大鼠脑内C6胶质瘤模型的建立

目的建立稳定可靠的大鼠脑内C6胶质瘤模型。方法取20只健康成年SD大鼠,用立体定向技术将含1×106个C6胶质瘤细胞的悬液15μl缓慢接种于大鼠左侧尾状核区;接种后3周时随机挑选其中10只大鼠麻醉后行MRI检查,观察肿瘤影像学表现,随后处死大鼠取出肿瘤组织,行免疫组化及HE染色检测肿瘤生长情况;另外9只(麻醉死亡1只)大鼠继续饲养直至死亡,观察其生存时间。结果1只接种肿瘤细胞后出现短暂体重下降,但未出现明显神经系统体征,饲养60 d后尸检未见肿瘤生长;其余18只大鼠颅内均有肿瘤生长且无颅外转移现象,病理学检查结果显示脑内肿瘤呈浸润生长,可见新生血管,胶质纤维酸性蛋白表达阳性。荷瘤鼠生存时间为25~47 d,平均(32.78±2.34)d。结论采用立体定向方法向大鼠尾状核区种植C6细胞可获得较为稳定、可靠的胶质瘤动物模型,能够满足胶质瘤动物实验研究的需要。     

Experimental brain tumor in adult mongrel cat

Small animal models such as the rat have serious limitations for multiple human scale instrumentation, surgical manipulations, and computerized tomographic (CT) evaluations, so that large animal models are required for the study using them. Although brain tumors induced with Rous sarcoma virus in neonatal beagle or adult monkey had been reported, these animals are very expensive ones for tumor research. A major drawback of virally induced brain tumor model is, moreover, the need for specialized viral facilities and safety precautions for laboratory personnel. In this paper, a cat glioma model implanted with C6 glioma cells derived from rats injected with N-nitrosomethylurea is reported. For an implantation dose of 5 x 10(5) cells/50 microliters, C6 glioma cells were suspended in modified Eagle medium supplemented with 10% fetal bovine serum and 0.5% agar. Twenty adult mongrel cats were injected with 5 x 10(5) C6 glioma cells intracerebrally. Implanted cats had brain tumors of about 10 mm in diameter with a yield of 80%. The mean survival was about 3 weeks after implantation. Tumors developed as spheroidal, hemorrhagic masses with central areas of necrosis and peripheral edema. They were located within the parenchyma of the implanted region. This tumor possessed many of the histological and radiological characteristics of human glioblastoma such as the following: Areas of hemorrhage and necrosis surrounded by pseudopallisading were observed within the tumor consisting of spindle-shaped cells with pleomorphic nuclei. A mass lesion with ring or garland-like enhancement surrounded by brain edema was shown on the CT scans.(ABSTRACT TRUNCATED AT 250 WORDS)     

三种鼠脑胶质瘤模型的建立与比较

目的 ;建立GL261胶质瘤细胞C57BL/6小鼠、C6胶质瘤细胞SD大鼠及BALB/c小鼠皮下动物模型,比较其肿瘤生长特点。方法借助动物立体定向仪,将体外培养小鼠GL261、大鼠C6胶质瘤细胞分别接种于C57BL/6小鼠及SD大鼠右侧尾状核区,C6胶质瘤细胞接种于BALB/c小鼠左前肢皮下。接种后观察不同种实验鼠的生存状态及肿瘤的生长特性,颅内模型于接种后7d、14d、21d、28d进行MRI检查,皮下模型测量体积,并绘制生长曲线。解剖标本,做组织病理学和胶质纤维酸性蛋(GFAP)免疫组化检查。结果 GL261胶质瘤细胞C57BL/6小鼠模型较之后两种模型在组织病理学上接近人脑胶质瘤,而且颅内生长稳定,成瘤率高,未见颅外转移病灶,实验周期短,重复性好。结论 GL261胶质瘤细胞C57BL/6小鼠模型,其肿瘤生长特性及病理特征与人脑胶质瘤相似,可作为临床胶质瘤基础研究的理想模型。     

