TITLE:
Effects of Human Insulin Gene Transfection on the Adipogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in Silk Fibroin Scaffolds in Vitro
AUTHORS:
Cheng Zhang, Yi Liu, Jun Tang, Meisi Xue, Sijia Min
KEYWORDS:
Tissue Engineering Adipose, Human Umbilical Cord Mesenchymal Stem Cells, Silk Fibroin, Insulin, Recombinant Lentivirus, Gene Transfection
JOURNAL NAME:
Open Journal of Regenerative Medicine,
Vol.4 No.2,
June
29,
2015
ABSTRACT: The resorption of
the transplanted fat over time limited the use of autologous fat for the reconstruction
of soft tissue defect. Tissue engineering (TE) adipose with silk fibroin
scaffold could be a promising substitute for soft tissue filling. In this
study, we try to develop a tissue engineering adipose in vitro by seeding silk fibroin scaffold with human umbilical cord
mesenchymal stem cells (hUCMSCs) after transfected with recombinant human insulin
gene lentivirus. Our aim was to observe the effects of the insulin gene
transfection on the adipogenesis of hUCMSCs when cultured with silk fibroin
scaffolds. The hUCMSCs infected with recombinant lentiviral pLenti6.3-insulin-IRES-EGFP
were seeded on silk fibroin scaffolds and cultured in adipogenic differentiation
medium for 5 - 7 days. The expression of adipogenic gene PPARγ-2 was tested by RT-PCR after 7 days
culture of adipogenic induction. The accumulation of cytoplasmic droplets of
neutral lipids was assessed by Oil Red O staining. The RNA and protein
expression of transfected insulin gene in hUCMSCs were detected by QPCR and western
blot. The effect of recombinant lentivirus transfection on the growth and
proliferation 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 300.25
times higher than that in the untransfected group. The western blot showed that
a 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 in
silk fibroin scaffold; round adipose droplets showed intracellularly; the
size of the adipocytes was not homogenous, and the density of adipocytes in transfected
group was significantly higher than that in untransfected group (P = 0.007, P γ-2 in transfected group was much
stronger than that in untransfected group. MTT test showed that there was no significant
difference in optical density (A) at each time point between transfected group
and nontransfected group (P = 0.056, P > 0.05). And there was also no significant
difference in optical density (A) between cell group and cell-scalffold group
(P = 0.066, P > 0.05). We concluded that insulin gene could obviously
promote the adipogenic differentiation of hUCMSCs, and a tissue engineering adipose
could be constructed by the silk fibroin scaffolds seeded with human insulin
gene-modified hUCMSCs effectively in
vitro.