CAJ | 학술논문

目的对已构建的突变型人低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)真核表达载体pcDNA3.1+/HIF-1α-564Ala(单突变)和pcDNA3.1+/HIF-1α-564Ala-803Ala(双突变)进行进一步的功能鉴定.方法将pcDNA3.1+/HIF-1α、pcDNA3.1+/HIF-1α-564Ala和pcDNA3.1+/HIF-1α-564Ala-803Ala分别用脂质体法短暂转染人胚肾上皮细胞(human embryo kidney 293,HEK293);Western blotting法检测正常氧、低氧无钙离子或有钙离子条件下各组转染细胞HIF-1α蛋白水平:RT-PCR法检测正常氧条件各组转染细胞血管内皮生长因子(VEGF)mRNA表达.结果正常氧条件下,转染突变体的细胞较转染野生型HIF-1α载体的细胞HIF-1α蛋白和VEGFmRNA水平增高;低氧条件下,转染野生型HIF-1α载体的HEK293细胞HIF-1α蛋白水平增高:Ca~(2+)刺激减少低氧时转染野生型HIF-1α载体细胞HIF-1α蛋白水平,但对转染突变体细胞HIF-1α蛋白水平无明显影响.结论 pcDNA3.1+/HIF-1α-564Ala和pcDNA3.1+/HIF-1α-564Ala-803Ala两种突变体产物在蛋白水平上具耐氧和耐蛋白酶降解的特性.
목적 대이구건적돌변형인저양유도인자-1α(hypoxia-inducible factor-1α,HIF-1α)진핵표체재체pcDNA3.1+/HIF-1α-564Ala(단돌변)화pcDNA3.1+/HIF-1α-564Ala-803Ala(쌍돌변)진행진일보적공능감정.방법 장pcDNA3.1+/HIF-1α、pcDNA3.1+/HIF-1α-564Ala화pcDNA3.1+/HIF-1α-564Ala-803Ala분별용지질체법단잠전염인배신상피세포(human embryo kidney 293,HEK293);Western blotting법검측정상양、저양무개리자혹유개리자조건하각조전염세포HIF-1α단백수평:RT-PCR법검측정상양조건각조전염세포혈관내피생장인자(VEGF)mRNA표체.결과 정상양조건하,전염돌변체적세포교전염야생형HIF-1α재체적세포HIF-1α단백화VEGFmRNA수평증고;저양조건하,전염야생형HIF-1α재체적HEK293세포HIF-1α단백수평증고:Ca~(2+)자격감소저양시전염야생형HIF-1α재체세포HIF-1α단백수평,단대전염돌변체세포HIF-1α단백수평무명현영향.결론 pcDNA3.1+/HIF-1α-564Ala화pcDNA3.1+/HIF-1α-564Ala-803Ala량충돌변체산물재단백수평상구내양화내단백매강해적특성.
Objective To study the effects of oxygen and calcium on the expression of eukaryotic vectors harboring wild-type or mutated hypoxia-inducible factor-1α(HIF-1α)in HEK293 cells.Methods HEK293 cells were transiently transfected with pcDNA3.1+/HIF-1α,pcDNA3.1+/HIF-1α-564Ala and pcDNA3.1+/HIF-1α-564Ala-803Ala via lipofectin.Western blotting were used to detect HIF-1α protein after normoxic or hypoxic exposure of the transfected HEK293 cells in the presence or absence of Ca~(2+).The levels of vascular endothelial growth factor(VEGF)mRNA in the transfected cells in normoxic condition was detected using RT-PCR.Results The levels of HIF-1α protein and VEGF mRNA increased in HEK293 cells transfected with the vectors harboring mutated HIF-1α, but not in the cells transfected with wild-type HIF-1α vectors in normoxia.Hyooxia increased the levels of HIF-1α protein in the cells transfected with wild-type HIF-1α vectors,which was inhibited by the application of Ca~(2+). Ca~(2+) showed no inhibitory effect on HIF-1α levels in HEK293 cells transfected with the vectors containing mutated HIF-1α. Conclusion The protein products of pcDNA3.1+/HIF-1α-564Ala and pcDNA3.1+/HIF-1α-564Ala-803Ala in HEK293 cells enhance the cell tolerance to oxygen and protease.