体外IDH1^(R132H)突变模型建立与生物学表型研究

Establishment of IDH1^(R132H) mutant model in vitro and study of biological phenotype

目的研究体外异柠檬酸脱氢酶1(IDH1^(R132H))突变对胶质瘤细胞的影响。方法首先挖掘数据库信息,分析IDH1^(R132H)突变胶质瘤患者的临床预后生存情况。之后利用慢病毒转染技术,构建两株胶质瘤细胞SF295和A172体外IDH1^(R132H)突变细胞模型,包括IDH1^(R132H)突变组(IDH1 Mut组)和对照组(IDH1 NC组)。通过对胶质瘤细胞基因突变位点测序,Western blot实验检测突变基因表达的蛋白和IDH1^(R132H)突变代谢产物2-羟基戊二酸(D2HG)。比较两株胶质瘤细胞IDH1 Mut组和IDH1 NC组细胞的增殖情况,Transwell实验检测两组细胞的侵袭能力,细胞划痕实验分析两组细胞的迁移能力。结果首先成功构建了两株IDH1^(R132H)突变胶质瘤细胞模型SF295和A172,测序结果显示两株胶质瘤细胞中IDH1 Mut组的IDH1基因位点突变成功,并且成功检测到突变基因蛋白IDH1的表达和突变代谢产物D2HG。之后细胞表型实验显示,与IDH1 NC组比较,IDH1 Mut组细胞增殖速度明显变慢,细胞侵袭能力下降,细胞迁移能力显著降低,差异均有统计学意义(P<0.05)。结论体外实验表明,IDH1^(R132H)突变能够抑制胶质瘤细胞增殖、侵袭、迁移表型,显示出IDH1^(R132H)突变在胶质瘤分子治疗中的潜力。

Objective To study the effect of isocitrate dehydrogenase 1(IDH1^(R132H))mutation on glioma cell model in vitro.Methods Database information was excavated for the analysis of the clinical prognosis and survival of patients with IDH1^(R132H)mutant glioma.Lentiviral transfection technology was used to construct two in vitro IDH1^(R132H)mutant cell models of glioma cells SF295 and A172,including IDH1^(R132H)mutant group(IDH1 Mut group)and control group(IDH1 NC group).DNA sequencing was used to detect the gene mutation sites of glioma cells;Western blot experiment was used to detect the protein expressed by the mutated gene,and IDH1^(R132H)mutant metabolite 2-hydroxyglutarate(D-2-Hydroxyglutarate,D2 HG);through these three aspects,the constructed glioma mutant cell model was identified.After the identification of the glioma cell mutation model was completed,the proliferation of the two glioma cells IDH1 Mut group and IDH1 NC group was compared.Transwell experiment was used to detect the invasion ability of two groups of cells.Cell scratch experiment was used to analyze the migration ability of the two groups of cells.Results Two IDH1^(R132H)mutant glioma cell models SF295 and A172 were successfully constructed.The sequencing results showed that the IDH1 gene locus of the IDH1 Mut group in the two glioma cells was successfully mutated;the expression of the mutant gene protein IDH1 and the mutant metabolite D2 HG were detected.Cell phenotyping experiments showed that compared with IDH1 NC group,the cell proliferation speed of IDH1 Mut group was significantly slower,cell invasion ability was decreased,and the cell migration ability was significantly reduced(P all<0.05).Conclusion In vitro experiments had shown that IDH1^(R132H)mutation could inhibit the proliferation,invasion,and migration phenotypes of glioma cells,showed the potential of IDH1^(R132H)mutation in molecular therapy of glioma.