脂肪干细胞移植对兔正张力皮瓣回缩率的影响

Effect of local transplantation of adipose stem cells on the recovery rate of positive tension skin flaps in rabbits

目的观察脂肪干细胞移植对兔正张力皮瓣回缩率的影响。方法取新西兰大白兔16只,无菌条件下分别取腹股沟脂肪,酶消化法提取脂肪干细胞(ADSCs)并培养至第3代,流式细胞仪器检测特异性标志物,成脂、成骨诱导鉴定其分化潜能。对移植ADSCs行CM-Di I荧光染色。于兔臀背部制作正张力皮瓣,皮瓣表面设计2 cm×2 cm注射干细胞区域。将实验动物随机分为2组,实验组注射1×105 U ADSCs悬液1 ml,对照组注射同剂量的培养基。分别于第3、7、14天测量设计区域面积,计算回缩率;第14天处死动物,取移植皮瓣ADSCs注射区域组织行苏木精-伊红(HE)染色,酶联免疫吸附试验(ELISA)法检测组织中血管内皮生长因子(VEGF)的表达,免疫组织化学法检测组织中CD31表达并计算微血管数量。结果16只动物模型均成功,术后第7、14天实验组ADSCs注射区域面积回缩率分别为(24.84±4.67)%和(28.41±4.54)%,对照组为(30.22±3.35)%和(34.91±3.65)%,组间差异有统计学意义(t=2.650,P<0.05;t=3.160,P<0.05)。实验组移植皮瓣组织中依CD31表达计算微血管数目为(8.63±2.00)个/视野,对照组为(5.13±1.13)个/视野,组间差异有统计学意义(t=4.320,P<0.01)。实验组移植皮瓣组织中VEGF的含量为(213.95±26.05) pg/mg,对照组为(169.40±24.94) pg/mg,组间差异有统计学意义(t=3.500,P<0.01)。荧光示踪可见ADSCs可在皮瓣皮肤组织成活。结论移植ADSCs可于大白兔皮瓣中成活,并通过促进正张力皮瓣血管再生的方式,缩小皮瓣回缩率。

Objective To investigate the effect of adipose-derived stem cells (ADSCs) transplantation on the retraction rate of positive tension skin flaps of rabbits. Methods Sixteen New Zealand white rabbits were taken for inguinal fat extraction under aseptic conditions. ADSCs were extracted by enzymatic digestion and cultured to the third generation. Flow cytometry was used to detect its specific markers, and adipogenic and osteogenic induction were used to identify its differentiation potential. The ADSCs were stained with CM-Di I fluorescence. Positive tension flap on the buttock and back of rabbits were Designed, and the skin flap surface design was 2 cm×2 cm with stem cell injection area. The rabbits were randomly divided into two groups, and the experimental group was injected with 1×105 units of stem cell suspension (1ml), and the control group was injected with the same amount of culture medium. The area of the design area was recorded at 3, 7 and 14 days respectively to calculate the skin flap retraction rate. On the 14th day, the animals were executed and the regional tissue of the designed skin flaps was taken out. Hematoxylin and eosin (HE) staining was used to observe the histology of expanded skin flaps, and enzyme linked immunosorbent assay (ELISA) was used to detect the expression of vascular endothelial growth factor (VEGF) in tissues. Immunohistochemical staining was used to detect the expression of CD31 in tissues and calculate the number of microvessels. Cell tracing was performed by fluorescent staining of tissues. Results On the 7th and 14th day, the retraction rate of the experimental group was significantly lower than that of the control group (P<0.05). The expression of CD31 in the experimental group was significantly higher than that in the control group and the microvessel density, and the content of vascular endothelial growth factor in the experimental group was significantly higher than that in the control group (P<0.05). Fluorescence tracing showed that the adipose stem cell family survived in the skin tissue of the flaps. Conclusion The ADSCs can survive in the flaps, and local transplantation of ADSCs could significantly reduce the contraction rate of positive tension skin flaps by promoting vascular regeneration.