【作者】 邓林；

【导师】 李刚；

【作者基本信息】 山东大学 ， 神经外科， 2010， 博士

【摘要】 研究背景和目的人脑胶质瘤是人类颅内肿瘤中发病率最高的一类肿瘤,其中胶质母细胞瘤是最常见、恶性程度最高的组织学类型。目前治疗脑胶质瘤的方法主要包括了手术切除、放疗和化疗。但是,即使在临床手术技术日益提高的今日,脑胶质瘤患者的预后依然很差。因此,在对人脑胶质瘤的治疗研究中,寻找一种更加有效的治疗方法成为一项刻不容缓的课题。在已经完成的实验研究中,发现胶质瘤细胞的高度增殖能力和侵袭性是导致肿瘤复发和治疗失败的主要原因。尽管人脑胶质瘤很少在全身其他系统中转移,但其细胞对周围组织的浸润和脑组织中较远部位的侵袭是胶质瘤自然病程发展的必然结局。肿瘤细胞为了能够侵入周围组织,必须经历以下几步：从其周围的组织和细胞上脱离下来,穿过基底膜,在细胞外基质中迁移,到达新的组织部位并在此增殖,最终形成新肿瘤。在这一过程中,分解细胞外基质并在其中迁移是一个关键步骤。而基质金属蛋白酶(MMPs)又在分解细胞外基质中的过程中发挥了关键作用。MMPs是一族结构与功能上相关的内肽水解酶类,属于Metzincin超家族,具有分解各种细胞外基质蛋白的能力。在最近的研究中表明,MMPs在多种肿瘤细胞的侵袭和迁移过程中扮演了非常重要的角色,其中MMP-2和MMP-9在人脑胶质瘤侵袭性的相关研究中最受关注。Rho是一种小G结合蛋白,在细胞功能中发挥着多重作用,其中比较重要的一个作用就是维持肌球蛋白丝的收缩功能。这种收缩功能形成了细胞的张力纤维并维持着与细胞间的相互粘附。Rho下游有多种效应蛋白,最重要的是丝氨酸／苏氨酸激酶,即Rho蛋白相关激酶(ROCK)。有研究证实,在人脑胶质母细胞瘤细胞系中(T98G、U251) ROCK呈高表达；在对临床标本的分析研究中,ROCK的表达量与胶质瘤的恶性程度呈正相关。ROCK已经成为胶质瘤分子生物学研究的热点之一,但大多数研究局限于其在细胞骨架结构中的作用及其对人脑胶质瘤细胞迁移的影响。法舒地尔(Fasudil)作为一种特异性的ROCK阻断剂,被证实能够通过改变平滑肌细胞中的肌球蛋白轻链的磷酸化而达到抑制血管痉挛的作用。在临床治疗中被用于控制蛛网膜下腔出血导致的脑血管痉挛以及相关的中枢神经系统缺血症状。在一系列临床前期研究中,Fasudil可能对多种心脑血管疾病有治疗作用,如冠状动脉痉挛引起的心绞痛、高血压、肺动脉高压、脑卒中和心衰等。目前,未见有Fasudil对人脑胶质母细胞瘤作用的的相关研究报道。方法和结果为了阐明Fasudil对胶质母细胞瘤细胞的作用及其内在机制,两种人类胶质母细胞瘤细胞系T98G和U251作为本课题的研究对象。用不同浓度的Fasudil分别处理这两种细胞,并观察由Fasudil引起的细胞生物学特性的变化,包括细胞形态、增殖、侵袭和凋亡等方面。由于假定MMPs在胶质母细胞瘤细胞的迁移和侵袭中起到了关键作用,因此检测了Fasudil是否影响MMPs和MMPs的负性调节因子TIMPs的表达。为了进一步探索Fasudil这一已经用于临床治疗的药物在体内是否也发挥了同样的作用,本课题建立了颅内绿色荧光标记的胶质母细胞瘤小鼠动物模型,通过尾静脉给药对体内肿瘤进行处理,将肿瘤组织进行冰冻切片镜下观察并绘制荷瘤鼠生存曲线。一、ROCK抑制剂法舒地尔抑制人脑胶质瘤的体外实验研究1.法舒地尔改变胶质瘤细胞形态用不同浓度的法舒地尔处理T98G和U251细胞后发现,24h后细胞形态改变,细胞突触增多增长且胞体变细。胶质瘤细胞的这种变化表现为法舒地尔的浓度依赖性和时间依赖性。在100μM的高浓度法舒地尔处理组,细胞完全失去正常形态,突触长达胞体的数倍之多。为了验证法舒地尔的这种作用是否为可逆过程,在处理完24h后进行了恢复实验。实验结果显示,经过24h不同浓度法舒地尔处理,低浓度组的胶质瘤细胞在更换全新培养基之后的24h内,恢复了正常的细胞形态；而高浓度组的细胞则无法从拉伸变长、突触增多的星形形态中恢复至正常。2.法舒地尔对胶质瘤细胞迁移和侵袭的影响2.1法舒地尔抑制GBM的迁移损伤修复实验被用来测定胶质瘤细胞的运动迁移能力。用10μL移液器吸头在胶质瘤单细胞层上作出一道划痕,并以不同浓度的法舒地尔及阳性对照进行处理,显微镜下将划痕宽度的变化进行拍照记录。实验结果显示,法舒地尔浓度依赖的抑制了两种胶质瘤细胞的迁移。2.2法舒地尔抑制GBM的侵袭Transwell(?)小室被用来检测胶质瘤细胞在法舒地尔的作用下侵袭能力的变化。经过24h的侵袭,将Transwell(?)小室下表面的已穿膜细胞染色并计数,从而得到胶质瘤细胞相对侵袭力的数据。实验结果显示,法舒地尔能浓度依赖的抑制T98G和U251细胞的侵袭能力。但数据分析发现,法舒地尔对侵袭的抑制作用要高于其对迁移的抑制作用。3.法舒地尔抑制胶质瘤细胞MMP-2的表达RT-PCR法检测法舒地尔对侵袭相关基因转录水平的影响。选择20μM的法舒地尔和siRNA分别处理两种胶质瘤细胞,提取总RNA并进行扩增,结果发现在MMP-2/-9、TIMP1/2/3等侵袭相关基因中,只有MMP-2转录水平发生了变化,且随法舒地尔的浓度升高,MMP-2的转录水平相应降低。之后为了进一步确认法舒地尔对MMP-2的抑制作用,又运用Western Blot和明胶酶谱法从蛋白水平和分泌水平上验证了RT-PCR的结果。4.法舒地尔对胶质瘤细胞增殖和凋亡的影响用[3H]脱氧胸腺嘧啶核苷酸掺入法测定了法舒地尔对胶质瘤细胞增殖的作用。结果发现,法舒地尔在高浓度下处理胶质瘤细胞48h能浓度依赖的抑制T98G和U251细胞的增殖。用流式细胞术检测法舒地尔对胶质瘤细胞早期凋亡率的影响,结果表明在低浓度下法舒地尔在24h的处理后也可以引起细胞的凋亡,且成浓度依赖性。二、ROCK抑制剂法舒地尔抑制人脑胶质瘤的体内实验研究1.建立稳定表达增强型绿色荧光蛋白eGFP-T98G人脑胶质瘤裸鼠颅内原位移植模型用LipofectamineTM 2000向T98G中转染带有表达eGFP和G418抗性的质粒,G418加压筛选阳性细胞,通过有限稀释法将阳性细胞传代于96孔板中,挑选单克隆细胞株,分离培养并在荧光显微镜下观察鉴定为稳定表达eGFP的T98G细胞系(eGFP-T98G)。细胞传代培养扩增后,选取合适的细胞浓度,用鼠脑立体定向仪将eGFP-T98G细胞悬液种植于裸鼠尾状核。体视荧光显微镜下观察裸鼠移植瘤模型的成瘤情况。2.法舒地尔抑制人脑胶质瘤体内的增殖和侵袭在裸鼠接种成瘤第14天,分别对治疗组和对照组经尾静脉给药(法舒地尔浓度50μg/100μL)。通过大体观察测量、鼠脑冰冻切片和绘制生存曲线等方式检测法舒地尔对人脑胶质瘤体内生长的作用。结果显示,法舒地尔能有效抑制人脑胶质瘤在裸鼠体内的生长和对周围组织的侵袭,接受治疗的裸鼠生存时间较对照组有显著延长。结论在本研究中,我们首先发现并确认Fasudil能够引起人脑胶质瘤细胞的形态学改变,并能降低T98G和U251细胞的迁移和侵袭。但Fasudil对肿瘤细胞的迁移和侵袭抑制作用的强弱并不一致,这种差异引导本课题进行了进一步的研究,即Fasudil对胶质瘤细胞的迁移和侵袭抑制的机制研究。通过一系列的实验我们发现,在常见的肿瘤侵袭相关基因中(MMPs和TIMPs),只有MMP-2的表达能够被Fasudil抑制,这一结果同样在其他的ROCK抑制剂和ROCK-siRNA的阳性对照实验中得到了验证。我们又发现,Fasudil能够明显促进胶质瘤细胞的凋亡,并在高浓度条件下抑制其增殖。通过冰冻切片和生存期曲线等体内实验进一步证明,使用Fasudil阻断ROCK能在体外和体内抑制人脑胶质瘤的恶性进展。因此,我们得到以下结论：Fasudil通过抑制细胞侵袭和促进凋亡降低了人脑胶质瘤的恶性特征; Rho/ROCK信号通路是治疗人脑胶质瘤的有效靶点；对其机制的深入研究有助于发现Fasudil的潜在药理作用,并有可能作为一种已应用于临床的药物成为胶质瘤化学治疗候选药物之一。更多还原

