。5.CXCR4特异性拮抗剂AMD3100抑制GSCs移植瘤生长和血管生成。①AMD3100治疗1 w时,肿瘤体积与空白对照组和PBS对照组无明显差别。而后,AMD3100治疗组移植瘤体积显著小于对照组,观察期截止时,AMD3100治疗组移植瘤体积为0.23±0.06 cm3,与空白对照组(2.21±0.32 cm3)和PBS对照组(2.16±0.37 cm3)相比有显著统计学差异(P<0.05),空白对照组与PBS对照组之间肿瘤体积无显著性统计学差异(P>0.05)。②AMD3100治疗后肿瘤微血管密度明显降低,与PBS对照组比较具有显著统计学意义(P<0.05)。AMD3100治疗组移植瘤血管生成因子VEGF和IL-8的表达显著低于对照组(P<0.05)。③AMD3100不影响GSCs移植瘤中CXCL12和CXCR4表达(P>0.05)。④AMD3100治疗组与对照组GSCs移植瘤中CD133~+细胞和nestin~+细胞比例无显著统计学差异(P>0.05)。综上所述,CXCR4活化可促进人恶性胶质瘤细胞系U87迁移及产生血管生成因子,诺帝能够抑制U87细胞CXCR4蛋白表达及活化后的效应;胶质瘤细胞表达的CXCR4具有非均一性,优势表达于GSCs,后者存在于人原代胶质瘤组织、胶质瘤细胞系及其移植瘤中;CXCR4活化在介导GSCs诱导胶质瘤血管生成中起重要作用,而抑制CXCR4可阻抑胶质瘤血管生成从而抑制胶质瘤生长与侵袭。结果表明,CXCR4可能是恶性胶质瘤治疗的有效靶分子,而GSCs可能是其中更为重要的靶标。更多还原

【Abstract】 Malignant gliomas are common primary tumors within the central nervous system, which are characterized by invasive growth, aberrant neovascularizion and rapid expansion. The active neovascularization not only contributes to the rapid growth of tumor cells by providing them nutrient and oxygen, but also facilitates these cells to invade into normal brain tissues, resulting in the difficulties in malignat glioma therapies. Thus, it is of great importance to further investigate the mechanism of angiogenesis for developing new antiangiogenesis strategy to treat such kind of malignancy.Tumor angiogenesis is regulated by aberrant production of proangiogenic and antiangiogenic factors produced by malignant tumor cells as well as the infiltrating leukocytes. VEGF and IL-8 are two important angiogenic factors. They promote endothelial cell proliferation, migration and tubule formation, which are cruiel steps in angiogenesis. Recently, it was reported that the CXC chemokine receptor CXCR4 regulated pathological angiogenesis, tumor invasion and metastasis. Our previous studies showed that CXCR4 expression was correlated directly with the degree of malignancy and microvessel density (MVD) of human gliomas. However, the machenism of CXCR4-induced angiogenesis remains to be clarified.Cancer stem cells (CSCs), or tumor initiating cells, are becoming fascinating because of their special biologic behaviors and the important roles in carcinogenesis. Although these cells account for only a small fraction of cancer cell population, they exclusively exhibit capacities of self-renewal, multipotent differentiation and tumor initiation. CSCs are also resistant to chemo- and radiotherapy, thus possibly responsible for cancer recurrence after treatment and metastasis. The great progress of study on CSCs is enormously challenging traditional theory of tumor vascularization, for instance, whether and how CSCs initiate angiogenesis. Our previous results showed that glioma stem cells (GSCs) produced higher levels of VEGF and IL-8 compared to the differentiated tumor cells. However, it is unclear whether GSCs produce VEGF and IL-8 constitutively or the production is induced by factors present in tumor microenvironment. Recently, we found high level of CXCR4 expression by GSCs with unknown function.In this study, we hypothezed that CXCR4 activation might initiate GSCs-mediated angiogenesis by promoting the production of VEGF and IL-8. We firstly decteted the effect of CXCR4 activation on a malignant glioma cell line U87 cell invasion and the production of angiogenic factors, and explored the inhibitory effect of Nordy, a sythesized chiral compound based on the structure of natural nordihydroguaiaretic acid (NDGA), on the response of U87 cells to CXCR4 agonist CXCL12. We then detected the distribution of GSCs in primary glioma tissues, isolated and characterized GSCs from the tissues, glioma cell lines and their xenografts. We further investigated the activation of CXCR4 on GSCs-induced angiogenesis and the potential mechanisms. Finally, we explored the therapeutic significance of inhibiting CXCR4 with GSCs xenograft model. The main results and conclusions are as follows:1. CXCR4 activation promoted U87 cells to migrate and produce VEGF and IL-8. (1) Stimulation of U87 cells with CXCL12, the CXCR4 ligand, caused a rapid increase in intracellular calcium mobilization with optimal effect of CXCL12 at 50 ng/ml. The effect of CXCL12 was abolished by the CXCR4 antagonist AMD3100. (2) CXCL12 promoted but AMD3100 inhibited the chemotactic response of U87, which might be attributed to the actin polymerization induced by CXCL12. (3) CXCL12 increased