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Development of gene therapy for schwannomas associated with neurofibromatosis type 2

2 型神经纤维瘤相关神经鞘瘤基因疗法的开发

基本信息

批准号：
12671351
负责人：
SAITO Kiyoshi
金额：
$ 1.98万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671351/
关键词：
Neurofibromatosis type 1 Schwannoma Gene therapy VEGF Neuroendoscope PPARγ

项目摘要

Schwannomas associated with neurofibromatosis type 2 (NF2) are difficult to treat because they are multiple and tend to recur. We performed basic researches to develop gene therapy for schwannomas with NF2.Surgically resected sporadic or NF2-associated schwannomas were primarily cultured. Cultured cells were positive for S-100 immunostaining. B- galactosidase gene was transfected into cultured cells using adenovirus vector. More than 50% cells expressed the gene at MOI = 1, and almost 100% cells at MOI = 10.Vascular endothelial growth factor (VEGF) has been reported to act as a survival factor for Schwann cells. We therefore investigated VEGF expression. VEGF immunostaining was present in sporadic and NF2-associated schwannomas. VEGF mRNA was detected in all 4 schwannomas tested from the NF2 group and in 1 of 4 sporadic schwannomas. MIB-1 labeling index and microvascular density were higher in the NF2 than the sporadic group. VEGF was also detected in the culture medium of schwannoma cells at the concentration of 100〜1180 pg/ml.These results suggested that VEGF might be a factor affecting proliferative potential of schwannomas. Then, gene therapy to bloc the function of VEGF was planned. Adenovirus vector expressing excess extracellular domain of Flt-1 (soluble VEGF receptor) was prepared.Expression of peroxisome proliferatior-activated receptorγ(PPARγ) was confirmed in gliomas. Administration of its ligand suppressed the growth of glioma cell lines. New mechanism to inhibit the tumor growth was suggested.For injection of gene into multiple cranial or spinal schwannomas in NF2 patients, neuroendoscope must be utilized. We performed preliminary research using dogs. After suboccipital craniotomy or lumber laminectomy, spinal subarachnoid space was investigated using a fine neuroendoscope. Although spinal nerves were observed, injection was difficult. Further slender neuroendoscope with a channel for drug injection needs to be designed.

与神经纤维瘤病2型（NF 2）相关的神经鞘瘤很难治疗，因为它们是多发性的，而且容易复发。本研究为NF 2相关神经鞘瘤的基因治疗做了基础研究。培养细胞S-100免疫组化染色阳性。用腺病毒载体将B-半乳糖苷酶基因转染到培养的细胞中。当MOI = 1时，超过50%的细胞表达该基因，当MOI = 10时，几乎100%的细胞表达该基因。因此，我们研究了VEGF的表达。VEGF免疫染色存在于散发性和NF 2相关神经鞘瘤。VEGF mRNA在NF 2组的所有4个神经鞘瘤和4个散发性神经鞘瘤中的1个中检测到。NF 2中的MIB-1标记指数和微血管密度高于散发组。在神经鞘瘤细胞培养液中也检测到VEGF的表达，浓度为100 ~ 1180 pg/ml，提示VEGF可能是影响神经鞘瘤增殖能力的一个因素。然后，计划基因治疗以阻断VEGF的功能。制备了表达Flt-1胞外区（可溶性VEGF受体）的腺病毒载体，证实了过氧化物酶体增殖物激活受体γ（PPARγ）在胶质瘤中的表达。其配体的施用抑制胶质瘤细胞系的生长。对多发性神经鞘瘤NF 2患者进行基因注射治疗时，必须借助神经内镜。我们用狗做了初步研究。经枕下开颅或椎板切除术后，使用精细神经内窥镜检查脊髓蛛网膜下腔。虽然观察到脊神经，但注射困难。需要设计具有用于药物注射的通道的进一步细长的神经内窥镜。