Inhibition of glioma angiogenesis and invasion by anti-microtubule agent in a rat brain tumor model.

主要完成了以下两个方面的研究。(1)目的：通过定量示踪增强型绿色荧光蛋白(EGFP)标记的人脑恶性胶质瘤细胞系，分析抗基质金属蛋白酶(抗基质金属蛋白酶)药物SI-27的抗侵袭作用。方法：利用pEGFP-C1载体，在人恶性胶质瘤细胞克隆(U87 MG、U251 MG和U373 MG)中建立绿色荧光蛋白的持续表达。将1微升Matrigel中的肿瘤球体植入严重联合免疫缺陷小鼠脑片的尾状核-壳核内。为了定量评估肿瘤细胞的侵袭性，在不同浓度的SI-27(0、1、10、50或100微g/ml)存在的情况下，在第1、3、5和7天用荧光立体显微镜和图像分析仪测量侵袭面积指数。结果：对照组(0微克/毫升)所有胶质瘤细胞系均呈指状侵袭，并到达对侧…。更多的是通过老茧体来观察。SI-27浓度为10、50和100微g/ml时，对第5、7天的侵袭面积指数有明显的抑制作用，且呈剂量依赖关系，而1微g/ml的SI-27对侵袭面积指数无明显影响。透射电子显微镜和激光共聚焦显微镜显示肿瘤细胞已穿透脑片，脑内正常结构的完整性一直维持到第7天。结论：该模型能够对正常脑组织中单个侵袭性肿瘤细胞进行明确的周期性追踪。非细胞毒性浓度的SI-27对脑胶质瘤细胞的侵袭有明显的抑制作用，提示抗基质金属蛋白酶治疗可能是未来脑恶性肿瘤治疗的重要策略。(2)目的：基质金属蛋白酶抑制剂SI-27因具有抗脑胶质瘤侵袭和血管生成的作用而得到广泛的发展。然而，之前的研究都是在体外进行的。本工作研究了SI-27在啮齿动物脑瘤模型中抑制肿瘤侵袭、减缓血管生成和延长生存的潜力。方法：将稳定表达增强型绿色荧光蛋白的人脑胶质瘤细胞系U251 MG经环孢菌素A、SI-27(1 mg/kg或10 mg/kg)免疫抑制后，立体定向异种移植到Fischer 944大鼠的前纹状体和胼胝体周围，连续3d腹腔注射SI-27或载体液，3周后用定量图像分析检测肿瘤侵袭和血管生成情况。这是在通过直接观察增强的绿色荧光蛋白表达的胶质瘤细胞或通过附加免疫组织化学方法检测内皮细胞的全脑切片上进行的，冰冻切片上的原位酶谱用于检测基质金属蛋白酶的活性。结果：与接受携带者的对照组相比，共接受30 mg/kg剂量组的存活时间显著延长(P&lt；0.001)(中位生存期分别为47.3d和32.6d)。此外，基质金属蛋白酶活性、肿瘤细胞侵袭和新生血管也有所减少。相比之下，给予3毫克/公斤的动物并没有表现出这些差异。结论：全身应用抗MMP剂SI-27对脑胶质瘤动物模型的治疗是有效的。较少

We mainly achieved the following two investigations.(1)OBJECTIVE : We aimed to analyze the anti-invasive effect of the anti-matrix metalloproteinase (anti-MMP) agent SI-27 by quantitative tracking of enhanced green fluorescent protein(EGFP)-labeled human malignant glioma cell lines in a brain slice model. METHODS : Persistent expression of EGFP in human malignant glioma cell clones (U87MG, U251MG, and U373MG) was established with the use of the pEGFP-C1 vector. Tumor spheroid in 1 microl Matrigel was implanted into the caudate nucleus-putamen of a severe combined immunodeficient mouse brain slice. To allow the quantitative assessment of tumor cell invasion, the invasion area index was measured on Days 1,3,5,and 7 with a fluorescence stereomicroscope and an image analyzer in the presence of various concentrations of SI-27(0,1,10,50,or 100 microg/ml). RESULTS : In the control group (0 microg/ml), all glioma cell lines invaded in a fingerlike fashion and reached the contralateral hemisphe … More re through the corpus callosum. SI-27 at concentrations of 10,50,and 100 microg/ml significantly suppressed the invasion area index on Days 5 and 7 in a dose-dependent manner, whereas 1 microg/ml had no effect. Transmission electron microscopy and laser confocal microscopy indicated that the tumor cells had penetrated the brain slice and that the normal structural integrity of the brain was maintained until Day 7. CONCLUSION : This model enabled unequivocal periodic tracking of individual invading tumor cells in normal brain. The significant suppression of glioma cell invasion by noncytotoxic concentrations of SI-27 indicates that anti-MMP treatment may represent an important future therapeutic strategy for malignant cerebral neoplasms.(2)OBJECTIVE : The matrix metalloproteinase(MMP) inhibitor SI-27 has undergone extensive development because of its effectiveness against glioma invasion and angiogenesis. However, previous studies have been performed in vitro. The present work investigates the potential of SI-27 to inhibit tumor invasion, slow angiogenesis, and prolong survival in rodent brain tumor models. METHODS: Stable enhanced green fluorescent protein-expressing clones of a human malignant glioma cell line, U251MG, were stereotactically xenografted into the periphery of the anterior striatum and corpus callosum of Fischer 944 rats after immunosuppression with cyclosporin A, SI-27(1 or 10 mg/kg) or carrier solution was administered on three successive days by intraperitoneal injection, and tumor invasion and angiogenesis were assessed 3 weeks later by quantitative image analysis. This was performed on whole brain sections analyzed either by direct observation of enhanced green fluorescent protein-expressing glioma cells or by additional immunohistochemistry to detect the endothelial cells with anti-factor VIII monoclonal antibody In situ zymography on frozen sections was used to detect MMP activity. RESULTS : The group receiving a total of 30 mg/kg showed a statistically significant (P<0.001) increase in survival time compared with the controls receiving carrier (median survival, 47.3 versus 32.6 d). There was also a decrease in MMP activity, tumor cell invasion, and neovascularization. In contrast, animals given 3 mg/kg did not show these differences. CONCLUSION : Systemic administration of the anti-MMP agent SI-27 is effective in the treatment of glioma in an animal model. Less