Ⅱ型神经纤维瘤病肿瘤性许旺细胞提取与纯化的体外研究

Extraction and purification of tumorigenic Schwann cells in vitro from neurofibromatosis type 2 tissues

目的建立简便易行的Ⅱ型神经纤维瘤病肿瘤性许旺细胞体外提取并纯化方法,以获取能够满足实验需求的Ⅱ型神经纤维瘤病肿瘤性许旺细胞,提高原代培养成功率。方法采集6例Ⅱ型神经纤维瘤病患者肿瘤组织标本作为实验材料,Ⅰ型胶原酶消化培养法提取肿瘤性许旺细胞,低浓度酶快速消化法、差数贴壁法和克隆筛选技术纯化肿瘤性许旺细胞。倒置相差显微镜观察细胞形态变化,免疫细胞化学染色检测纯化细胞S-100蛋白表达水平,结合细胞形态鉴定经体外培养并纯化的细胞为肿瘤性许旺细胞。结果自3例肿瘤组织标本培养液中成功获得肿瘤性许旺细胞。肿瘤细胞原代培养至第3天,可见大量双极梭形或三角形细胞,以低浓度酶快速消化法和差数贴壁法纯化细胞;培养至第3代,倒置相差显微镜观察可见4～5个克隆细胞,免疫细胞化学染色S-100蛋白表达阳性,肿瘤性许旺细胞纯度>95%,活力良好,可以稳定传代培养。结论应用体外原代培养方法结合低浓度酶快速消化法、差数贴壁法和克隆筛选技术能够有效剔除纤维母细胞,获得纯度高、活力良好的肿瘤性许旺细胞,可用于Ⅱ型神经纤维瘤病发病机制及治疗研究。

Objective To establish a simple method to extract and purify tumorigenic Schwann ceils （TSCs） from neurofibromatosis type 2 （NF2） tumor tissues in vitro, in order to improve the successful rate of primary culture and obtain a large number of TSCs fi＇om NF2 patients for further study. Methods Six fresh tumor tissues from patients with NF2 were obtained fi＇om 2009 to 2010 in our hospital. TSCs were extracted after collagenase digestion, followed by cell purification through rapid digestion with low trypsin concentration, differential detachment and clone screening. Morphological changes of TSCs were observed under inverted microscope and cultured TSCs were identified immunocytochemically by S- 100 protein staining. Results TSCs were successfully cultured from 3 NF2 specimens. A large number of bipolar spindle or triangular-shaped cells could be seen on the third day of primary culture and 4 to 5 clones were formed after the third passage in each successfully cultured specimen. Purified TSCs expressed S-100 protein detected by immunocytochemistry with the rate of positive cells of above 95%, and their cell activity was fine and were able to be passaged stably in the subculture. Conclusion It is a successful culture method to extract and purify TSCs, combining primary culture with rapid trypsin digestion with low concentration and differential detachment. After clone screening, the purified and energetic TSCs can be obtained and applied to clinical study for NF2 pathogenesis and treatment. Fibroblasts （FBs） can be removed effectively as well.