血管内皮细胞生长因子基因转染血管内皮祖细胞移植促进缺血皮瓣存活的实验研究

目的探讨VEGF165基因转染血管内皮祖细胞(EPCs)移植促进缺血皮瓣的血管新生,提高皮瓣存活率。方法体外分离、培养人脐血中EPCs,利用脂质体介导血管内皮细胞生长因子165(VEGF165)基因体外转染EPCs,然后移植于裸鼠随意皮瓣,皮瓣早期断蒂。结果脐血中分离培养的EPCs表达CD34、KDR及CD133,VEGF165基因转染EPCs体外及体内检测均有VEGF165蛋白的表达。转染VEGF165基因的EPCs组和EPCs组移植裸鼠皮瓣后,EPCs整合到缺血部位新生血管中,与对照组的皮瓣存活率分别为97 2% ±2 8%、60 3% ±2 1%、34 2% ±1 8% (P<0 05 ),而且前二组毛细血管密度、血流灌注差异均有统计学意义(P<0 05),较对照组均有明显改善(P<0 05)。术后第7d时三组皮瓣中的EPCs密度分别为136个/mm2 ±10个/mm2、75个/mm2 ±6个/mm2、0个/mm2 (P<0 05 ); 第11天时EPCs密度分别为305个/mm2 ±26个/mm2、199个/mm2 ±18个/mm2、0个/mm2 (P<0 05)。结论脐血中的EPCs体外培养后移植体内可促进缺血皮瓣的血管新生,提高存活率,而转染VEGF165基因的EPCs具有更强大的促血管新生的作用。

Promotion of the survival of ischemic skin flap by transplanted endothelial progenitor cells transfected with VEGF165 gene: an experimental study with mice

OBJECTIVE: To investigate the feasibility of transplanted endothelial progenitor cells transfected with VEGF165 gene to ischemic flap with increased neovascularization and augmented the survival areas.METHODS: EPCs were isolated from human cord blood and cultured in vitro. Plasmid PcDNA3.1(-)/VEGF165 containing VEGF gene was transfected into the EPCs. EPCs transfected with blank plasmid, and EPCs without transfection were used as controls. ELISA was used to detect the expression of VEGF protein in the culture fluids. The EPCs were dyed with CM-DiI 7 days later. Ischemic skin flaps were made on the backs of 27 nude mice. The mice were randomly divided into 3 equal groups with their skin flaps being transplanted with EPCs transfected with 3.1(-)/VEGF165 plasmid, EPCs not transfected with 3.1(-)/VEGF165 plasmid, and injected with M199 medium at the basal part. Four days after the peduncles of the skin flaps were cut. Seven days after the cutting-off of the peduncles the survival rate of skin flap was observed and the blood perfusion was observed with laser Doppler flowmetry, 10 days after the density of capillary arteries were observed with microcirculation microscope. Three specimens of skin flap were taken 7 and 11 days after the skin flaps were made to undergo histological examination to detect the density of capillary arteries by CD34 immunohistochemistry and to observe the proliferation of EPCs with fluorescence microscopy. Peripheral blood samples were collected 1, 4, 7, 14, and 28 days after the skin flaps were made to undergo ELISA to detect the levels of VEGF protein RESULTS: The VEGF levels in the culture supernatants of the groups A, B, and C were 352 ng/L +/- 35 ng/L, 45 ng/L +/- 5 ng/L, and 0 ng/L respectively with significant difference between any 2 groups (all P < 0.05). The skin flap survival rates of the three groups were 97.2%, 60.3%, and 34.2% respectively with significant difference between any 2 groups (all P < 0.05) and the survival quality of the group A was the best. The capillary density of the group A was greater than those of the groups B and C. The VEGF levels at any time point of the group A were all significantly higher than those of the group B and C (all P < 0.05) and there was not a significant difference between the groups B and C. The capillary density levels at different time points decreased progressively in the order of groups A, B, and C with significant difference between any 2 groups (all P < 0.05). No EPC was shown by fluorescence microscopy in the skin flaps of the group C. The EPC density in skin flap 7 and 11 days after the flaps were made were 136 +/- 10 and 75 +/- 6/mm(2) and 305 +/- 26 and 199 +/- 18/mm(2) respectively with significant differences between the groups A and B. (both P < 0.05). CONCLUSION: The EPCs from human cord blood, especially those transfected with VEGF165 gene increases the neovascularization in ischemic skin flaps and augments their survival rate.