Development of gene therapy for schwannomas associated with neurofibromatosis type 2

与2型神经纤维瘤病(NF2)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。我们开展了NF2基因治疗神经鞘瘤的基础研究，手术切除的散发性或NF2相关的神经鞘瘤进行了原代培养。培养细胞S-100免疫染色阳性。用腺病毒载体将β-半乳糖苷酶基因导入培养细胞。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。血管内皮生长因子(VEGF)已被报道为雪旺细胞的生存因子。因此，我们研究了血管内皮生长因子的表达。散发性神经鞘瘤和NF2相关神经鞘瘤中均可见血管内皮生长因子免疫染色。NF2组检测的4例神经鞘瘤和1例散发性神经鞘瘤均可检测到VEGFmRNA。NF2组MIB-1标记指数和微血管密度均高于散发性组。神经鞘瘤细胞培养上清液中也检测到100~1180pg/ml的血管内皮生长因子，提示血管内皮生长因子可能是影响神经鞘瘤细胞增殖潜能的一个因素。然后，计划进行基因治疗来阻断血管内皮生长因子的功能。制备了表达过量Flt-1胞外区(可溶性血管内皮生长因子受体)的腺病毒载体，证实其在胶质瘤中表达了过氧化体增殖物激活受体γ(PPARγ)。给予其配体可抑制胶质瘤细胞系的生长。提出了抑制肿瘤生长的新机制。对于NF2患者的多发性颅脑或脊髓神经鞘瘤，必须使用神经内窥镜进行基因注射。我们用狗进行了初步研究。枕下开颅或腰椎板切除术后，使用精细神经内窥镜检查脊柱蛛网膜下腔。虽然观察到脊神经，但注射困难。此外，还需要设计具有注射药物通道的细长神经内窥镜。

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.