摘要
目的探讨腺病毒介导的人诱变型低氧诱导因子1(简称Ad-H564)在大鼠急性后肢缺血模型中的表达及促进血管新生的作用。方法①重组腺病毒在HEK293A细胞内大量扩增,氯化铯浓度梯度离心,透析纯化后用分光光度计法测定病毒滴度。②建立大鼠急性后肢缺血动物模型,将72只大鼠随机均分为4组:Ad-LacZ组、Ad-HIF1α0组、Ad-HIF1α564组和对照组,其中在测定HIF-1αmRNA表达时取生理盐水组作为对照组,在血管造影和铸型时取假手术组作为对照组。实验组分别以Ad-LacZ、Ad-H0和Ad-H564转染术侧后肢骨骼肌,对照组注射生理盐水,假手术组不做任何处理。③于转染后1、3、5、7d,以生理盐水组为对照,采用RT-PCR法测定肌肉中的HIF-1 mRNA表达;④基因转染后28d,以假手术组为对照,选择性后肢动脉造影及血管铸型观察血管密度。结果①经PCR及基因测序鉴定,扩增纯化后的腺病毒所携带的目的基因信息无丢失或变异,病毒滴度达1011OPU/ml。②RT-PCR结果显示:Ad-H564组基因相对表达量最高(平均0.5447±0.1412),与生理盐水组,Ad-LacZ组相比差异有统计学意义(P=0.000),但与Ad-H0组(平均0.5330±0.0416)的差异不显著(P=0.368);各组均以基因转染后7d表达量最高,Ad-H564组与其他各组对照间差异有统计学意义(P=0.000)。③基因转染28d后造影结果显示:Ad-H564组和Ad-H0组的侧支血管密度均大于对照组,但两组间无显著差异。动脉血管铸型结果显示:Ad-H564组的微小血管密度较其他各组明显增多。结论单一位点诱变型人HIF-1α基因能够增强局部肌肉内基因表达,促进缺血组织中血管新生,但与野生型基因相比差异无统计学意义。
Objective To explore the expression of recombinant adenovirus hypoxia-inducing factor-1α （HIF-1α） and its effects on the angiogenesis in a rat model of acute hindlimb ischemia. Methods （1） Recombinant adenovirus HIF-1α-Alα564 （Ad-H564 for short） was amplified in large scale in HEK293A cells and concentration gradient centrifuged in CsCl, purified by dialysis and then its titer was determined by spectrophotometry. （2） A model of acute ischemia in rats＇ hind limb was reproduced, and 72 rats were randomly allocated into Ad-LacZ group, Ad-H0 group, Ad-H564 group and control group （18 rats for each group）. After the HIF-1α mRNA expression was detected, six rats of control group were intramuscularly injected with normal saline （NS group） to serve as control. Angiography and vascular cast were carried out in another two groups with in each six rats to serve as sham operated controls. In to hind limb skeletal muscles of rats of Ad-LacZ, Ad-H0 and Ad-H564 groups Ad-LacZ, Ad-H0 and Ad-H564 were respectively injected, normal saline was given to the rats of NS group, and nothing was given to the rats in sham group. （3） HIF-1α mRNA expression was determined by RT-PCR on the 1st, 3rd, 5th and 7th day after injection. （4） Selective arteriography and vascular cast were performed to evaluate the vascular density. Results （1） PCR and gene sequencing suggested that correct recombinant adenovirus HIF-1α was obtained after being amplified and purified, and the titer of adenovirus was 1011OPU/ml. （2） RT-PCR showed that the highest expression of HIF-1α mRNA （0.544 7±0.141 2） was seen in Ad-H564 group, and the value was significantly different compared with that of normal group and Ad-LacZ group （P=0.000）, but it did not show significant difference compared with that of Ad-H564 group and Ad-H0 group （0.533 0±0.041 6, P=0.368）. The expression of HIF-1α mRNA reached the highest value on the 7th day in all the groups. （3） Arteriography on the 28th day after injection showed that the density of the collateral vessels was higher in Ad-H564 group than that in Ad-H0 group, but no significant difference was found. Vascular casts showed denser microvasculature in Ad-H564 group than that in the other groups. Conclusions The present study suggests that single site mutation may enhance the expression of human HIF-1α gene in the local muscles, facilitate angiogenesis in the ischemic tissue, but no significant difference was found between the wild-type and the mutated HIF-1α genes.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2008年第5期576-578,581,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金资助项目(30370587)