【Abstract】 IntroductionGlioma has the highest incidence among human intracranial tumors, and glioblastoma (GBM) is the most frequent and malignant histological type. Despite aggressive surgical resection, radiation, and chemo-therapy, the prognosis of patients remains poor. Therefore, more effective therapeutic methods are urgently required for the treatment of GBM. According to existing research, the hyperproliferation and inva-siveness of GBM cells are the major reasons for treatment failure and tumor recurrence.Although it is rarely metastasized systemically, GBM cells infiltrate contiguous and distant regions of the brain. In order to invade foreign tissue, a neoplastic cell must interrupt its adhesion with surrounding cells or tissue, cross the basal membrane, migrate through the extracellular matrix, proliferate, and generate its own capillary network. Matrix metalloproteinases (MMPs), such as collagenases, stromelysins, and gelatinases, belong to a larger family of proteases known as the metzincin superfamily. They are capable of degrading all kinds of extracellular matrix proteins. Recently, a growing number of evidence showed that MMPs play an important role in tumor invasion and migration. In particular, two gelatinases, MMP-2 and MMP-9, have attracted the most attention in terms of glioma invasion.Rho, a small GTP-binding protein, has various effects on cellular functions, including regulating the formation of contractile actin-myosin filaments, which form stress fibers and maintain focal adhesions at the rear of the cell. One important Rho target is Ser/Thr kinase, Rho-kinase (ROCK). Studies have shown that the expression of Rho/ROCK increases in GBM cell lines, T98G and U251, and correlates positively with the degree of malignancy in astrocytomas. The ROCK inhibitor, Y27632, suppresses the migration and proliferation of GBM cells. However, for the mechanism of Rho/ ROCK-dependent migration in tumor biology, previous reports mainly focused on the changes in cytoskeletal structure.Fasudil [1-(5-isoquinolinesulfonyl)-homopiperazine] is a ROCK inhibitor which has been proved to modify myosin light chain phosphorylation in smooth muscle cells. It has been used for the clinical treatment of cerebral vasospasms that occur after subarachnoid hemorrhage and associated cerebral ischemic symptoms. In addition, it is reported that fasudil may be useful for cardiovascular diseases, including angina pectoris, hypertension, pulmonary hypertension, stroke, and heart failure. However, the effect of fasudil treatment for GBM has not been investigated.To determine the effect of fasudil on GBM cells and reveal its underlying mechanisms, two human GBM cell lines, T98G and U251, were treated with fasudil at different doses. The biological properties of these tumor