Inhibition of H-ras as a treatment for experimental brain C6 glioma

The oncogene H-ras plays an important role in tumor growth and maintenance and could serve as a target treatment for brain tumors. In this study, diallyl disulfide (DADS), an inhibitor of H-ras was used to treat experimental brain glioma in a rat model. One hundred and twenty-five Sprague-Dawley rats (175-200 g) were implanted with 2 x 10(5) C6 glioma cells into the intra striatal region of the brain. Animals were treated with DADS (33 micromol) either before or after implantation of tumor cells. Control animals received soybean oil. Treatment outcome was evaluated based on H-ras expression in tumor tissue, animal's neurological status, tumor size, and life span. Application of DADS 7 days before implantation of tumor cells reduced the tumor size (P<0.05), improved neurological status (P<0.05), and increased the animal life span (P<0.05) when compared to the control group (no treatment). The expression of H-ras was significantly (P<0.05) reduced in brain tumor tissue of animals treated with DADS before implantation. Application of DADS after tumor implantation failed to improve clinical status or life span. This study demonstrates that pretreatment with DADS is capable of inhibiting the expression of H-ras in experimental brain C6 glioma which leads to an improved neurological status and an extended life span in the rat. Higher doses of DADS or other more potent inhibitors need to be used after tumor has been implanted.     

Spontaneous regression of experimental gliomas--an immunohistochemical and MRI study of the C6 glioma spheroid implantation model

OBJECTIVE: The orthotopic C6 glioma spheroid implantation model has been used to examine factors of neoangiogenesis, growth factor release, and protease expression as well the effect of antitumor agents. The present study systematically investigates the long-term course of orthotopically implanted C6 spheroid gliomas. METHODS: Reaggregated C6 spheroid tumors were implanted into the forebrain of 48 male Sprague-Dawley rats (32 immunocompetent, 16 thymectomized). The animals were examined by MRI at postoperative day (POD) 7, 14, 21, 28, 32, 45, 60, and 70. The MRI protocol included a T2-w and T1-w SE sequence before and after application of contrast medium and a CISS 3D sequence for volumetry. A total of six animals were selected after each MR exam from both groups and sacrificed for HE light microscopy and CD8+ T-lymphocyte, ED1+ macrophage, CD31+ endothelial cell immunohistochemistry. RESULTS: The tumors progressed to reach a maximum volume on day 28: 0.23 +/- 0.05 ml in the thymectomized and 0.16 +/- 0.021 ml in the immunocompetent group. Tumors then consistently regressed to vanish completely by POD 70. The influx of cytotoxic CD8+ T-lymphocytes correlated with tumor progression and the tumors reached a larger size in the thymectomized group. However, the time course of tumor regression was the same for both groups. CONCLUSION: The present data suggest that the orthotopic C6 glioma implanted into Sprague-Dawley rats will progress within a time span of approximately 4 weeks and can then retrogress again spontaneously. This finding has to be taken into account when deciding on a study protocol and the appropriate animal model. The C6 glioma model may be suitable to study the cell biological steps involved in the phenomenon of spontaneous tumor regression.     

鼠脑胶质瘤模型的建立

目的：建立可重复性大鼠脑胶质瘤模型。方法：雄性ＳＤ大鼠３０只，体重１５０～２５０克，分为脑胶质瘤组（２０只）和对照组（１０只）。于前囟前１ｍｍ、矢状缝右旁开３ｍｍ处用牙钻钻一骨孔。用静脉注射套管针立体定向注射１０ｕｌ含前约１０＾５个Ｃ６细胞的混悬液于鼠脑右侧层状核内，建立鼠脑胶质瘤模型。对照组手术过程同上，但只注射１０ｕｌ细胞培养基，两组大鼠术后第１６、２５天分别作ＭＲＩ测量胶质瘤大小、位置，术后第２５天处死大鼠，立刻取脑，制作病理切片，观察组织形态学变化，脑胶质瘤组的石蜡切片经免疫组织化学染色检测Ｓ－１００蛋白，结果：脑胶质瘤组，２０只大鼠经ＭＲＩ平扫，增强扫描和形态学检查证实均前单灶肿块，且肿瘤部位、大小、形态基本一致。无脑外转移和颅外扩散，经免疫组织化学染色检测Ｓ－１００蛋白均为阳性，对照组均无瘤体。结论     

