不同无血清培养方式提取SHG44人脑胶质瘤干细胞的比较研究

Comparison of different culture methods for SHG44 human brain glioma stem cells with serum-free medium

目的筛选较理想的SHG44人脑胶质瘤干细胞的培养方法。方法采用无血清培养基(含EGF、FGF、B27)以培养皿、培养瓶及悬浮细胞培养板为载体培养SHG44人脑胶质瘤干细胞,通过CD133、Nestin联合Bcl-2标记进行免疫荧光鉴定,并对比其倒置显微镜下细胞形态及干细胞球的形态、大小及数目。结果培养皿和培养瓶初次富集的SHG44胶质瘤干细胞球呈较大不规则形,经二、三次纯化后发现第二与三次纯化后的大部分胶质瘤干细胞呈不规则分化状,大部分胶质瘤干细胞球呈分散、较小、不规则形生长,但第三次比第一、二次有更少的胶质瘤干细胞球却呈更规则球形生长;悬浮细胞培养板提取、纯化的干细胞呈类圆形,基本全部形成胶质瘤干细胞球,且第三次比第一、二次纯化的干细胞球呈更大更规则球形生长;经统计分析悬浮培养板培养纯化的胶质瘤干细胞球较培养皿和培养瓶纯化的胶质瘤干细胞球大且数目多(P<0.05);对于悬浮培养板,随着纯化次数增加,胶质瘤干细胞球更大、数目更多(P<0.05),且呈更规则球形。结论无血清培养基以悬浮细胞培养板为载体纯化胶质瘤干细胞是较理想的方法。

Objective To determine the ideal culture method for SHG44 human brain glioma stem cells. Methods SHG44 human brain glioma stem cells were cultured in the serum-free medium supplemented with epidermal growth factor,fibroblast growth factor,and B27. According to different culture instruments,they were divided into three groups： culture dish group,culture flask group,and suspension cell culture plate group. The SHG44 human brain glioma stem cells were identified by the immunofluorescence staining of CD133,Nestin,and Bcl-2. The morphology,size,and counts of stem cells and stem cell spheres under an inverted microscope were compared between the three groups. Results In the culture dish group and the culture flask group,the first enriched SHG44 glioma stem cell spheres were large and not regularly shaped. After the second or third purification,most of the glioma stem cells were irregular,and most of the glioma stem cell spheres were scattered,small,and irregular. Compared with the first and second purification,after the third purification,there were fewer glioma stem cell spheres,but which were regularly spherical. In the suspension cell culture plate group,the glioma stem cells were round,and almost all glioma stem cells formed glioma stem cell spheres. And compared with the first and second purification,after the third purification,the stem cell spheres were larger and more regularly spherical. The statistical analysis showed that the suspension cell culture plate group had significantly larger and more glioma stem cell spheres than the culture dish group and the culture flask group（ P〈0. 05）. For the suspension culture plate group,the glioma stem cell spheres were larger and more numerous（ P〈0. 05） and more regular with the increase in times of purification. Conclusions It is an ideal method to purify glioma stem cells using suspension cell culture plate.