脊髓弥漫性星形细胞瘤干细胞的培养和建模

孙建军¹ 王振宇¹ 李凌松² 余海燕³ 徐永胜³罗艺¹ 刘斌¹ 毛锦龙⁴ 沈莉² 林英杰²

1 北京大学第三医院神经外科, 北京大学神经系统恶性肿瘤干细胞研究中心, 北京 100191;2 北京大学干细胞研究中心;3 北京大学第三医院临床干细胞中心;4 北京协和医院神经外科；

目的:提取并培养脊髓弥漫性星形细胞瘤原代细胞、CD133标记分选提取肿瘤干细胞，建立裸鼠脑内模型。

方法：从离体的脊髓弥漫性星形细胞瘤组织中提取肿瘤干细胞，悬浮培养、传代、扩增后，对细胞进行流式鉴定并定量。CD133抗体标记后，以磁珠分选为阳性和阴性细胞组，按每只裸鼠10000个细胞，分两组接种入10只4周龄雌性裸鼠脑内建模，CD133阳性细胞组为实验组（Tumor组），CD133阴性细胞组为对照组（Control组）。裸鼠接种后6周时，开始每周2次测量体重和头围，7周后，选择头围增大明显和体重波动较大的裸鼠进行头颅MRI平扫+增强，扫描后次日断椎处死裸鼠，解剖出整个脑子后，解剖显微镜下观察脑皮层和颅骨的大体形态，然后标本取材、固定、切片，行HE、GFAP等染色。每周对阳性和阴性各1只裸鼠进行MRI扫描和处死，分5次检查并处死裸鼠。

结果：细胞在特定的干细胞培养基内呈悬浮细胞球样生长、至2代时，细胞鉴定CD133阳性比率为49.44%。至3代时，细胞数量足以进行接种。CD133阳性组，裸鼠成瘤明显，在解剖显微镜下，有的可见红色的瘤结节、血运丰富；有的呈内陷性生长，皮层表面针眼周红褐色改变；组织学切片显示部分瘤细胞向脑白质内浸润性生长，在MRI影像上瘤体有不同程度强化表现。CD133阴性组，大部分未见肿瘤生长，仅见接种的针道；瘤细胞接种9周鼠（A329 C3），虽大体标本的脑表面未见异常改变，但脑白质内深方可见小瘤体。

结论：CD133抗体不是脊髓弥漫性星形细胞瘤干细胞唯一的标记物，呈阴性表达的肿瘤细胞也可增殖成瘤。

关键词：胶质瘤；星形细胞瘤；肿瘤干细胞；CD133抗体；裸鼠模型

Culture the spinal cord diffuse astrocytoma cells and establish nude mice model

Jianjun Sun¹, Zhenyu Wang¹, Lingsong Li², Haiyan Yu³, Yongsheng Xu³, Yi Luo¹, Bin Liu¹, Jinlong Mao⁴, Li Shen², Yingjie Lin²

1 Neurosurgical Department, Peking University Third Hospital, Neural Malignant Tumor Stem Cell Research Center, Peking University, Beijing 100191, China; 2 China Stem Cell Research Center; Peking University Health Science Center; 3 Clinical Stem Cell Center, Peking University Third Hospital; 4 Neurosurgical Department, Peking Union Medical College Hospital

Objective This basic study was to isolate and culture original spinal cord diffuse astrocytoma cells, and to extract CD133+ glioma stem cells, and to initiate glioma in the brain of nude mice model.

Methods The original astrocytoma cells were isolated from a surgical specimen of a patient with recurrent intramedullary spinal cord diffuse astrocytoma. The CD133- negative and CD133+ positive cells were separated by magnetic sorting using the CD133 Microbead kit. The nude mice model with brain glioma was established with different kind glioma cells. 10000 cells were implanted in brain for every mouse. Five mice implanted with CD133+ glioma cells were grouped as Tumor, and another five mice implanted with CD133- glioma cells as Control. In the six weeks, weigh and head circumference of mice were measured for two times every week. Between seven and eleven weeks, a couple of mice, including one Tumor mouse and one Control mouse, were selected to accept MRI scan according to its obviously enlarged head circumference and fluctuated weigh every week. Whole skull and brain of mice was checked under anatomy microscope. The slice of mice brain was stained with HE and immunohistochemistry.

Results Glioma stem cells cultured in special culture media and aggregated in suspension cells ball. Up to second generation, the rate of CD133 positive cells in whole glioma cells was evaluated in 49.44 percent. Up to third generation, the number of cells was enough to be implanted for nude mice model. In Tumor group with CD133+ cells, the xenograft formed surely. Under anatomy microscope, some show red xenograft nodule with abundance blood supply, and some show implicitly with red-brown lesion around the insert spot. On the histological slice, some glioma cells grow infiltrative into Alba. On the MRI scan, the xenograft was shown in contrast. In Control group with CD133- cells, no xenograft was observed in most mice brain, only inserted spot kept there. However, small xenograft was found into deep Alba in No. A329C3 mouse.

Conclusion CD133 antibody is not the unique spinal cord astrocytomas stem cells marker, CD 133 negative tumor cells can also be proliferation into the xenograft.

Key Words: Glioma; Astrocytoma; Cancer stem cell; CD133 antibody; Nude mice model