人源胶质瘤细胞株的建立及其生物学特性分析  

Establishment of a new human glioma cell line and analysis of its biological characteristics

作　　者：陈桂林[1] 李炎炎[1] 谢学顺[1] 陈金明[1] 吴庭枫[1] 李学涛[1] 王杭州[1] 周幽心[1] 杜子威[1] 

机构地区：[1]苏州大学附属第一医院神经外科暨脑神经研究室,215006

出　　处：《中华肿瘤杂志》2015年第2期84-90,共7页Chinese Journal of Oncology

基　　金：国家自然科学基金（NSFC81372689）;江苏省卫生厅重点科研课题（K201106）;江苏省“六大人才高峰”第九批高层次项目（WS-050）

摘　　要：目的：建立新的胶质瘤细胞株,为肿瘤研究提供具备新特性的细胞工具。方法临床手术来源的胶质瘤组织进行培养传代,第10代后克隆建立细胞株（ SHG139）。经免疫荧光细胞化学技术检测蛋白表达,流式细胞术（ FCM）分析细胞增殖和细胞周期,染色体核型分析SHG139细胞的生物学特性。应用神经干细胞培养液（ NSCM ）培养 SHG139细胞,使其转化成胶质瘤干细胞球（SHG139s）,并检测SHG139s细胞的干性标志物。诱导分化SHG139s细胞后,检测分化细胞的胶质前体神经节苷脂（A2B5）、胶质纤维酸性蛋白（GFAP）、β-Ⅲ tubulin和半乳糖神经酰胺（GalC）的表达情况,进一步比较SHG139和SHG139s细胞颅内原位移植瘤的病理特征。结果成功建立的SHG139第20代和第60代细胞A2B5、GalC、GFAP、酸性钙结合蛋白（S-100）和vimentin表达均为阳性,与肿瘤组织的免疫组化结果及病理特征一致。 SHG139细胞传代后24 h内增殖显著,染色体总数为68条,多倍体较多。 SHG139s细胞中A2B5、巢蛋白（nestin）和神经元-神经胶质2型抗原（NG2）阳性比例分别为（84.12±9.96）%、（73.86±5.01）%和（73.37±2.09）%。 SHG139s细胞诱导分化后,贴壁细胞中GFAP、β-Ⅲ tubulin 和 GalC 的阳性比例分别为（92.89±2.24）%、（64.85±4.09）%和（33.57±4.14）%。 SHG139s细胞颅内移植瘤呈胶质肉瘤和星形-少枝胶质细胞瘤的多种形态,肿瘤血管和瘤周血管以鼠源性为主。结论 SHG139为星形胶质瘤细胞株, NSCM 培养可成功获得 SHG139s。SHG139s为A2B5＋ CD133-亚群细胞,具有自我更新和多向分化潜能。与SHG139细胞颅内移植瘤比较,SHG139s细胞颅内移植瘤具有更强的恶性度和侵袭性。Objective To establish a new glioma cell line and analyze its biological characteristics, and to provide a useful cellular tool with new features for cancer research. Methods Glioma tissue was taken from surgical specimen clinical of a clinical patient. Primary culture was carried out, and a cell line （ SHG139 ） was established after 10 passages. Immunofluorescence staining was performed to detect the expression of proteins, and cell proliferation and cycle were detected by flow cytometry method （ FCM） . The biological characteristics of SHG139 cells were detected by chromosome karyotype analysis. SHG139s glioma cells derived from SHG139 glioma cell line were cultured with neural stem cell medium. Then stem cell markers were determined. SHG139s cells were induced with serum-containing medium, and their expression of A2B5, GFAP,β-Ⅲtubulin, and GalC was detected. Intracranial xenograft tumor of both SHG139 glioma cells and SHG139s glioma stem cell spheres was generated in rats. Results The expressions of A2B5, GalC, GFAP, S-100, and vimentin in the 20 and 60 passages of SHG139 cells were positive, consistent with the immunohistochemical results and pathological features. SHG139 cells proliferated significantly within 24 h after subculture, and their total number of chromosomes was 68 and mostly multiploid. They were positive for A2B5 （84. 12 ± 9. 96）%, nestin （73. 86 ± 5. 01）%, and NG2 （73. 37 ± 2. 09）%. SHG139s cells were induced, and the ratio of positive cells of GFAP, β-Ⅲ tubulin and GalC was （92. 89 ± 2. 24）%,（64. 85 ± 4. 09）% and （33. 57 ± 4. 14）%, respectively. Conclusions SHG139 is an astroglioma cell line, from which SHG139s cells can be successfully obtained by culture with NSCM. SHG139s cells are of A2B5^＋/CD133^-GSCs subgroup cells, with potentials of self-renewal and multi-directional differentiation. Compared with the intracranial SHG139 xenograft tumor, the intracranial SHG139s xenograft tumor is more malignant and aggressive.

关 键 词：神经胶质瘤 干细胞 细胞移植 CD133 胶质前体神经节苷脂 小鼠 裸 

分 类 号：R739.4[医药卫生—肿瘤]