人体前庭神经鞘瘤的原代培养
Summary
前庭神经鞘瘤（VSS）是雪旺细胞（SC）的原点非恶性肿瘤，与在NF2的肿瘤抑制基因的突变相关联。我们提出一个可重复的，高效的协议，用于原代人类VS细胞培养，允许分子和细胞实验操作和分析，概括人类疾病的异质性。
Abstract
前庭神经鞘瘤（VSS）代表雪旺氏细胞的前庭神经（SC）的肿瘤，危及所有颅内肿瘤的10％。 VSS发生在任何偶发或家族（神经纤维瘤病2型，NF2）形式，既在NF2肿瘤抑制基因失活的缺陷有关 基因。治疗VSS通常是手术切除或放射治疗，但此类程序的发病率，带动调查微创治疗。从历史上看，缺乏新鲜组织标本，事实上，神经鞘瘤细胞不会永生都显著阻碍了利用原代培养的神经鞘瘤肿瘤发生的研究。为了克服原代培养的供应有限，是由转导与HPV E6和E7癌基因产生的永生HEI193 VS细胞系。这种致癌转导介绍显著分子和表型改变的细胞，从而限制了其作为一种模式为人类的使用chwannoma肿瘤。因此，我们说明了一个简单的，可重复的协议，用于原代人类文化VS细胞。这很容易掌握技术允许分子和细胞研究能够更准确地概括了VS疾病的复杂性。
Introduction
前庭神经鞘瘤（VSS）代表雪旺氏细胞的前庭神经（SC）的肿瘤，危及所有颅内肿瘤1-3的10％。 VSS发生在任何偶发或家族（神经纤维瘤病2型，NF2）形式，既在NF2肿瘤抑制基因失活的缺陷有关 基因。治疗VSS通常是手术切除或放射治疗，但此类程序的发病率，包括耳聋，面部神经病变，脊髓液渗漏，不平衡，和肿瘤再生长1。另外不是所有的患者都可以接受手术或放疗的候选人。这种显著的发病率替代疗法，以及缺乏带动调查VSS的独特分子生物学在开发新的治疗方法3,4的希望。
细胞培养允许潜在的治疗COM的分子和细胞行为和筛选的快速，轻便，并深入分析磅。从历史上看，缺乏新鲜组织标本，事实上，神经鞘瘤细胞不会永生都显著阻碍了利用原代培养的神经鞘瘤肿瘤发生的研究。为了克服原代培养的供应有限，是由转导与HPV E6和E7癌基因5所产生的永生HEI193 VS细胞系。这种致癌转导介绍显著分子和表型改变的细胞，从而限制了其作为一种模式为人类神经鞘瘤的使用。从缺乏功能性NF2基因的转基因小鼠系衍生的资深大律师代表的文化研究在体外NF2依赖SC肿瘤发生另一种选择。然而，这些文化不能概括人类VSS或帐户人类特定行为的异质性。以往大多数VS培养技术需要相对较长的处理时间和复杂的选择性培养技术6-8。在这里，我们提出了一个简单的，可重复的协议，用于原发性VS细胞培养与完整的处理下3小时，用95％的肿瘤细胞的纯度，通过免疫测定。
Protocol
Representative Results
Discussion
在体外培养神经鞘瘤史
努力建立体外培养神经鞘瘤的最初听神经瘤的开放手术切除发生于1894年12开始后短短的几十年，第一次有记载的文化是由Kredel在20世纪20年代，谁不成功地试图用“悬滴法”12成长切碎的肿瘤。后来，博士。穆雷和烈性黑啤酒发表了他们在体外用生长在凝结人类血浆神经鞘瘤的文化体验。有趣的是，这种?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
技术支持：NIDCD R01DC009801，P30DC010362，5T32DC000040-17
Materials
| Poly-L-Ornithine, 0.01% Solution | Sigma | P4957 | Cell Culture Surface Treatment |
| Laminin Mouse Protein | Gibco | 23017-015 / L2020 | Cell Culture Surface Treatment |
| 0.2% Collagenase (dissolved in HBSS -/-) | Sigma | C2674 | Dissociation Reagent |
| 0.25% Trypsin | Gibco | 25200-056 | Dissociation Reagent |
| TPS 100mm Round Culture Dish | Midwest Scientific | TP93100 | |
| Permanox 4 well slide | Fisher Scientific | 1256521 | |
| Permanox 8 well slide | Fisher Scientific | 1256522 | |
| Suction Filter Flask | Midwest Scientific | TP99500 | |
| 15ml Conical Tube | Midwest Scientific | TP91015 | |
| 50ml Conical Tube | Midwest Scientific | TP91050 | |
| 2mL Round Bottom Tube | USA Scientific | 1620-2700 | |
| Small Scissor | FST | 14058-11 | |
| Small Forceps | FST | 11251-20 | |
| Scalpel Handle | FST | 10004-13 | |
| #11 Scalpel Blade | Roboz | RS-9801-11 | |
| Non-Tissue Culture 100mm Round Petri Dish | Fisher Scientific | 50-820-904 | |
| P1000 Pipetteman | Bioexpress | P3963-1000 | |
| Serological Pipetteman | Bioexpress | R3073-2P | |
| Sterile, Non-Filtered P1000 Pipette Tips | Midwest Scientific | TD1250R | |
| Insulated Ice Cooler | |||
| Culture Hood | Baker | ||
| Centrifuge | Eppendorf -5810R | ||
| Hanks Balanced Salt Solution (HBSS) +/+ (w/ Ca2+, Mg2+) | Gibco | 24020-117 | Schwannoma Culture and Media Components |
| Hanks Balanced Salt Solution (HBSS) -/- (w/out Ca2+, Mg2+) | Gibco | 14170-112 | Schwannoma Culture and Media Components |
| Dulbecco’s Modified Eagle Medium (DMEM), High Glucose, w/ Phenol Red | Gibco | 11965-092 | Schwannoma Culture and Media Components |
| N2 Supplement | Gibco | 17502-048 | Schwannoma Culture and Media Components |
| Fetal Bovine Serum | Gibco | 26140-079 | Schwannoma Culture and Media Components |
| Penicillin – Streptomycin | Gibco | 15140-163 | Schwannoma Culture and Media Components |
| Bovine Insulin (1mg/ml 200x) | Sigma | I6634 | Schwannoma Culture and Media Components |
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