cells, including their morphology, proliferation, invasion, and apoptosis, were also studied. Assuming that MMPs play a critical role in tumor invasion and migration, the regulatory effect of fasudil on MMPs and their negative regulator tissue inhibitor of metalloproteinases (TIMPs) were investigated. The intra-cranial xenograft models were established. Cryosection of the tumor and survival curve analysis were used to determine the effect of fasudil on GBM in vivo.Methods and Results Part 11. Effect of fasudil on the morphological changes of GBM cellsThe T98G and U251 cells treated with various concentrations of fasudil in 24 h lost their original polygonal morphology and adopted more of a stellate morphology with an increasing number of cell processes. The cell body became thinner, and the processes presented as filopodia. These changes in GBM cells occurred in a time-and dose-dependent manner. At a higher concentration of fasudil (100μM), the cell showed complete loss of normal morphology, and the processes increased several times the length of the cell body.To determine if the effects of fasudil on GBM cell morphology were reversible, a recovery experiment was performed. It indicated that GBM cells treated with 5μM fasudil completely regained their original polygonal morphology, similar to what was observed in untreated control cells. However, cells treated at higher doses (20 and 100μM) after 24 h incubation in DMEM medium with 10% FBS cannot completely recover their normal morphology. These results indicated that the effects of fasudil are reversible at a lower dose after 24 h of fasudil treatment and suggested that high concentrations of fasudil may have a toxic effect on T98G and U251 cells.2. Effect of fasudil on migration and invasion of GBM cells2.1 Fasudil inhibits the motility of GBM cellsThe wound healing assay was used to assess the effects of fasudil on the migration capacity of GBM cells. The sites of the wound line were photographed immediately after scratching and 12 h later. The width of the wound line was measured, and the migration distances were calculated relative to the control group. According to our data, fasudil inhibited the motility of T98G and U251 in a dose-dependent manner. The inhibition effect of fasudil on GBM cell migration was dose-dependent. Although there was no difference in the low-dose group compared to the non-treated controls, a higher concentration of fasudil (20μM) inhibited T98G migration by at least 30%, while 100μM fasudil robustly inhibited migration by 50%. Similar outcomes were obtained in the migration of U251. 2.2 Fasudil inhibits invasion of GBM cellsThe matrigel transwell assay was conducted to determine the effect of fasudil on the invasion of GBM cells. After 24 h of invasion, the images of the cells affixed on the lower side of the membrane were taken and the cells were counted. All the groups were presented as the percentage of the control. After the cells were treated with various concentrations of fasudil, a dose-dependent effect of fasudil was observed on tumor cell invasion