Experimental Study On Construction Of Tissue Engineering Adipose With Human Umbilical Cord Mesenchymal Stem Cells Transfected By Recombinant Human Insulin Gene And Silk Fibroin Scaffold

Objective:To study the feasibility to construct tissue engineering adipose in vitro and vivo with silk fibroin scaffold and human umbilical cord mesenchymal stem cells (hUCMSCs) transfected by lentiviral vector pLenti6.3-insulin-IRES-EGFP carrying recombinant insulin gene.Methods: l.hUCMSCs infected with recombinant lentiviral pLenti6.3-insulin-IRES-EGFP (Group A) at the optimal MOI(MOI=10)were seeded in silk fibroin scaffold; cell adhesion and growth were observed by fluorescent inverted phase contrast microscope and scanning electron microscope; hUCMSCs transfected by EGFP gene (Group B) was regarded as negative control. The cell-scaffold complexes were cultured in adipogenic defferentiation medium for5-7days, then which were detected by oil red O stain and RT-PCR.2. MTT test was used to detect whether recombinant lentiviral vector pLenti6.3-insulin-IRES-EGFP affected the growth and proliferation of hUCMSCs, it was devided into two groups, transfected group and nontransfected group.3. MTT test was employed to detect the growth activity of hUCMSCs seeded in silk fibroin scaffold which had been transfected by insulin-IRES-EGFP gene, it was devided into two groups, cell group and cell-scaffold group.4. hUCMSCs infected with recombinant lentiviral (transfected group) by the best MOI (MOI=10) were seeded in silk fibroin scaffold incubating in10%FBS of LG-DMEM. The nontransfected hUCMSCs were regarded as control. Incubated for1-2days, the medium was changed to adipogenic defferentiation medium for5-7days, and then the cell-scaffold complexes were implanted under subcutaneouslayer in female Wistar rats.12weeks later, the transplants were taken out, HE staning, oil red O staning, scanning electron microscope and fluorescence in situ hybridization were employed respectively to examinate and identify them.Results:1. Afeter5-7days for adipogenic culture, hUCMSCs in group A and group B adhensioned well in the silk fibroin scaffold and secreted a lot of matrix observed by scanning electron microscope. Stained by oil red O. there were fat-like cells exsited in the silk fibroin scaffold, and the numbers of fat-like cells in group A were significantly more than those in group B(P=0.007, P＜0.01). RT-PCR results showed that hUCMSCs both in group A and group B had adipogenic-specific gene expression of PPARy-2, and hUCMSCs in group A expressed much stronger than that in group B, at the same time, in negative control group, there was not any expression observed.2. MTT test showed that there was no significant difference in optical density (D) at each time between transfected group and nontransfected group (P=0.056, P>0.05); it implied that transfecting recombinant lentiviral vector pLenti6.3-insulin-IRES-EGFP hadn’t obviously affected the growth and proliferation of hUCMSCs.3. MTT test demonstrated that there was no significant difference in optical density (D) between cell group and cell-scaffold group (P=0.066, P＞0.05); it implied that the silk fibroin scaffold was not harmful to hUCMSCs.4. After the cell-scaffold complexes were implanted for12weeks, both in transfected group and control group, it suggested the positive results stained by oil red O were observed that adipose tissue is formed in transplants. What’s more, a number of fat-like cells in transfected group were significantly more than those in control group(P＜O.O1). HE staning showed significant angiogenesis appeared in or around the new formed tissue, the structure of which was similar to natural adipose tissue,and the undegradated biomaterials was surrounded by inflammatory cell infiltration. Silk fibroin in transfected group degradated significantly, in this group, the number of angiogenesis was more than that in control group, and inflammatory cell infiltration was significantly less than control group. The gross structure of silk fibroin in control group could still be observed. Scanning electron microscopy results showed that fat-like cells and angiogenesis exsisted in two groups, the structure of transplants in transfected group was more similar to natural adipose tissue.Conclusion:1. Insulin gene could promot hUCMSCs getting into adipose; silk fibroin scaffolds and hUCMSCs carrying recombinant human insulin gene lentiviral vector could effectively construct tissue engineering adipose in vitro.2. hUCMSCs carrying recombinant human insulin gene lentiviral vector and silk fibroin scaffolds could construct tissue engineering adipose in vivo, and its construction is similar to natural adipose.