基因转染脐带间充质干细胞与丝素蛋白构建组织工程脂肪

背景：目前国内外构建组织工程脂肪的方法并不完善，虽然构建出了脂肪，但效果不理想。目的：探讨一种新的构建组织工程脂肪的方法，观察携带重组人胰岛素基因慢病毒载体转染的人脐带间充质干细胞与丝素蛋白复合后在Wistar大鼠体内构建组织工程脂肪的能力。方法：分离培养人脐带间充质干细胞，将携带重组人胰岛素基因慢病毒载体以最适MOI=10转染人脐带间充质干细胞，将转染组与未转染组（对照组）的人脐带间充质干细胞分别接种于丝素蛋白支架，移植于Wistar大鼠背部皮下。移植后12周取材，行荧光原位杂交技术鉴定、组织形态学和扫描电镜观察。结果与结论：①油红O染色显示，两组移植物均呈阳性，证明移植物己在体内合成为脂肪组织，转染组脂肪样细胞数量明显高于对照组（P〈0．01）。②苏木精一伊红染色显示，两组移植物组织结构与正常脂肪组织相似，可见明显新生血管。转染组支架材料降解明显，炎性细胞浸润明显少于对照组，新生血管数量也多于对照组。③扫描电镜观察提示，转染组移植物内脂肪样细胞聚集成团，其结构与正常脂肪组织相似；对照组脂肪样细胞散在分布于支架孔隙内。④提示胰岛素基因能明显促进人脐带间充质干细胞成脂肪化，携带重组人胰岛素基因慢病毒载体转染的人脐带间充质干细胞与丝素蛋白支架复合后，在Wistar大鼠体内能构建出组织工程化脂肪组织，其结构类似正常脂肪组织。

Constructing tissue-engineered adipose with the combination of gene-transfected human umbilical cord mesenchymal stem cells and silk fibroin scaffold

BACKGROUND： The method of construction of tissue-engineered adipose is imperfect at present. Although the tissue-engineered adipose can be constructed, the efficacy is not satisfactory. OBJECTIVE： To study the new method of construction of tissue-engineered adipose, in order to observecapacity of human umbilical cord mesenchymal stem cells transferred with recombinant insulin gene lentivira vector combined with silk fibroin scaffold in the construction of tissue engineering adipose in Wistar rats. METHODS： Human umbilical cord mesenchymal stem cells were separated and cultured, and then transfected with recombinant insulin gene lentiviral vector （transfected group） by the best multiplicity of infection =10. The nontransfected human umbilical cord mesenchymal stem cells were regarded as control group. The human umbilical cord mesenchymal stem cells in the transfected group and the control group were seeded onto the silk fibroin scaffold and implanted into the subcutaneous layer of Wistar rats. At 12 weeks after implantation, the transplants were taken, and then identified with fluorescence in situ hybridization and observed with histomorphology and scanning electron microscopy. RESULTS AND CONCLUSION： Oil red O staining showed the transplants in two groups were positive, suggesting that the transplants were synthesized in adipose tissue, and the number of fat-like cells in the transfected group was significantly higher than that in the control group （P 〈 0.01）. Hematoxylin-eosin staining showed significant angiogenesis appeared in or around the new formed tissue, the structure of which was similar to natural adipose tissue. Silk fibroin scaffold in the transfected group was degraded significantly, the number of new vessels in the transfected group was more than that in the control group, and inflammatory cell infiltration in the transfected group was significantly less than that in the control group. Scanning electron microscopy results showed that fat-like cells in the trasnfected group congregated and the structure was similar to that of the normal adipose tissue; the fat-like cells in the control group scattered in the pore of the scaffold. Insulin gene could obviously promote human umbilical cord mesenchymal stem cells to differentiate into adipose; human umbilical cord mesenchymal stem cells transferred with recombinant human insulin gene lentiviral vector composite with silk fibroin scaffolds can construct tissue-engineered adipose in the Wistar rats, and its structure is similar to natural adipose.