ability. Interestingly, unlike the migration assay, even the low-dose fasudil (5μM) repressed cell invasion. This inhibition was much more significant in high-dose groups. Thus, although low-dose fasudil had no effect on cell migration, it did inhibit cell invasion, indicating that migration inhibition may not be sufficient to explain how fasudil represses GBM invasion.3. Effect of fasudil on MMP-2 expression of GBM cells3.1 Effect of fasudil on mRNA expression of invasion-related genesTo determine the effect of fasudil on invasion-related gene expression, RT-PCR experiments were performed. After treat-ment with 20μM fasudil and with ROCK siRNA for 24 h to serve as positive controls, the total mRNA of T98G and U251 was extracted. Since MMPs and TIMPs are involved in the invasion and metastasis of tumor cells and ECM degradation is mainly mediated by the balance between them, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were detected using RT-PCR. The MMP-2 mRNA levels of T98G treated with fasudil and ROCK siRNA were apparently down-regulated by 50% compared to the untreated group. There was no significant difference in MMP-9, TIMP-1, TIMP-2, and TIMP-3 between the treated and untreated groups. U251 had similar results. Gelatin zymogram further confirmed that only MMP-2, not MMP-9, may be responsible for fasudil bioavailability in repressing GBM invasion.3.2 Fasudil inhibits MMP-2 expression of GBM cells To further confirm that fasudil inhibited MMP-2 expression, RT-PCR, western-blot analysis, and gelatin zymogram were performed. Exposure to fasudil for 24 h significantly de-creased the MMP-2 mRNA level. Detection of the corresponding proteins by western-blot and gelatin zymogram confirmed this finding. Quantitations of the active forms of MMP-2 were suppressed in a dose-dependent manner by fasudil treatment. A gelatin zymogram detecting MMP-2 in the supernatant of cultured cells and directly reflecting MMP-2 expression of T98G and U251 showed consistent results concerning the marked dose-dependent inhibition effect of fasudil. Various concentrations of fasudil down-regulated MMP-2 levels by 15.3,41.4, and 46.5% compared to the control in T98G, and by 11.7,16, and 32.4% compared to the control in U251, respectively, for 5,20,100μM. These results suggest that fasudil has a dose-dependent inhibition effect on MMP-2 expression of GBM cells.4. Effect of fasudil on cell proliferation and apoptosis of GBM cells Fasudil inhibits cell proliferation of GBM cellsTo examine the effect of fasudil on the growth of GBM cells, [3H]-thymidine incorporation was performed. According to the results, fasudil contributed to potential growth inhibition in the T98G and U251 cell lines in a dose-dependent manner. At higher concentrations (20 and 100μM), fasudil suppressed cell proliferation by 65.7 and 76.7% compared to the control in T98G and by 65.6 and 70.2% compared to the control in U251, respectively.Fasudil induces cell apoptosis of GBM cellsSince the Rho/ROCK pathway involves diverse cellular responses, including cell growth, differentiation, and