超声分子成像评价三突变型低氧诱导因子-1α在老年小鼠缺血后肢的血管新生效应

Ultrasound molecular imaging of the angiogenic response induced by triple-point mutations of hypoxia-inducible factor-1α in aged mice with hind-limb ischemia

目的应用携αv-整合素分子探针的超声分子成像评价三突变型低氧诱导因子-1α（hypoxia inducible factor-1α，HIF-1α）老年小鼠缺血后肢的血管新生效应。方法24只老年小鼠结扎一侧后肢股动脉制备后肢缺血模型并随机等分为两组，分别经肌肉注射腺病毒包裹的β-半乳糖苷酶基因（Ad—LacZ）、腺病毒包裹的Pro402、Pro564、Asn803三位点突变型HIF-1α基因（Ad—triple mutant HIF-1α，Ad—TM）。术前、术后0、7、14、21、28d分别对小鼠双侧后肢骨骼肌（每组6只）行超声灌注成像评价缺血后肢骨骼肌的血流情况。超声分子成像于术后7d进行，每组小鼠随机先后（间隔30min）经尾静脉注入携抗小鼠αv-整合素单抗的靶向微泡（MBav）和携同型抗体的对照微泡（MBiso），并于8min后行超声造影检查，测量双侧后肢的显影强度（VI）。超声分子成像结束后处死小鼠并取后肢骨骼肌行免疫荧光检查。结果术前双侧后肢血流量基本相等，术后0d两组的血流量均下降至15％左右（P〉0．05）。随着时间的推移，两组的血流均有所恢复，其中，Ad—TM组的血流恢复快于Ad—LacZ组；术后28d，Ad—TM组中缺血后肢的血流量达到对侧非缺血后肢血流量的（65．6±8．0）％，较Ad—LacZ组的（30．7±4．5）％显著增加（P〈0．01）。对于非缺血后肢，MBav和MBiso经过8min的循环时间后均未见明显的超声显影，其VI值分别为（5．2±1．5）U、（5．8±2．2）U（P〉0．05）；对于缺血后肢，MBiso经过8min的循环时间后可见轻度的超声显影，Ad—TM组和Ad—LacZ组的VI值分别为（7．9±1．6）U、（8．1±2．0）U（P〉0．05）；而MBav经过8min的循环时间后可见明显的超声显影，而且Ad—TM组的显影强度较Ad—LacZ组明显增强（P〈0．01）。免疫荧光检查显示非缺血后肢未见αv-整合素表达，缺血后肢可见αv-整合素表达，而且Ad—TM组的表达明显多于Ad—LacZ组。结论三突变型HIF-1α基因可有效促进老年小鼠缺血后肢血管新生。

Objective To evaluate the angiogenic response of the HIF-1α with triple-point mutations in aged mice with hind-limb ischemia by ultrasound molecular imaging of αv-integrin expression. Methods Twenty four aged mice under unilateral artery ligation were equally divided into 2 test groups and adenovirus （Ad） vector particles containing the gene for LacZ （Ad-LacZ） or HIF-1α 402/564/803（triple-point mutant, Ad-TM） were injected intramuscularly into the ischemic hind limb. Serial contrast-enhanced ultrasound perfusion imaging of the proximal adductor muscles in the ischemic and contralateral non-ischemic hind limbs was performed （ n = 6 per group at each time）. Ultrasound molecular imaging of the endothelial αv-integrin expression was carried out in mice（ n = 6 per group） on day 7 after surgery at 8 minutes after intravenous injection of either αv-integrin targeting microbubbles or isotype control microbubbles in random with 30 min interval and the video intensity （VI） was measured. Following ultrasound imaging, the hind- limb was harvested for histological examination of αv-integrin expression. Results Immediately after femoral artery ligation, blood flow （as a percentage of that in the non isehemia hind limb） was reduced to 15% in the 2 test groups （ P〉0.05）. Although blood flow gradually recovered over time, recovery was persistently better in the Ad-TM group as compared with the Ad-LacZ group. On postoperative day 28, the blood flow ratio was significantly increased in the Ad-TM group （65.6 ± 8.0） % as compared with the Ad- LaeZ group （30.7 ±4.5）% （ P 〈0.01）. Minimal ultrasound signals were obtained with both MBαv[VI, （5.2 ± 1.5） U] and MBiso [VI, （5.8 ± 2.2） U] in the non ischemie hind-limb. Progressive signal enhancement was noted in the isehemie hind limbs with MBav in the order of the Ad-lacZ group [VI, （15.9 ±2.8）U ], Ad-TM group [VI, （30.8± 5.1 ）U ], whereas no significant change of signals was noted in the Ad-TM group[VI, （7.9± 1.6）U] and Ad-LacZ group[VI, （8.1 ±2.0）U] with MBiso. No αv-integrin expression was observed in the non-isehemie hind-limb, a marked endothelial αv-integrin expression in ischemie hind-limb was confirmed by immunofluorescenee, especially in the Ad TM group. Conclusions The angiogenic response is enhanced in the aged mice with hind-limb ischemia by using HIF-1α with triple- point mutations.