人脂肪间充质干细胞与京尼平交联的Ⅰ型胶原蛋白体外构建组织工程脂肪的实验研究

Experimental study on construction of tissue engineered adipose with human adipose-drived mesenchymal stem cells and Genipin cross-linked type Ⅰ sponge collagen scaffold in vitro

目的探讨人脂肪间充质干细胞（h ADSCs）与京尼平交联的Ⅰ型胶原蛋白体外构建组织工程脂肪的可行性。方法选取生长良好的第3代h ADSCs,成血管内皮细胞诱导分化。h ADSCs和诱导的血管内皮细胞按4∶1接种于支架材料上共培养作为A组,单纯接种h ADSCs为B组,单纯接种诱导的血管内皮细胞诱导为C组。三组细胞接种于支架材料后,成脂诱导5~7 d,细胞—支架复合物行油红O染色,反转录-PCR检测成脂特异性基因过氧化物酶体增殖物激活受体γ-2（PPARγ-2）的表达,扫描电镜观察支架材料上细胞的生长情况。结果三组细胞在材料上均生长良好。反转录-PCR可检测到A、B两组复合物表达PPARγ-2,C组则无PPARγ-2表达;油红O染色表明成脂诱导后,A、B两组均成功诱导生成大量成熟脂肪样细胞,C组则无脂肪细胞生成,A、B两组和C组支架材料上的脂肪细胞数量差异有统计学意义（P〈0.01）。结论 h ADSCs成血管内皮细胞诱导分化后与h ADSCs接种于支架材料上共培养,成脂诱导后,能在京尼平交联的Ⅰ型胶原蛋白支架材料生成成熟脂肪样细胞。

Objective To investigate the feasibility of the construction of tissue engineering adipose with human adipose derived stem cells （hADSCs） and Genipin crosslinked type Ⅰ collagen in vitro. Methods The well-grown third generation hADSCs were selected and induced to differentiate into vascular endothelial cells, hADSCs and the vascular endothelial cells who were implanted and co-cultured on Genipin crosslinked type I collagen at a rate of 4 ： 1 were taken as group A, Pure hADSCs and pure vascular endothelial cells were respectively seeded on scaffolds as control Group B and C respectively. After three groups of cells were seeded on scaffolds, they were cultured with adipogenic differentiation medium for 5 - 7 days to induce the lipid formation. Oil red O was used to stain the complexs of cells and scaffolds, RT-PCR was performed to detect the expression of adipogenic specific gene PPARγ-2. Cells growth status was observed through scanning electron microscopy. Results The cells of Group A, B and C all grew well on scaffolds. PPARγ-2 could be detected in Group A and B by reverse transcription -PCR, while no expression was detected in Group C. Oil red O staining showed a large number of mature adipose were successfully induced in Group A and B while no apparent adipose cells were found in Group C. There was a significant difference in the number of adipose cells between Group A, B and Group C （P 〈 0.01）. Conclusion hADSCs induced and differentiated into vascular endothelial co-cultured with hADSCs on the Genipin crosslinked type I collagen could generate mature adipose after adipogenie induction.