Establishment of experimental glioma models at the intrinsic brainstem region of the rats

OBJECTIVES: As the treatment of human intrinsic brainstem gliomas remains challenging, experimental glioma models are needed. METHODS: We developed a rat model of intrinsic brain stem glioma that uses a stereotactic frame to fix the head for the delivery of C6 glioma cells to target sites via a permanently implanted cannula. We inoculated the rat midbrain, pons or cerebral cortex with 5 x 10(4) cells suspended in 1 microl culture medium over the course of 2 minutes. RESULTS: Three days post-implantation, tumor formation was visible in the periaqueductal gray matter in the midbrain and the tegmentum of the pons. On the tenth day, the tumor diameter exceeded over 2 mm; there was no tumor cell seeding into the cerebrospinal fluid space. The tumor manifested the histological features typical of glioblastoma; Ki-67 labeling index was 32%. DISCUSSION: Because in our model the cannula is permanently implanted, additional inocula can be delivered. Here we detail our rat brainstem glioma model and discuss its usefulness for the investigation of these tumor in humans.     

血管内皮生长因子抗体对胶质瘤细胞凋亡的影响

目的 观察血管内皮生长因子(VEGF)抗体对大鼠C6胶质瘤细胞凋亡的影响及对肿瘤生长的抑制作用。方法 皮下接种建立异位大鼠C6胶质瘤模型，实验组每3d局部应用VEGF抗体，对照组局部应用PBS溶液，每5d量取肿瘤的长径和宽径，15d后处死动物，称取肿瘤湿重，肿瘤组织行H—E及TUNEL法原位凋亡检测。结果 实验组动物肿瘤生长速度明显减慢，光镜下观察实验组肿瘤标本见到较多的坏死灶，TUNEL法原位凋亡检测发现实验组肿瘤组织内肿瘤细胞凋亡。结论 应用VEGF抗体后大鼠C6胶质瘤生长速度减慢，肿瘤细胞发生凋亡。抗血管形成疗法不仅能使肿瘤坏死增加，而且可以诱导肿瘤细胞凋亡。     

CDglyTK双自杀基因杀伤C6胶质瘤细胞的体内实验研究

目的 观察CDgtyTK双自杀基因对C6实体胶质瘤生长的抑制作用. 方法 利用逆转录病毒介导的大肠杆菌胞嘧啶脱氨酶(CD)和单纯疱疹病毒胸苷激酶(HSV-TK)融合基因转染小鼠C6实体胶质瘤,RT-PCR检测分析融合基因的表达,并观察对照组和治疗组肿瘤体积、重量、抑瘤牢及生存期的变化,同时观察其对C6胶质瘤细胞凋亡的影响,分析CDglyTK双自杀基因系统对肿瘤的抑制作用. 结果 RT-PCR检测显示逆转录病毒介导的COgtyTK双自杀基因在C6胶质瘤中有表达.对照组第4周肿瘤已长至20 mmx30 mm大小.并出现小鼠死亡现象,至第6周末全部死亡;治疗组肿瘤体积未增长,约5 mm大小.部分肿瘤消失.肉眼观察可见对照组肿瘤体积大,色红,血供丰富,治疗组肿瘤体积减小或消失.21 d后处死取瘤称重,对照组[(2.51±0.58)g]与治疗组肿瘤质量[(0.35±0.26)g]比较差异有统计学意义(P<0.05),治疗组抑瘤牢为86.1%.流式细胞仪检测可见治疗组细胞凋亡率(34.41%±5.20%)明显高于对照组(2.92%±1.30%),差异有统计学意义(P<0.05).电镜观察可见治疗组肿瘤细胞凋亡小体形成. 结论 效 CDglyTK双自杀基因联合双前药治疗能取得显著的抗胶质瘤作用.     

