细胞移植术治疗兔下肢缺血模型的影像学评价

目的:利用血管内皮生长因子(VEGF_(165))基因转染体外诱导的血管内皮祖细胞(EPCs)治疗慢性下肢缺血兔模型,探讨CTA、DSA及多普勒超声检查对缺血下肢侧支循环形成评价的敏感性,并比较各种检查方法的差异性。方法:①制作高脂血兔,梯度离心法分离兔骨髓单个核细胞(MNCs),用内皮细胞专业培养基(EGM-2)诱导培养EPCs,脂质体介导转染携带增强型绿色荧光蛋白EGFP标记的VEGF_(165)质粒转染EPCs,流式细胞仪检测整体转染率。②制作兔单侧下肢缺血模型,随机分为A、B、C组,分别移植注射未转染EPCs(EPCs组)、VEGF_(165)转染的EPCs(EPC/VEGF组)、EGM-2培养基(EGM-2组),多种方法检测移植效果。结果:①自兔骨髓诱导出的梭形贴壁细胞为EPCs。②流式细胞仪检测其总体转染率约22.5%。③DSA、CTA、多普勒超声及免疫组化显示移植基因修饰后的EPCs组比未转染EPCs组能更好地促进缺血肢体新生血管形成,改善缺血肢体血运。DSA较CTA对新生血管显示更清晰,各处理组内对2种检查结果进行比较,结果均具有统计学意义(P0.05)。结论:CTA、DSA及多普勒超声均能客观评价缺血下肢侧支循环建立情况,DSA对新生血管的显示上优于CTA,但DSA为有创检查,技术要求高,CTA为无创性检查,对缺血下肢评价时宜首选CTA检查,超声多普勒用于对治疗效果的长期随访。

Imaging evaluation of transplantation of autologous EPCs for lower limb ischemia

Objective： To explore the efficacy of the transplantation of autologous EPCs transfected with VEGF165 in the treatment of lower limb ischemia, and to explore the sensitivity of CTA, DSA and Doppler ultrasound examinations in the evaluation of collateral vessels formation in lower limb ischemia, and compare their differences. Methods.. Making hypercholesterolemic rabbit model. Rabbit bone marrow mononuclear cells were isolated by density gradient centrifugation,then they were cultivated with EGM-2 culture medium in order to induce them to EPCs, EGFP-N1-VEGF165 transfected into EPCs mediated by lipofectamineTM2000 , VEGF165 transfection efficiency was detected by flow cytometry. We made unilateral hindlimb ischemia rabbit models. The rabbits were randomly divided into three groups which transplanted with EPCs, EPCs transfected with VEGF165 （EPC/VEGF） and EGM-2 respectively, the effection of transplantation was evaluated by different ways. Results： Mononuclear cells isolated were induced successfully; The transduction efficiency in VEGF transduced EPCs was 22.5% detected by flow cytometry;The results of DSA. CTA, Doppler and immunohisto- chemisty indicated that the effects of transplantation with EPCs transfected was better VEGF165 than the other groups. There were sig- nificant differences in every group （ P 〈0. 01）, the neovasculars were showed more clarity in DSA than that in CTA. There were significant differences between the two examinations in every group （ P 〈0. 05）. Conclusion： CTA, DSA and Doppler ultrasound can objectively evaluate the situation of collateral circulation in lower ischemic limbs, the neovasculars were showed more clarity in DSA than that in CTA. However, DSA is an invasive device and with high technical requirement. CTA and Doppler ultrasound were non-invasive examinations, so we preferred CTA to evaluate the lower ischemic limb first, Doppler ultrasound for the treatment of long-term follow-up.