CAJ | 학술논문

目的为了在常氧条件下观察低氧诱导因子-1α( HIF-1 α)在骨缺损部位促新血管生成作用,构建能够同时表达突变型HIF-1α和人源化绿色荧光蛋白(hrGFP)的腺病毒载体并检测其在兔骨髓间充质干细胞(MSCs)中的表达。方法利用聚合酶链式反应(PCR)定点突变人HIF-1α编码区(CDS)第402、564和803位氨基酸,将突变后HIF-1α和hrGFP重组入pAdEasy-1系统,包装病毒并测定滴度。以感染复数(MOI)=100将腺病毒转染MSCs,逆转录-聚合酶链反应(RT-PCR)和Western blot方法检测转染细胞中HIF-1αmRNA和蛋白表达。结果 (1)第402、564和803位氨基酸均定点突变为丙氨酸。(2)A、B两组HIF-1α mRNA表达量明显高于C、D两组,差异有统计学意义(P＜0.01);A组HIF-1α蛋白表达量明显高于其他3组,差异有统计学意义(P＜0.01)。结论 (1)腺病毒载体Ad-HIF-1 αmu-IRES-hrGFP-1构建成功。(2)突变后HIF-1α基因能够在常氧条件下大量且高效表达。
목적 위료재상양조건하관찰저양유도인자-1α( HIF-1 α)재골결손부위촉신혈관생성작용,구건능구동시표체돌변형HIF-1α화인원화록색형광단백(hrGFP)적선병독재체병검측기재토골수간충질간세포(MSCs)중적표체。방법 이용취합매련식반응(PCR)정점돌변인HIF-1α편마구(CDS)제402、564화803위안기산,장돌변후HIF-1α화hrGFP중조입pAdEasy-1계통,포장병독병측정적도。이감염복수(MOI)=100장선병독전염MSCs,역전록-취합매련반응(RT-PCR)화Western blot방법검측전염세포중HIF-1αmRNA화단백표체。결과 (1)제402、564화803위안기산균정점돌변위병안산。(2)A、B량조HIF-1α mRNA표체량명현고우C、D량조,차이유통계학의의(P＜0.01);A조HIF-1α단백표체량명현고우기타3조,차이유통계학의의(P＜0.01)。결론 (1)선병독재체Ad-HIF-1 αmu-IRES-hrGFP-1구건성공。(2)돌변후HIF-1α기인능구재상양조건하대량차고효표체。
Objective To study the angiogenesis promoting effect of hypoxia inducible tactor (HIF)-lα gene at bone detect and loss location under normoxic conditions, construct a new adenovims vector which can express mutant hypoxia HIF-lo and human renilla reniformis green fluorescent protein (hrGFP) simultaneously and detect the expression of the vector in rabbit marrow stromal cells (MSCs).Methods Site-directed mutagenesis of the 402,564 and 803 position amino acids in HIF-1 α gene coding sequence (CDS) was done by using polymerase chain reaction (PCR) technique. The mutant HIF-1αt and hrGFP gene were recombined into pAdEasy-1 adenovirus system, the viruses were packed and the titer was assayed. The recombinant adenovirus was transfected into MSCs following MOI =100, and the HIF-1omRNA and protein expression was detected by using reverse transcription (RT)-PCR and Western blotting.Results ( 1 ) The 402, 564 and 803 amino acids all had site-directed mutagenesis into 2-aminopropionic acid. (2) There was statistically significant difference in the HIF-1α mRNA expression between group A or B and group C or D (P ＜0. 01 ). The HIF-1α protein expression in group A was significantly higher than other three groups (P ＜ 0. 01 ). Conclusion ( 1 ) A novel adenovirus vector namely Ad-CMV-HIF-1α muIRES-hrGFP-1 was constructed successfully; (2) The expression of HIF-1α gene after mutation is abundant and efficient under the normoxic conditions.