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目的:利用β-雌二醇受体的性质,构建Fas与β-雌二醇受体融合基因,使转染该融合基因的胶质瘤细胞能在激素的诱导下发生凋亡.方法:利用PCR技术及基因重组手段,将人Fas基因的跨膜区、胞内区和人β-雌二醇受体的激素结合结构域基因片断融合,并插入高效真核表达载体peDNA3.脂质体法转染人胶质瘤细胞BT325.结果:加入G4186周后筛选出的转化细胞,经Westem blot检测证明,该融合基因在BT325细胞中得到较高表达.MTT法检测表明,在培养基中加入一定浓度的β-雌二醇.可有效地杀死转化细胞,IC_(50)为10~(-9)mol/L DNA Ladder检测进一步证明,转化细胞被诱导发生凋亡.结论:Fas与β-雌二醇受体融合基因转染的胶质瘤细胞,其调亡严格受激素调控.
OBJECTIVE: To construct a fusion gene between Fas and β-estradiol receptor by using the properties of β-estradiol receptor, so that glioma cells transfected with the fusion gene can induce apoptosis under the condition of hormone.Methods: PCR technique and gene recombination method, the human Fas gene transmembrane region, intracellular domain and human β-estradiol receptor hormone binding domain gene fragments were fused and inserted into the highly efficient eukaryotic expression vector peDNA3. Liposome method Transfection of human glioma cells BT325.Results: Transformed cells selected by G4186weeks after Westem blot test showed that the fusion gene was highly expressed in BT325 cells.MTT assay showed that adding certain Concentration of β-estradiol can effectively kill the transformed cells.It is further proved by DNA Ladder assay that the IC50 of 10 ~ (-9) mol / L is detected.Conclusion: Fas and β- Estradiol receptor fusion gene transfected glioma cells, its regulation strictly regulated by hormones.