阳离子聚合物携带HIF-1α基因传递对小鼠后肢缺血的影响

PC mediated HIF-1α gene delivery for treatment of hind limb ischemia in mouse model

目的 研究阳离子聚合物(PC)携带缺氧诱导因子1α(HIF-1α)基因传递对C57BL/6小鼠后肢缺血模型血管再生的影响。方法 PC经静电作用结合HIF-1α为PC-HIF-1α,在人脐静脉血管内皮细胞(HUVECs)完成细胞转染实验,18只C57BL/6小鼠构建后肢缺血模型成功后,按随机数字表法分为PBS组、PC组和PC-HIF-1α组,每组6只。术后第1、3、5天,PBS组在小鼠左后肢腓肠肌肌肉注射100μL PBS,在同样的时间和部位,PC组肌肉注射100μL PC溶液,PC-HIF-1α组肌肉注射100μL PC-HIF-1α。7 d后,取小腿缺血侧腓肠肌,行血小板-内皮细胞黏附分子CD31免疫荧光染色。观察PC-HIF-1α粒径及HUVECs细胞对PC-HIF-1α的摄取情况以判断3组小鼠缺血后的血流恢复情况。结果 PC-HIF-1α粒径为150~200 nm;PCHIF-1α的细胞摄取效率较高,且依赖于细胞骨架。PC-HIF-1α组左后肢与右后肢的血流灌注比恢复到86.00%,而PBS组和PC组仅为50.17%和51.00%,与PBS和PC组相比,PC-HIF-1α组血流灌注比显著增加(P<0.01);在显微镜同一视野下,与PBS和PC组相比,PC-HIF-1α组血管数量显著增加(P<0.01),且缺血区域纤维母细胞增生。结论 PC-HIF-1α治疗可以促进缺血肢体侧支循环的建立。

Objective To investigate the effect of hypoxia-inducible factors 1 alpha(HIF 1α)gene delivered by cationic polymer(PC)for treatment of hind limb ischemia in mice.Methods The PC was electrostatically bound to HIF-1αto form PC-HIF-1α.The hind limb ischemia model was established in 18 C57BL/6 mice and the blood flow of the model mice was examined by laser Doppler at d1 and d7 after modeling.The model mice were randomly divided into PBS,PC and PC HIF 1αgroups with 6 mice in each group,100μL PBS,PC solution or PC-HIF 1αwere injected at d1,d3 and d5 after modeling,respectively.After 7 d,the ischemic gastrocnemius muscle was taken for CD31 immunofluorescence staining.The size and the intracellular trafficking of PC-HIF-1αwere determined to evaluate the blood perfusion of the ischemic limb in the 3 groups of mice.Results The PC-HIF 1αhad an average size of 150-200 nm.The PC-HIF 1αefficiently mediated cellular uptake and trafficking,which was dependent on cytoskeleton.The recovering of blood perfusion in the hind limb of PC-HIF-1αgroup was significantly higher than that of the PBS group and PC group at d7(86.00%vs.50.17%and 86.00%vs.51.00%,respectively;both P<0.01).Intramuscular injection of PC-HIF-1αsignificantly increased the number of vessels/per field and the expansion of fibroblasts(both P<0.01).Conclusion PC-HIF-1αcan stimulate the early establishment of effective collateral circulation in mice with hind limb ischemia.