Effects of Human Insulin Gene Transfection on the Adipogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in Silk Fibroin Scaffolds in VitroReportar como inadecuado




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The resorption ofthe transplanted fat over time limited the use of autologous fat for the reconstructionof soft tissue defect. Tissue engineering TE adipose with silk fibroinscaffold could be a promising substitute for soft tissue filling. In thisstudy, we try to develop a tissue engineering adipose in vitro by seeding silk fibroin scaffold with human umbilical cordmesenchymal stem cells hUCMSCs after transfected with recombinant human insulingene lentivirus. Our aim was to observe the effects of the insulin genetransfection on the adipogenesis of hUCMSCs when cultured with silk fibroinscaffolds. The hUCMSCs infected with recombinant lentiviral pLenti6.3-insulin-IRES-EGFPwere seeded on silk fibroin scaffolds and cultured in adipogenic differentiationmedium for 5 - 7 days. The expression of adipogenic gene PPARγ-2 was tested by RT-PCR after 7 daysculture of adipogenic induction. The accumulation of cytoplasmic droplets ofneutral lipids was assessed by Oil Red O staining. The RNA and proteinexpression of transfected insulin gene in hUCMSCs were detected by QPCR and westernblot. The effect of recombinant lentivirus transfection on the growth andproliferation of hUCMSCs was observed by MTT test. We observed that the 2-ΔΔCt value of insulin gene expression of hUCMSCs in the transfected group was 300.25times higher than that in the untransfected group. The western blot showed thata positive band was discerned at the site of a relative molecular mass of 8 ×103 Dalton in transfected group. After adipogenic culture for 7 days,under the fluorescent inverted phase-contrast microscope, after Oil Red O staining, a lot of adipocytes appeared insilk fibroin scaffold; round adipose droplets showed intracellularly; thesize of the adipocytes was not homogenous, and the density of adipocytes in transfectedgroup was significantly higher than that in untransfected group P = 0.007, P <0.01. RT-PCR results showed that the expression of adipogenic gene PPARγ-2 in transfected group was muchstronger than that in untransfected group. MTT test showed that there was no significantdifference in optical density A at each time point between transfected groupand nontransfected group P = 0.056, P > 0.05. And there was also no significantdifference in optical density A between cell group and cell-scalffold groupP = 0.066, P > 0.05. We concluded that insulin gene could obviouslypromote the adipogenic differentiation of hUCMSCs, and a tissue engineering adiposecould be constructed by the silk fibroin scaffolds seeded with human insulingene-modified hUCMSCs effectively invitro.

KEYWORDS

Tissue Engineering Adipose, Human Umbilical Cord Mesenchymal Stem Cells, Silk Fibroin, Insulin, Recombinant Lentivirus, Gene Transfection

Cite this paper

Zhang, C. , Liu, Y. , Tang, J. , Xue, M. and Min, S. 2015 Effects of Human Insulin Gene Transfection on the Adipogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in Silk Fibroin Scaffolds in Vitro. Open Journal of Regenerative Medicine, 4, 15-25. doi: 10.4236-ojrm.2015.42003.





Autor: Cheng Zhang1, Yi Liu1*, Jun Tang1, Meisi Xue1, Sijia Min2

Fuente: http://www.scirp.org/



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