小鼠神经干细胞对胶质瘤趋向性实验研究

目的 探讨小鼠神经干细胞（NSCs）在体外和体内对胶质瘤细胞的趋向性。方法 将原代培养6d的小鼠NSCs悬液离心、收集备用。制备C6胶质瘤细胞、3T3成纤维细胞爬片，随后与NSCs共同培养3d。用荧光染料Hoeehst33342标记NSCs，借助立体定向技术于胶质瘤动物模型内接种NSCs，观察NSCs在胶质瘤动物模型脑内的分布情况。结果 小鼠NSCs对C6细胞有明显的趋向性。体内实验见肿瘤组织内满布标记的NSCs。结论 小鼠NSCs无论在体外或体内对胶质瘤细胞均有一定的趋向性。     

利用C6胶质瘤干细胞立体定向建立大鼠脑胶质瘤模型

目的利用C6胶质瘤干细胞建立wistar大鼠脑胶质瘤模型,观察肿瘤生长规律及病理特征,探讨利用肿瘤干细胞构建模型的优势。方法体外提取并培养c6细胞系中胶质瘤干细胞,应用立体定向法在鼠脑右侧尾状核区接种10^4个胶质瘤干细胞,术后连续观察大鼠生存状态和生存时间,分时段行鼠脑MRI检查、病理HE切片及GFAP免疫组织化学检查,观察肿瘤生长及病理特征。结果利用本方法建立模型成瘤率为100％,术后大鼠恢复快,术后反应轻,未见颅外转移,生存期稳定,重复性好,肿瘤生长规律、影像学特征及组织病理特点与人脑胶质瘤相似。结论利用胶质瘤干细胞建立的大鼠脑胶质瘤模型稳定可靠,肿瘤生长更贴近胶质瘤在脑内自然发生的特点,且较传统方法脑内注射体积小,模型制作时间短,术后恢复快,成瘤率更高等优点,为研究胶质瘤干细胞在肿瘤形成过程中的生长方式、分子机制及实验性治疗提供了一个较为理想的模型。     

大鼠C6脑胶质瘤模型的病理特征与MRI的观察

目的建立SD大鼠C6胶质瘤模型并对其病理特征及MRI进行观察。方法50只SD大鼠随机分成5组，每组10只，c6胶质瘤细胞悬液立体定向接种于大鼠的右侧尾状核，接种后观察大鼠的生活状态、生存期；分别于接种后不同时段进行MRI观察肿瘤生长特性及肿瘤体积的测量；取不同时段组大鼠脑标本行脑组织HE染色、透射电镜(transmission electron microscope，TEM)、脑组织含水量测量(与10只正常大鼠对照)、胶质纤维酸性蛋白(GFAP)免疫组化检查。结果立体定向颅内接种成功率97．5％，未见远处及颅外转移，肿瘤在一定时期内牛长较快，脑水肿随肿瘤的生长明显加重，生存期观察组中7只荷瘤鼠死亡，3只肿瘤自发部分消退。结论立体定向建立大鼠C6胶质瘤模型成功率高，接种后颅内肿瘤呈浸润性生长，与人脑胶质瘤具有相似性，由于生存期观察组中有部分荷瘤鼠出现肿瘤自发部分消退，故应用该模型评价治疗效果时应慎重；TIWI增强扫描可清晰显示肿瘤影像，且能更早发现肿瘤；MRI联合病理可较好反映肿瘤生长方式及发展过程。     

