【摘要】 目的比较两种不同肌筋膜瓣包裹脂肪来源干细胞(adipose-derived stromal cells,ADSCs)与胶原蛋白支架复合物在体内成脂效率的差异,为临床游离脂肪组织的高效移植提供实验依据。方法3～4月龄新西兰大白兔10只,雌雄不限,体重2.0～2.5 kg。取兔颈部皮下脂肪组织分离ADSCs并行原代及传代培养,待第3代细胞生长至瓶底70%～80%面积时,以BrdU(10μg/mL)标记细胞48 h,行免疫荧光染色观察;成脂诱导2周后行油红O染色观察;成骨诱导3周后行茜素红染色观察;成软骨诱导2周后行阿利辛蓝染色观察。另取第3代经BrdU标记的ADSCs成脂诱导2周后,以1×107个/块接种于10 mm×10 mm×5 mm大小的ColⅠ中,制备细胞载体复合物。实验分为两组,A组由带血管蒂的右侧背阔肌筋膜瓣包裹复合物,B组由无特定血管蒂的右侧臀大肌筋膜瓣包裹复合物,每只动物均采用自体ADSCs移植及自身对照。术后8周取出移植物,经HE染色和免疫组织化学染色鉴定新生组织,测定两组新生组织的湿重和微血管数,并采用免疫荧光染色鉴定新生组织及微血管内皮细胞的来源。结果荧光显微镜下BrdU标记为阳性的ADSCs胞核呈绿色荧光,ADSCs阳性标记率>90%。第3代ADSCs成脂诱导2周,可见细胞内有红色脂滴形成;成骨诱导3周,可见红色钙结节样改变;成软骨诱导2周,可见高度聚集生长的细胞团,阿利辛蓝染色呈蓝色。术后8周取材,A组可见血管增生并长入材料,未见明显纤维包裹;B组有少量血管长入材料。A组新生组织湿重为(0.149 5±0.017 3)g,B组为(0.095 3±0.012 7)g,比较差异有统计学意义(P<0.01)。HE染色均可见移植物中有新生脂肪组织形成和不同程度微血管长入,支架材料已基本降解吸收。A组微血管数为(31.2±4.5)根,B组为(19.3±2.6)根,比较差异有统计学意义(P<0.01)。术后8周新生组织免疫荧光染色示,A、B组新生脂肪细胞的胞核及部分微血管内皮细胞的胞核呈绿色荧光。结论带血管蒂的背阔肌筋膜瓣包裹ADSCs与胶原蛋白支架复合物在体内的成脂效率较无特定血管蒂的臀大肌筋膜瓣高。更多还原

【Abstract】 Objective To compare two kinds of myofascial flap encapsulating adipose-derived stromal cells(ADSCs) in adipogenic e cacy in vivo,and to provide experimental basis for the e cient transplantation of free adipose tissue.Methods ADSCs were isolated from the subcutaneous adipose tissue in the neck of 10 New Zealand rabbits(aged 3-4 months old,male and female,weighing 2.0-2.5 kg),and primary culture and subculture of ADSCs were conducted.When the cells at passage 3 covered 70%-80% of the bottom of the culture ask,BrdU(10 μg/mL) was applied to label the cells for 48 hours before performing immuno uorescence staining.Oil red O staining observation was conducted to those cells 2 weeks after being induced towards adipocyte,alizarin red staining observation was performed 3 weeks after being induced towards osteoblast,and alcian blue staining was conducted 2 weeks after being induced towards chondrocyte.Besides,after being induced towards adipocyte for 2 weeks,1 × 107 ADSCs/piece at passage 3 labeled by BrdU was seeded into Col I(10 mm × 10 mm × 5 mm/piece) to prepare cell carrier complex.The experiment was divided into two groups: group A in which vascular pedicled dextral latissimus dorsi fascial ap was adopted to encapsulate the complex;group B in which dextral gluteus maximus fascial ap with no speci c vessel pedicle was applied to encapsulate the complex.Rabbits in each group went through autogenous ADSCs transplant and self control.The implants were dislodged 8 weeks after operation,HE staining and immunohistochemistry staining were performed to testify cambium,the wet weight and micro vessel count of the cambium in each group were tested,immuno uorescence staining was performed to determine the origin of cambium and microvascular endothelium.Results The nucleus of ADSCs positive for BrdU labeling showed green uorescence under uorescence microscope,with the positive labeling ratio of ADSCs above 90%.For ADSCs at passage 3,the formation of red lipid droplets within cells was observed 2 weeks after being induced towards adipocyte,red calcium nodules were evident 3 weeks after being induced towards osteoblast,and highly congregated cell mass positive for alcian blue staining appeared 2 weeks after being induced towards chondrocyte.Eight weeks after operation,neogenetic blood vessel grew into sca olds and no obvious bre encapsulation was observed in group A,while few blood vessel grew into sca olds in group B.The wet weight of cambium in group A and B was(0.149 5 ± 0.017 3) g and(0.095 3 ± 0.012 7) g,respectively,indicating there was a signi cant di erence between two groups(P < 0.01).HE staining showed the formation of neogenetic adipose tissue and the growth of micrangium in the implant,and the degradation and absorption of sca old.The micro vessel count of group A and B was 31.2 ± 4.5 and 19.3 ± 2.6,respectively,indicating there was a signi cant di erence between two groups(P < 0.01).Eight weeks after operation,the immuno uorescence staining of cambium showed that the cell nucleus of regenerated adipocytes and partial capillary endothelium in groups A and B presented green fluorescence.Conclusion ADSCs encapsulated by vascular pedicled latissimus dorsi fascial ap and collagen protein sca old complex has a higher adipogenic e cacy in vivo than the gluteus maximus fascial ap with no speci c vessel pedicle.更多还原