VEGF and IL-8 in U87 cells at both protein and mRNA levels. The effect of CXCL12 was bolcked by AMD3100.2. Nordy inhibited functional expression of CXCR4 on malignant glioma cells. (1) Immunofluorescence staining showed the decreased expression of CXCR4 after treatment with Nordy in a time-dependent manner. However, Nordy had no significant effects on CXCR4 mRNA expression. Futhermore, CXCR4 expression in U87 xenografts with Nordy treatment decreased as compared with control xenografts. (2) CXCL12-induced migration and actin polymerization of glioma cells were inhibited after pretreatment with Nordy. (3) Nordy also inhibited production of IL-8 and VEGF at either protein or mRNA level induced by CXCL12.3. CD133~+/nestin~+ GSCs were found in both primary and xengrafted gliomas. (1) CD133~+ and/or nestin~+ tumor cells were observed in primary human gliomas, human glioma cell lines and their xenografts. (2) CD133~+/nestin~+glioma cells were GSCs. When the CD133~+ cells were cultured in neural stem cell medium, they could form neurosphere-like spheroids expressing CD133~+/nestin~+. When seeded in differentiation conditions (medium containing serum), the spheroids generated differentiated cells with expression of GFAP, MBP and/orβ-tubulin III. Moreover, CD133~+/nestin~+ glioma cells initiated xenografts in nude mice. (3) Such kind of GSCs could be isolated and enriched with other methods, including colony heterogeneity-based and invasive potential-based methods for enrichment, and drug resistance-based selection. Moreover, increasing the concentration of serum-free neural stem cell medium was used as a new strategy for enrichment of GSCs.4. GSCs expressed higher levels of functional CXCR4, which promoted angiogenesis by producing VEGF and IL-8. (1) Compared with committed tumor cells, the GSCs expressed higher levels of CXCR4. (2) Stimulation of GSCs with CXCL12 caused a rapid increase of intracellular calcium mobilization with optimal effect of CXCL12 at 50 ng/ml, which could be abolished by pretreament with AMD3100. (3) The colony forming efficacy (CFE) of GSCs was increased when CXCR4 on the GSCs was activated. In the absence of AMD3100, the CFE of GSCs was 8.2±1.3 %, and increased to 17.4±4.8% after CXCL12 stimulation. After treatment with AMD3100, CFE of GSCs with or without CXCL12 stimulation was significantly inhibited. (4) CXCL12 could induce VEGF and IL-8 at both mRNA and protein levels produced by GSCs, while AMD3100 inhibited the effects. (5) The conditioned medium of GSCs presented more prowerful capability of promoting proliferation of umbilical vein endothelial cells than the CD133- conterparter. (6) CD133~+/nestin~+ cells were detected in the tissues adjacent to capillaries, which were revealed by CD31, in human primary glioma tissues. (7) The differential expression of microRNA was found between GSCs and committed differentiated cells, which might be responsible for their differential CXCR4 expression.5. AMD3100 suppressed growth and angiogenesis of xenografts formed by GSCs. (1) The xenografts in the mice treated with AMD3100 were significantly smaller (0.23±0.06 cm3) than those in the mice without any treatment (2.21±0.32 cm3) or with PBS treatment group (2.16±0.37 cm3). (2) Immunofluorescent staining with anti-CD31 antibody indicated that tumor specimens from AMD3100-treated mice contained significantly reduced MVD. This was associated with lower levels of VEGF and IL-8 in tumors from AMD3100 treatment group. (3) There was no significant difference in the expression of either CXCL12 or CXCR4 between AMD3100 treatment group and the control group. (4) AMD3100 did not change the ratio of CD133~+ or nestin~+ cells in GSCs xenografts in vivo.In summary, these results suggest that (1) activation of CXCR4 promoted U87 cells to migrate and produce VEGF and IL-8. Nordy inhibited the expression and effect of CXCR4 in U87 cells. (2) There were GSCs in human primary gliomas, human glioma cell lines and glioma xenografts. These cells expressed higher levels of CXCR4, activation of which promoted angiogenesis by producing VEGF and IL-8. A CXCR4 inhibitor AMD3100 suppressed GSCs xenograft growth and angiogenesis. Thus, CXCR4/CXCL12 axis is crucial for neovascularization of gliomas and may represent therapeutic targets for developing novel anti-GSCs agents to improve glioma treatment.更多还原