Min Xu, Dingchao Xiang, Xun Zhu, Cunzu Wang
Kunshan TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Department of Neurosurgery, Kunshan, Jiangsu Province, China.
Turk Neurosurg. 2022;32(3):357-363. doi: 10.5137/1019-5149.JTN.26278-19.2.
To study the relationship between temozolomide (TMZ) chemotherapy-resistant cells and stem cells in gliomas.
The U251 glioma cell line was exposed to TMZ to generate TMZ-resistant colonies (U251/TMZ cell line) using the pulse drug method. The TMZ sensitivity of U251/TMZ and parental cells was examined using an MTT assay. The cell growth curve was drawn to show the growth of the two kinds of cells. Glioma stem cells (GSCs) were cultured and differentiated in vitro. Immunofluorescence assays were used to identify the expression of CD133, Nestin, and ABCG2 in U251/TM and U251 cells. Western blot analysis was used to analyse protein expression levels.
The U251/TMZ cell line was successfully cultured in vitro. The IC50 value of the U251/TMZ cell line is 8.1 times that of the parental U251 cell line (t=-63.28, p=0.00). The doubling time of U251/TMZ cells was long compared with the parental cells. GSC tumour spheres were successfully cultured in vitro, and they differentiated in medium containing serum. The expression of CD133, Nestin, and ABCG2 in U251/TMZ cells was significantly higher than that in the parental U251 cells (t=43.35, p=0.00; t=12.31, p=0.00; t=11.49, p=0.00). Immunofluorescence staining of CD133, Nestin, and ABCG2 was significantly higher in U251/TMZ than in the parental U251 cells (t=43.35, p=0.00; t=12.31, p=0.00; t=11.49, p=0.00). Moreover, Western blot results showed that CD133, Nestin, and ABCG2 expression was significantly higher in U251/TMZ cells than that in the parental U251 cells (t=17.76, p=0.00; t=18.78, p=0.00; t=6.19, p=0.00).
The U251/TMZ cell line has the biological characteristics of GSCs. The relationship between GSCs and chemotherapy-resistant cells has been preliminary proven to be partially overlapping, which can provide a new perspective when using appropriate cell subpopulations as targets for glioma.
研究替莫唑胺（TMZ）化疗耐药细胞与胶质瘤干细胞之间的关系。
采用脉冲给药法，将U251胶质瘤细胞系暴露于TMZ以产生TMZ耐药集落（U251/TMZ细胞系）。采用MTT法检测U251/TMZ细胞系和亲本细胞对TMZ的敏感性。绘制细胞生长曲线以显示两种细胞的生长情况。体外培养并分化胶质瘤干细胞（GSCs）。采用免疫荧光法检测U251/TMZ和U251细胞中CD133、巢蛋白和ABCG2的表达。采用蛋白质免疫印迹分析来分析蛋白质表达水平。
成功在体外培养出U251/TMZ细胞系。U251/TMZ细胞系的IC50值是亲本U251细胞系的8.1倍（t=-63.28，p=0.00）。与亲本细胞相比，U251/TMZ细胞的倍增时间较长。成功在体外培养出GSC肿瘤球，并且它们在含血清的培养基中分化。U251/TMZ细胞中CD133、巢蛋白和ABCG2的表达显著高于亲本U251细胞（t=43.35，p=0.00；t=12.31，p=0.00；t=11.49，p=0.00）。U251/TMZ细胞中CD133、巢蛋白和ABCG2的免疫荧光染色显著高于亲本U251细胞（t=43.35，p=0.00；t=12.31，p=0.00；t=11.49，p=0.00）。此外，蛋白质免疫印迹结果显示，U251/TMZ细胞中CD133、巢蛋白和ABCG2的表达显著高于亲本U251细胞（t=17.76，p=0.00；t=18.78，p=0.00；t=6.19，p=0.00）。
U251/TMZ细胞系具有GSCs的生物学特性。初步证明GSCs与化疗耐药细胞之间的关系存在部分重叠，这可为将合适的细胞亚群作为胶质瘤治疗靶点提供新的视角。