survival, the ability of the fasudil to induce apoptosis was therefore examined. An annexin V/PI staining assay was used for the T98G and U251 cells. Y27632 and ROCK siRNA were used as positive controls, which remarkably induced cell apoptosis. At higher concentrations (20 and 100μM), fasudil markedly induced apoptosis of both GBM cell lines in a dose-dependent manner compared to the control group and the low-dose group. There was a 7.1-fold increased induction at 20μM and a 9.75-fold increased induction at 100μM for T98G, and a 6.5-fold induction at 20μM and 10.45-fold induction at 100μM for U251.Part 2Fasudil suppresses GBM growth and invasion in vivoTo evaluate whether fasudil inhibits GBM progression in vivo, eGFP-T98G cells were inoculated into the left caudate putamen of the nude mice. When the tumors were established, the mice were treated with fasudil (50μg/100μL PBS via tail vein) or PBS (100μL) as control. After the 14th day of administration, the animals were sacrificed and brain specimens were cryosectioned into 25μM thick sections in the coronal plane. When the eGFP-T98G glioma cells were inoculated into the caudate-putamen, GFP-expressing tumor cells were found to have infiltrated the surrounding brain tissue and most of the cerebral cortex. In contrast, the demarcation between the tumor and the surrounding tissues was clear in the fasudil treatment. The Kaplan-Meier survival curve and log rank test analysis revealed statistically significant prolongation of survival in the group receiving fasudil (median survival time,42 d) compared with the control group receiving only PBS (median survival time,35 d; log-rank test, P＜0.05). These results briefly indicate that fasudil could inhibit glioma invasion and growth in vivo.ConclusionIn the present study, we first confirmed that fasudil markedly induced the morphological changes and down-regulated the migration and invasion capability of T98G and U251. The intriguing difference caused by fasudil on migration and invasion led us to further investi- gate the underlying mechanism of how fasudil regulates GBM invasion. Then, we found that, among the common invasion-related genes (i.e., MMPs and TIMPs), MMP-2 expression was suppressed by ROCK inhibitors. In addition, the inhibitor significantly increased the rate of apoptosis and inhibited the prolife-ration of GBM cells. The cryosection of tumor and the survival time of mice further suggested that fasudil, as a well-known ROCK inhibitor, suppresses the progression of GBM cell lines in vitro and in vivo by inhibiting ROCK. In conclusion, our results demonstrated that fasudil treatment suppressed GBM progression by inhibiting invasion and inducing apoptosis in vitro and in vivo. The Rho/ROCK signaling pathway is a promising target for GBM treatment. Further research of the mechanism can lead to the discovery of the underlying pharmacological action of fasudil. The fact that fasudil is approved for human use and is tolerated without serious adverse reactions makes it an attractive anti-tumor drug candidate for the treatment of GBM.更多还原