SLC22A18基因转染人胶质瘤U251细胞对放疗敏感性的影响

目的：探讨SLC22A18基因转染人胶质瘤U251细胞对放疗敏感性的影响。方法：用脂质体介导的转染技术将pIRES2-EGFP-SLC22A18表达重组质粒导入U251细胞,通过逆转录-聚合酶链反应（RT-PCR）和免疫印迹（Western blot）检测SLC22A18基因和蛋白的表达。U251细胞分为4组：对照组、转染组、放疗组和转染联合放疗组。集落形成实验检测各组U251细胞集落形成数,用四甲基偶氮唑盐微量酶反应比色法（MTT法）和流式细胞仪检测各组U251细胞生长抑制率和凋亡率,进一步应用裸鼠皮下荷瘤模型观察脂质体介导的pIRES2-SLC22A18表达重组质粒转染U251细胞对放疗敏感性的影响。结果：重组质粒转染组通过RT-PCR和Western blot证实了SLC22A18基因和蛋白在U251细胞中表达。集落形成实验检测发现SLC22A18基因对U251细胞的集落形成数为（60.6±5.2）个,当放射剂量为3、6和9 Gy时,U251细胞的集落形成数分别为（30.0±3.6）、（13.0±3.0）和（4.0±1.0）个。MTT检测发现SLC22A18基因对U251细胞的生长抑制率为（80.12±5.75）%。当放射剂量为3、6和9 Gy时,U251细胞的生长抑制率分别为（17.05±4.24）%、（17.34±1.62）%和（18.71±4.59）%。当SLC22A18基因与放疗（3、6和9 Gy）联合作用时,抑制率分别为（81.45±5.32）%、（90.45±1.65）%和（92.62±2.12）%。SLC22A18基因转染所产生的U251细胞凋亡率为17.68%。放疗（3、6和9 Gy）引起的细胞凋亡率分别为4.64%、4.87%和5.42%。当SLC22A18基因与放疗（3、6和9 Gy）联合作用时,凋亡率分别为18.42%、21.48%和23.92%。体内实验结果显示,SLC22A18基因对U251细胞6 Gy照射的抑瘤率比较为1.81。结论：SLC22A18基因转染增加了人胶质瘤U251细胞对放疗的敏感性。

Influence of introducing SLC22A18 gene on the radiosensitivity of human glioma U251 cells

Objective：To study the effect of SLC22A18 gene on the radiosensitivity of human glioma U251 cells.Methods：The eukaryotic expression plasmid pIRES2-EGFP-SLC22A18 was constructed and introduced by Lipofectamine 2000 into cultured U251 cells.SLC22A18 mRNA and protein expression was detected by RT-PCR and Western blot assay.U251 cells were divided into 4 groups：control group,transfected group,radiation group and combined treatmemt group.The number of colonies was assessed using a clonogenic assay.The cell growth inhibitition and apoptosis was assessed by MTT and flow cytometry.The tumor growth inhibition effect was further studied in vivo.The pIRES2-SLC22A18 was injected iniratumorally into established subcutaneous U251 glioma in nude mice mediated by Lipofectamine 2000.Results：The transfection of SLC22A18 gene into U251 cells was confirmed by RT-PCR and Western blot assay.The number of colonies was assessed using a clonogenic assay in the transfected group（60.6 ± 5.2）.That induced by radiation was decreased [（30.0 ± 3.6）,（13.0 ± 3.0）,（4.0 ± 1.0） ]with the increase of radiation doses（3,6,9 Gy）.MTT showed that SLC22A18 gene by itself induced strong inhibition effect on the growth of U251 cells [inhibition rate,IR（80.12 ± 5.75） ].The killing effect of radiation by itself on U251 cells was not strong [IR（17.05 ± 4.24） %,（17.34 ± 1.62） %,（18.71 ± 4.59） %]and increased with the increase of radiation doses（3,6,9 Gy）.When combined treatment of SLC22A18 gene transfection and radiation was used,that was significantly increased [IR（81.45 ± 5.32） %,（90.45 ± 1.65） %,（92.62 ± 2.12） %].The apoptotic rate of U251 cells induced by SLC22A18 gene transfection was 17.68%.That induced by radiation was increased（4.64%,4.87%,5.42%） with the increase of radiation doses（3,6,9 Gy）.The apoptotic rate was also significantly increased（18.42%,21.48%,23.92%） after combined treatment of SLC22A18 and radiation with different doses（3,6,9 Gy）.The antitumor enhancement ratio of SLC22A18 at 6 Gy was 1.81 for U251 cells in vivo.Conclusion：Introduction of SLC22A18 gene into human glioma U251 cells can increase their radiosensitivity.