Experimental Study Of The Feasibility Of Promoting The Formation Of Tissue Engineered Fat With Human Umbilical Cord Mesenchymal Stem Cells Transfected By Human Insulin Gene

Objective:To study the feasibility of promoting the formation of tissue engineered fat with human umbilical cord mesenchymal stem cells (hUCMSCs) transfected by human insulin gene.Methods:1.Passage4hUCMSCs were collected and transfected with adenovirus particles with the multiplicity of infection of20. The experiment was divided into four groups. Passage4hUCMSCs were included in the control group (C group). Passage4hUCMSCs tranfected with adenovirus particles were included in experimental group1(E1group), passage4hUCMSCs+adipogenic liquid were used in the experimental group2(E2group), and passage4hUCMSCs transfected with adenovirus particles+adipogenic liquid were used in experimental group3(E3group). After transfected for72hours, the experimental groups were cultured in adipogenic differentiation medium for14days, then which were detected by oil red O.2. hUCMSCs infected with Ade-insulin-EGFP were seeded in fibroin3D scaffolds with uniform50-60mmpore size. Silk fibroin scaffolds with untransfected hUCMSCs were used as control. They were cultured for4days in adipogenic medium and transplanted under the dorsal skins of female Wistar rats after the hUCMSCs had been labelled with Chloromethylbenzamido-1,1’-Dioctadecyl-3,3,3’3’-Tetramethylindocarbocyanine Perchlorate (CM-Dil). Macroscopical impression, fluorescence observation, histology, and scanning electron microscope (SEM) were used for assessment after transplantation at8and12weeks.Results:1. After transfected for48hours, weak fluorescence in human umbilical cord mesenchymal stem cells was observed in experimental groups1and3through the fluorescence microscope and strong fluorescence appeared after transfected for72hours.After cultured with adipose-inducing culture medium for14days, the cells in the experimental groups1,2,3were red under microscope after oil red O staining, no lipid droplets were observed in the control group; the lipid droplets in the experimental group3were larger and more than those in the experimental group.Quantitative analysis showed the positive area of oil red O staining and the absorbance value in the experimental group3were greater than those in the experimental group2(P<0.05).2. Fluorescence observation supported that the formed adipose tissue originated from seeded hUCMSCs rather than from possible infiltrating perivascular tissue. Oil red O staining of newly formed tissue showed that there was substantially more tissue regeneration in the experimental group than in the control group. SEM showed that experimental group cells had more fat-like cells, whose volumewas larger than that of the control group, and degradation of the silk fibroin scaffold was greater under SEM observation.Conclusion:1.Adenoviral transfection of the human insulin gene could promote adipogenic differentiation of the human umbilical cord mesenchymal stem cells.2. hUCMSCs tansfected by adenovirus particles have good compatibility with silk fibroin scaffold, and adenoviral ransfection of the human insulin gene could be used for the construction of tissue-engineered adipose.