血管内皮生长因子165基因修饰的人脂肪间充质干细胞与改性丝素蛋白体内构建血管化组织工程脂肪的研究

摘要

目的：探讨血管内皮生长因子165（VEGF165）基因修饰的人脂肪间充质干细胞（hADSCs）与壳聚糖修饰的丝素蛋白支架材料体内构建血管化组织工程脂肪的可行性。方法分离提取hADSCs，选取生长状态良好的第3代hADSCs，用携带重组人VEGF165基因的慢病毒进行感染（感染复数=50）。选用慢病毒感染和未感染的第3代hADSCs分别接种于支架材料上，记为实验组与对照组，同时成脂诱导5 d后，使用氯甲基苯甲酰胺标记细胞。取24只雌性Wistar大鼠，将上述实验组与对照组细胞—支架复合物移植于背部皮下，每组12只。移植后8，12周分别取材并行冰冻切片、HE染色、油红O染色及扫描电镜，同时观察支架材料的降解情况，观察移植物。免疫印迹法检测VEGF165及过氧化物酶体增殖体激活受体-γ2（PPARγ-2）蛋白的表达。结果移植后8，12周，免疫印迹法结果显示实验组移植物均表达VEGF165，对照组未见表达；实验组与对照组均表达PPARγ-2，实验组中的表达量明显多于对照组（P<0.05）。扫描电镜、油红O染色及HE染色均表明实验组与对照组生成大量脂肪样细胞，且实验组明显多于对照组（P<0.05）。结论携带重组人VEGF165基因的慢病毒感染的hADSCs与壳聚糖/丝素蛋白支架材料复合物在大鼠体内可持续表达VEGF165及PPARγ-2蛋白，能显著促进工程化脂肪组织的构建，为血管化组织工程脂肪的构建奠定了基础。%Objective To explore the feasibility to construct vascularized tissue engineered adipose with human adipose-derived mesenehymal stem cells (hADSCs) modified by human vascular endothelial growth factors 165(VEGF165) gene and a novel 3-D silk fibroin scaffold modified by chitosan (CS/SF) in vivo. Methods Isolated, cultured human adipose derived stem cells to the third generation(P3 hADSCs), then infected the cells with lentivirus vector carrying human recombinant VEGF165 gene(MOI=50). Infected and uninfected P3 hADSCs seeded on CS/SF were set as experimental group (E) and control group (C) respectively, both groups were induced by adipogenic differentiation medium for 5 days and labelled by CM-DIL. 24 female Wistar rats were divided into two groups, 12 in each one. Cell-scaffold complexs were transplanted under subcutaneous layer in the rats. 8 weeks and 12 weeks later, complexes in each group were taken out and ex-amined for frozen section, HE staining, oil red O staining and scanning electron microscopy, scaffold degra-dation was also observed at the same time. The expression of VEGF165 and PPARγ-2 were detected by Western bolt. Results 8 weeks and 12 weeks after implanted, Western blot results showed that the grafts in E group expressed VEGF165, while no expression were found in C group. Both E and C group expressed PPARγ-2, the expression levels of PPARγ-2 in E group was higher than those in C group (P<0.05). Scan-ning electron microscopy, oil red O staining and HE staining all showed that E and C groups both generated a lot of fat cells, furthermore the numbers of fat cells in E group was higher than that in C group (P<0.05). Conclusion Complexes of hADSCs infected by lentivirus vector carrying human recombinant VEGF165 gene and CS/SF could not only continuously express VEGF165 and PPARγ-2 in rats, but also promote to reconstruct tissue engineered adipose, this result might lay a solid foundation to reconstruct vascularized tissue engineered fat in vivo.