摘要
目的筛选鉴定胶质瘤干细胞新标志物CD98轻链蛋白L型氨基酸转运体1(LAT1).方法采用流式单色标记法检测U87及U251胶质瘤细胞中CD98重链蛋白CD98hc、CD98轻链蛋白LAT1以及CD133的表达,以选取胶质瘤细胞中可能的、潜在的干细胞标志物.采用流式细胞学技术分选LAT1阳性(LAT1^(+))和LAT1阴性(LAT1^(-))胶质瘤细胞,采用细胞免疫荧光检测、成球实验、体外极限稀释实验鉴定细胞干性.诱导分化培养胶质瘤干细胞,采用蛋白质免疫印迹法检测LAT1、分化细胞标志物β-tubulinⅢ和胶质纤维酸性蛋白(GFAP)的表达.通过构建裸鼠皮下荷瘤模型,观察LAT1^(+)和LAT1^(-)胶质瘤细胞在裸鼠皮下的成瘤情况(分别为LAT1^(+)组、LAT1^(-)组).结果流式细胞学结果显示,U87细胞LAT1(SLC7A5)阳性表达率为(1.99±0.18)%,CD133阳性表达率为(2.18±0.29)%;U251细胞LAT1(SLC7A5)阳性表达率为(6.24±0.69)%,CD133阳性表达率为(4.79±0.64)%.免疫荧光检测结果显示,LAT1^(+)-U251和LAT1^(+)-U87细胞的Sox2、Olig2和CD133阳性表达率均较高;LAT1^(-)-U251和LAT1^(-)-U87细胞的Sox2、Olig2和CD133阳性表达率均较低.成球实验和极限稀释实验显示,LAT1^(+)-U251细胞成球率优于LAT1^(+)-U87细胞(P=0.005);LAT1^(-)-U251和LAT1^(-)-U87细胞均未成球.蛋白质免疫印迹检测结果显示,LAT1^(-)-U251细胞未分化和诱导分化培养时,均较低表达β-tubulinⅢ、GFAP;LAT1^(+)-U251细胞分化前低表达β-tubulinⅢ、GFAP,而诱导分化后β-tubulinⅢ、GFAP表达显著增加(均P<0.05),LAT1表达显著降低(P=0.007).裸鼠成瘤模型结果显示,21 d和28 d LAT1^(+)组肿瘤体积均大于LAT1^(-)组,差异均具有统计学意义(均P<0.05);28 d时,LAT1^(+)组肿瘤重量大于LAT1^(-)组肿瘤重量,差异具有统计学意义(P=0.007).结论CD98轻链蛋白LAT1是潜在的胶质瘤干细胞标志物.
Objective To screen and identify CD98 light chain L-type amino acid transporter 1(LATI)as a new glioma stem cell marker.Methods The flow cytometric single-color labeling method was used to detect the expression of CD98 heavy chain protein CD98hc,CD98 light chain protein LATI and expression of CD133 in U87 and U251 glioma cells 1o seleet possible and potential stem cell markers in glioma cells.LATI^(+)and LAT1^(-)glioma cells were sorted by flow cytometry.Cellular immunoluorescence,sphere-formation assay,and in vitro limiting dilution assay were used to identify cell stemness.Differentiated glioma stem cells were cultured,and the expressions of B-tubulin II,GFAP,and LATI were detected by Western blot.By constructing a nude mouse subcutaneous tumor model,we observed the tumor formation of LATI^(+)and LATI^(-)glioma cells under the skin of nude mice(LATI^(+)group and LATI^(-)group respectively).Results Flow cytometry results showed that the positive rate of LATI(SLC7A5)in U87 cells was(1.99±0.18)%,the positive rate of CD133 in U87 cells was(2.18±0.29)%;the positive rate of LAT1(SLC7A5)in U251 cells was(6.24±0.69)%,and the positive rate of CD133 in U251 cells was(4.79±0.64)%.Immunofluorescence detection results showed that the positive expression rates of Sox2,Olig2 and CD133 in LAT1^(+)-U251 and LAT1^(-)-U87 cells were higher;the positive expression rates of Sox2,Olig2 and CD133 inLAT1^(-)-U251 and LATI^(+)-U87 cells were all lower.The sphere-formation assay and the limiting dilution assay showed that the sphere-formation rate of LATI^(+)-U251 cells was better than that of LATI-U87 cells(P=0.005);LAT1^(+)-U251 and LATI^(-)-U87 cells did not form spheroids.W estern botting results showed that LATI-U251 cells had low expression ofβ-tubulin II and GF AP when they were undifferentiated and culured without induction;LATI^(+)-U251 cells had low expression of B-tubulin Il and GFAP before differentiation,but after induced differentiation the expression ofβ-tubulin川l and GFAP inereased significantly(all P<0.05),and t
作者
王翔
刘翔浩
曾祥义
龙强友
毛庆
Wang Xiang;Liu Xianghao;Zeng Xiangyi;Long Qiangyou;Mao Qing(Department of Neurosurgery,West China Haspital of Sichuan University,Chengdu 610041,China)
出处
《中华神经外科杂志》
CSCD
北大核心
2021年第4期393-399,共7页
Chinese Journal of Neurosurgery