大鼠脑干胶质瘤模型的建立及MRI特点

目的 建立大鼠脑干胶质瘤模型,并研究成瘤鼠运动功能及胶质瘤MRI成像特点.方法 采用大鼠脑立体定向仪,将C6胶质瘤细胞接种至SD大鼠脑干内.MRI动态观察肿瘤生长情况,并观察接种C6胶质瘤细胞后大鼠的运动能力、生存周期等.结果 C6胶质瘤细胞接种成功率为100%.生存分析表明:大鼠于接种后第16～22天死亡.接种后第7天,MRI即可检出肿瘤生长,荷瘤鼠的运动能力随着成瘤时间的推移逐渐减弱.结论 大鼠脑干胶质瘤模型成瘤率高,有良好的可重复性和可预测性.MRI观察该模型具有脑干胶质瘤成像的特点,细胞接种后10～18 d是最佳观测期.     

大鼠脑干胶质瘤模型的建立

目的 建立大鼠脑干胶质瘤模型,为脑干胶质瘤的研究提供动物实验平台.方法 将1×10~6个大鼠C6胶质瘤细胞通过立体定向头架注射到大鼠脑桥,种植后2周对大鼠行磁共振扫描观察,然后行灌流固定取脑,HE染色.结果 磁共振检查均发现肿瘤生长,肿瘤为长T1和T2信号,经大鼠舌静脉注射Gd-DTPA信号明显增强.HE染色显示肿瘤为胶质母细胞瘤,呈浸润性生长,瘤内新生血管丰富,假栅栏样坏死明显.结论 大鼠C6胶质瘤细胞株可以建立脑干胶质瘤模型,成瘤率高,重复性好.     

Standardization and detailed characterization of the syngeneic Fischer/F98 glioma model

INTRODUCTION: Adequate animal glioma models are mandatory for the pursuit of preclinical research in neuro-oncology. Many implantation models have been described, but none perfectly emulate human malignant gliomas. This work reports our experience in standardizing, optimizing and characterizing the Fischer/F98 glioma model on the clinical, pathological, radiological and metabolic aspects. MATERIALS AND METHODS: F98 cells were implanted in 70 Fischer rats, varying the quantity of cells and volume of implantation solution, and using a micro-infusion pump to minimize implantation trauma, after adequate coordinates were established. Pathological analysis consisted in hematoxylin and eosin (H&E) staining and immunohistochemistry for GFAP, vimentin, albumin, TGF-bl, TGF-b2, CD3 and CD45. Twelve animals were used for MR imaging at 5, 10, 15 and 20 days. Corresponding MR images were compared with pathological slides. Two animals underwent 18F-FDG and 11C-acetate PET studies for metabolic characterization of the tumors. RESULTS: Implantation with 1 x 10(4) cells produced a median survival of 26 days and a tumor take of 100%. Large infiltrative neoplasms with a necrotic core were seen on H&E. Numerous mitosis, peritumoral infiltrative behavior, and neovascular proliferation were also obvious. GFAP and vimentin staining was positive inside the tumor cells. Albumin staining was observed in the extracellular space around the tumors. CD3 staining was negligible. The MR images correlated the pathologic findings. 18F-FDG uptake was strong in the tumors. CONCLUSION: The standardized model described in this study behaves in a predictable and reproducible fashion, and could be considered for future pre-clinical studies. It adequately mimics the behavior of human malignant astrocytomas.     

大鼠骨髓基质细胞移植治疗胶质瘤的实验研究

目的将Wistar大鼠骨髓基质细胞体外移植入C6胶质瘤大鼠模型,观察其生物学特性及大鼠存活状态。方法建立C6载瘤模型和骨髓基质细胞移植治疗模型;待术后生长至3w和4w时行MRI检测;不同时间段内大鼠脑标本行免疫组化染色。结果骨髓基质细胞移植组模型平均生存时间为4.03w;对照组平均生存时间为3.88w;且其平均肿瘤体积与对照组无明显差异。大鼠体内移植体外培养5月余的骨髓基质细胞未发现肿物生成。骨髓基质细胞包绕在胶质瘤周围,对胶质瘤细胞有定向靶向作用。结论骨髓基质细胞对胶质瘤有定向靶向作用,可以安全的作为生物载体转染目的基因治疗胶质瘤。     

