FOS样抗原1修饰间充质干细胞移植促进缺血肢体血管新生的实验研究

The experimental study of transplantation of FOS like antigen 1 modified mesenchymal stem cells promoting angiogenesis in ischemic limbs

目的 探讨FOS样抗原1(FOSL1)修饰的间充质干细胞(MSCs)体内移植后对缺血肢体血管新生的影响。方法 选用6~8周龄SPF级雄性NOD-SCID免疫缺陷小鼠30只,通过完全结扎股动脉构建动脉缺血模型。分别将1 mL PBS、 MSCs、FOSL1修饰的MSCs细胞悬液通过肌肉注射移植至缺血肢体,据此分为PBS组(n=10)、 MSCs组(n=10)和FOSL1组(n=10)。免疫荧光镜下观察组织内绿色荧光蛋白(GFP)与CD34双阳性细胞。免疫组化检测缺血组织血小板内皮细胞黏附分子-1的表达,并比较3组的微血管/肌纤维比值。通过Western blot观察缺血组织肝细胞生长因子(HGF)、成纤维细胞生长因子2(FGF2)及血管内皮生长因子(VEGF)的表达。激光散斑衬比分析(LASCA)3组缺血肢体的血流灌注情况。结果 完全结扎小鼠股动脉后,LASCA显示造模侧肢体几乎未见血流。体内移植后第2天,MSCs组与FOSL1组均有少量GFP与CD34双阳性细胞。体内移植后第14天,FOSL1组的微血管密度高于MSCs组[(1.95±0.28)vs(1.40±0.20),P<0.05]。FOSL1组缺血组织FGF2、 HGF和VEGF的表达高于MSCs组(均P<0.05)。FOSL1组血流灌注的恢复程度优于MSCs组(P<0.05)。结论 MSCs体内移植可能在缺血组织内向血管内皮细胞分化,FOSL1增强了MSCs促进缺血肢体的血管新生。

Objective To investigate the effects of transplantation of FOS like antigen 1(FOSL1) modified mesenchymal stem cells(MSCs) on angiogenesis in ischemic limbs in vivo. Methods Thirty SPF male NOD-SCID immunodeficient mice aged 6-8 weeks were selected. The model of arterial ischemia was established by complete ligation of femoral artery. 1 mL PBS, MSCs and FOSL1 modified MSCs cell suspension were respectively transplanted into ischemic limbs by intramuscular injection, and then the mice were divided into PBS group(n=10), MSCs group(n=10) and FOSL1 group(n=10). Double positive cells of green fluorescent protein(GFP) and CD34 were observed under immunofluorescence microscope. The expression level of platelet endothelial cell adhesion molecule-1 in the ischemic tissue was detected by immunohistochemistry assay, and the microvessel/muscle fiber ratio was compared among the three groups. Western blot was used to detect the expression levels of hepatocyte growth factor(HGF), fibroblast growth factor 2(FGF2) and vascular endothelial growth factor(VEGF) in the ischemic tissues. Laser speckle contrast analysis(LASCA) was used to analyze the blood perfusion of the ischemic limbs in 3 groups. Results After the femoral artery of mice was completely ligated, LASCA showed that there was almost no blood flow in the limb on the modeling side. On the second day after transplantation, a small number of GFP and CD34 double positive cells were found in both MSCs group and FOSL1 group. On the 14 th day after transplantation in vivo, the microvessel density of the FOSL1 group was higher than that of the MSCs group [(1.95±0.28) vs(1.40±0.20), P<0.05], the expression levels of FGF2, HGF and VEGF in the ischemic tissue of the FOSL1 group were higher than those of the MSCs group(all P<0.05), the degree of blood perfusion recovery of the FOSL1 group was better than that of the MSCs group(P<0.05). Conclusion MSCs transplanted in vivo may differentiate into vascular endothelial cells in ischemic tissues, and FOSL1 enhances MSCs to promote angiogenesis in ischemic limbs.