【作者】 王刚；

【导师】 刘毅；

【作者基本信息】 兰州大学 ， 外科学， 2014， 硕士

【摘要】 目的：探讨人脂肪间充质干细胞与京尼平交联的I型胶原蛋白支架材料构建组织工程脂肪的可行性。方法：1.分离培养人脂肪间充质干细胞(hADSCs),传代培养至第3代,接种于京尼平交联的I型胶原蛋白支架材料,测定细胞粘附率；MTT法评估hADSCs在支架材料上的粘附和增殖情况,支架材料对细胞的毒性作用；光镜和电镜分别观察hADSCs在支架材料上的粘附和生长,以及细胞形态学变化,评估]DSCs与京尼平交联的I型胶原蛋白材料的生物相容性。2.体外复合培养成脂和成血管内皮细胞诱导的hADSCs.实验分为3组,A组为实验组：：ADSCs和诱导的血管内皮细胞按4：1比例接种于支架材料；B、C组为对照组,B组：单纯接种1ADSCs:C组：单纯接种诱导的血管内皮细胞。细胞接种于支架材料并成脂诱导5-7天后,对细胞—支架复合物行油红O染色,RT-PCR检测成脂特异性基因PPAR γ-2的表达,扫描电镜观察支架材料上细胞的生长及成脂情况。3.体内实验：将上述A.B.C3组细胞-支架复合物及空白支架材料(D组)分别植入雌性Wistar大鼠大鼠背部皮下。12周后,取材并观察移植物,移植物行组织形态学和扫描电镜观察,]RT-PCR检测成脂特异性基因PPAR γ-2的表达。结果：1.hADSCs接种于支架材料后,能够迅速在材料上粘附、增殖,其平均黏附率为86.5%；光镜和扫描电镜显示hADSCs在支架材料上粘附性良好,随着时间的推移,细胞逐渐增多,可以迁移进入支架内部并均匀分布。2.体外实验：3组细胞在材料上均生长良好。RT-PCR检测到A、B两组成脂特异性基因PPAR γ-2的表达；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细胞,C组则没有脂肪细胞生成。A、B两组和C组支架材料上的脂肪细胞数量有显著差异(P<0.01)。3.体内实验：A、B两组移植物油红O染色均呈阳性,HE染色显示A组成熟脂肪细胞和新生血管数量均明显多于B组,有显著差异(P<0.01),C组、D组移植物内未见成熟脂肪细胞和新生血管形成；各组材料较植入时均明显降解,且A组材料较其它各组降解更为明显。结论：1、hADSCs在京尼平交联的Ⅰ型胶原蛋白支架材料上能很好地粘附、增殖,材料对hADSCs的毒性较低,交联后的支架材料与1ADSCs具有良好的体外生物相容性。2、hADSCs成血管内皮细胞诱导分化后,和hADSCs复合接种于支架材料,在体外能成功地构建组织工程化脂肪。3、复合移植在体内更能显著促进构建组织工程化脂肪,并能促进构建物内的血管新生。更多还原

【Abstract】 Objective To investigate the feasibility of constructing tissue engineered adipose with human adipose-derived stem cells (hADSCs)and Genipin cross-linked type I collagen sponge scaffoldMethods1. Isolated and cultured hADSCs to the third generation, and the cells were seeded on Genipin cross-linked type I collagen sponge scaffold. Determined cell adhesion rate, and MTT assay was used to evaluate the adhesion and proliferation of cells on the scaffold,and the toxic effects of Genipin cross-linked type I sponge collagen on hADSCs. Optical microscopy and scanning electron microscopy were utilized to observe the adhesion and growth process of hADSCs on the scaffold, and the morphological changes of cells.2Endothelial cells and adipogenic cells both induced from ADSCs were co-cultured on the scaffolds in vitro, the experiment was divided into three groups. In the experiment Group A, adipogenic cells and endothelial cells were seeded at a ratio of4:1. We seeded respectively endothelial cells and adipogenic cells on the scaffolds as Group B and C. After seeded the cells on the scaffolds,the cell-scaffold complexes were cultured in adipogenic introduce medium for5to7days, then Oil red O was adopted to detect adipogenic cells. The expression of specific gene PPARy-2was tested through RT-PCR. Then scanning electron microscope was performed to observe the cells’ growth on scaffolds.3Above three groups of complexes and another blank scaffold set as group D were transplanted under subcutaneous layer in female Wistar rats.12weeks after transplanted,the complexes were taken out from the rats and examined through the above methods. Simultaneously,the degradation was observed.Apart from that, the feasibility of contrUsting tissue engineered adipose in vivo and vascularization of tissue engineered adipose were evaluated. The expression of specific gene PPARy-2was tested through RT-PCRResults1hADSCs could adhere to the scaffold immediately after seeded and increase gradually on the scaffold,and the average adhesion rate of hADSCs on the scaffold was86.5%. Optical microscopy and scanning electron microscopy showed that hADSCs adhered on the material well. The cells increased gradually over time, and could migrate into the interior of scaffold, and distributed evenly with the passage of time when observed with optical microscopy.2In vitro experiment, cells could be seen in Group A, B and C, and they all grew well. The expression of PPARy-2was detected in Group A and B, while no expression was found in group C. Oil Red O staining result indicated that after adipogenic induction, Group A and B were successfully induced to generate a large number of mature adipocytes, meanwhile no adipocytes were found in Group C. The number of adipocytes in Group A and B showed a significant statistical difference when compared to Group C(P<0.01).3In vivo experiment,Oil red O staining was positive in Group A and B, the amount of adipocytes and new formed vessels in Group A were obviously more than that in Group B, there was a significant statistical difference between Group A and B(P<0.01). Scaffolds degraded significantly in four groups,and compared to other groups, scaffolds of Group A degraded more apparently.Conclusions1. hADSCs could adhere and proliferate well on Genipin cross-linked type I collagen sponge scaffold. The biomaterial possessed very low cytotoxicity to the cells, and the material showed outstanding biocompatibility in vitro.2. Endothelial cells induced from hADSCs and hADSCs co-cultured on Genipin cross-linked type I collagen sponges could generate mature adipocytes after adipogenic induction.3. Co-transplanting of endothelial cells and adipogenic cells both induced from hADSCs could significantly promote constructing tissue engineered adipose and angiogenesis.更多还原