Single doublecortin gene therapy significantly reduces glioma tumor volume

We employed lentivirus‐based doublecortin (DCX), as a glioma suppressor gene therapy in an intracranial glioma tumor xenograft model in nude rats. Single DCX‐expressing lentivirus was directly administered into the tumor on day 8 after U87 tumor cell implantation. DCX treatment significantly reduced U87 glioma tumor volume (～60%) on day 14 after DCX lentivirus injection and significantly improved median survival of tumor‐bearing nude rats. DCX synthesis induced neuronal markers MAP2, TUJ1, and PSA‐NCAM and the glial marker glial fibrillary acidic protein (GFAP) in the implanted U87 glioma tumors. DCX synthesis induced GFAP that colocalized with tubulin in the mitotic stage, inhibited cleavage furrow during cytokinesis, and blocked mitosis in glioma cells. DCX lentivirus infection did not induce apoptosis but significantly inhibited expression of the proliferation marker Ki‐67 and the blood vessel marker von‐Willebrand factor (vWF). U87 and other glioma cells except for brain tumor stem cells (BTSCs) do not express neuronal markers or both neuronal and glial markers. DCX‐synthesizing glioma cells express a phenotype of antiangiogenic BTSC‐like cells with terminal differentiation that causes remission of glioma cells by blocking mitosis via a novel DCX/GFAP pathway. Direct local delivery of lentivirus‐based DCX gene therapy is a potential differentiation‐based therapeutic approach for the treatment of glioma. © 2009 Wiley‐Liss, Inc.     

9L/F344与C6/Wistar大鼠脑胶质瘤模型局部细胞免疫反应的比较研究

目的对9L/F344和C6/Wistar两种大鼠脑胶质瘤模型肿瘤局部细胞免疫反应情况进行比较研究.方法采用立体定向技术建立两种大鼠脑胶质瘤模型.定期处死大鼠取脑,常规石蜡切片,苏木精-伊红染色行肿瘤组织病理学观察;冰冻切片,行免疫组织化学染色,观察大鼠脑内肿瘤淋巴细胞浸润情况.结果两种模型脑内肿瘤组织病理学均具有恶性脑胶质瘤的特点.C6/Wistar模型肿瘤周边和瘤内可见大量单个核细胞浸润,肿瘤周边血管呈现"淋巴细胞血管套"现象.肿瘤局部CD68、CD4、CD8阳性的淋巴细胞在C6/Wistar模型中多见;9L/F344模型中可见CD68阳性细胞,但数量较C6模型中为少,P＜0.01,且未见CD4、CD8阳性细胞.结论9L/F344模型肿瘤局部T淋巴细胞浸润少.C6/Wistar模型肿瘤周边及瘤内淋巴细胞浸润明显,存在强烈的细胞免疫反应,不适用于胶质瘤免疫治疗的实验研究.     

Cyclin D1/D2/D3在大鼠脑胶质瘤组织中的表达

目的 探讨cyclin D1/D2/D3在大鼠脑胶质瘤组织中的表达.方法 将体外培养的大鼠C6胶质瘤细胞(细胞数为3×105个)借助动物立体定向仪接种于Wistar大鼠左侧尾状核区,解剖标本,行组织病理学检查及GFAP、cyclin D1/D2/D3免疫组化检测.结果 cyclinD1/D2/D3均呈阳性表达.cyclin D1/D3主要在细胞胞核表达,cyclin D2主要在胞浆表达,部分在胞核表达,不同存活期荷瘤鼠瘤组织中cyclin D1/D2/D3表达量不同.结论 cyclinD1/D2/D3过分表达参与调节大鼠脑胶质瘤增殖.荷瘤鼠生存期与cyclin D家族蛋白的表达呈负相关关系.