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

Cheng Zhang; Yi Liu; Jun Tang; Meisi Xue; Sijia Min

doi:10.4236/ojrm.2015.42003

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OJRM> Vol.4 No.2, June 2015

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

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DOI: 10.4236/ojrm.2015.42003 3,813 Downloads 4,316 Views

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Cheng Zhang¹, Yi Liu^1*, Jun Tang¹, Meisi Xue¹, Sijia Min²

Affiliation(s)

¹Center of Burns and Plastic Surgery, Lanzhou General Hospital, Lanzhou, China.
²Department of Sericultural Science, Zhejiang University, Hangzhou, China.

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 < 0.01). RT-PCR results showed that the expression of adipogenic gene PPARγ-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.

KEYWORDS

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

Conflicts of Interest

The authors declare no conflicts of interest.

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.

References

[1]	Gomillion, C.T. and Burg, K.J.L. (2006) Stem Cells and Adipose Tissue Engineering. Biomaterials, 27, 6052-6063. http://dx.doi.org/10.1016/j.biomaterials.2006.07.033

[2]	Parker, A.M. and Katz, A.J. (2006) Adipose-Derived Stem Cells for the Regeneration of Damaged Tissues. Expert Opinion on Biological Therapy, 6, 567-578. http://dx.doi.org/10.1517/14712598.6.6.567

[3]	Wang, H.S., Hung, S.C., Peng, S.T., Huang, C.C., Wei, H.M., Guo, Y.J., et al. (2004) Mesenchymal Stem Cells in the Wharton’s Jelly of the Human Umbilical Cord. Stem Cells, 22, 1330-1337. http://dx.doi.org/10.1634/stemcells.2004-0013

[4]	Escribano, O., Arribas, M., Valverde, A.M. and Benito, M. (2007) IRS-3 Mediates Insulin-Induced Glucose Uptake in Differentiated IRS-2 Brown Adipocytes. Molecular and Cellular Endocrinology, 268, 1-9. http://dx.doi.org/10.1016/j.mce.2006.12.039

[5]	Wang, L. and Detamore, M.S. (2009) Insulin-Like Growth Factor-1 Improves Chondrogenesis of Predifferentiated Human Umbilical Cord Mesenchymal Stromal Cells. Journal of Orthopaedic Research, 27, 1109-1115. http://dx.doi.org/10.1002/jor.20848

[6]	Jiang, T., Zhang, D. and Nie, H. (2008) Construction and Expression of the Eukaryotic Expressed Plasmid of MIC3 Gene from Toxoplasma gondii in IBRS-2 Cells. Frontiers of Agriculture in China, 2, 498-501. http://dx.doi.org/10.1007/s11703-008-0075-6

[7]	Xue, M.S. and Liu, Y. (2010) Construction of Recombinant Human Insulin Gene Lentiviral Expression Vector and Virus Packaging. Journal of Clinical Rehabilitative Tissue Engineering Research, 14, 6133-6137.

[8]	Liu, Y. and Xue, M. (2010) Recombinant Human Insulin Gene Lentivirus Transfecting Human Umbilical Cord Mesenchymal Stem Cells in Vitro. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi (Chinese Journal of Reparative and Reconstructive Surgery), 24, 822-827.

[9]	Kim, H.J., Kim, H.S., Matsumoto, A., Chin, I.J., Jin, H.J. and Kaplan, D.L. (2005) Processing Windows for Forming Silk Fibroin Biomaterials into a 3D Porous Matrix. Australian Journal of Chemistry, 58, 716-720. http://dx.doi.org/10.1071/CH05170

[10]	Horan, R.L., Antle, K., Collette, A.L., Huang, Y.Z., Huang, J., Moreau, J.E., Volloch, V., Kaplan, D.L. and Altman, G.H. (2005) In Vitro Degradation of Silk Fibroin. Biomaterials, 26, 3385-3393. http://dx.doi.org/10.1016/j.biomaterials.2004.09.020

[11]	Wang, Y., Kim, H.J., Vunjak-Novakovic, G. and Kaplan, D.L. (2006) Stem Cell-Based Tissue Engineering with Silk Biomaterials. Biomaterials, 27, 6064-6082. http://dx.doi.org/10.1016/j.biomaterials.2006.07.008

[12]	Langer, R. and Tirrell, D.A. (2004) Designing Materials for Biology and Medicine. Nature, 428, 487-491. http://dx.doi.org/10.1038/nature02388

[13]	Yao, J., Nakazawa, Y. and Asakura, T. (2004) Structures of Bombyx mori and Samia cynthia ricini Silk Fibroins Studied with Solid-State NMR. Biomacromolecules, 5, 680-688. http://dx.doi.org/10.1021/bm034285u

[14]	Yang, M.Y., Shuai, Y.J., He, W., Min, S.J. and Zhu, L.J. (2012) Preparation of Porous Scaffolds from Silk Fibroin Extracted from the Silk Gland of Bombyx mori (B. mori). International Journal of Molecular Sciences, 13, 7762-7775. http://dx.doi.org/10.3390/ijms13067762

[15]	Liu, Y. and Xiao, H.T. (2010) The Co-Culture of Porus Silk Fibroin Scaffold with hUCMSCs. Chinese Journal of Medical Aesthetics and Cosmetology, 16, 45-48.

[16]	Yong, S. and David, J. (2009) Development of a Multiplex qPCR for Detection and Quantitation of Pathogenic Intestinal Spirochaetes in the Faeces of Pigs and Chickens. Veterinary Microbiology, 137, 129-136. http://dx.doi.org/10.1016/j.vetmic.2008.12.020

[17]	Le, L.P., Le, H.N., Nelson, A.R., Matthews, D.A., Yamamoto, M. and Curiel, D.T. (2006) Core Labeling of Adenovirus with EGFP. Virology, 351, 291-302. http://dx.doi.org/10.1016/j.virol.2006.03.042

[18]	Shao, Z., Young, R.J. and Vollrath, F. (1999) The Effect of Solvents on Spider Silk Studied by Mechanical Testing and Single-Fibre Raman Spectroscopy. International Journal of Biological Macromolecules, 24, 295-300. http://dx.doi.org/10.1016/S0141-8130(98)00093-2

[19]	Peng, X., Zhang, X. and Zeng, B. (2008) Locally Administered Lentivirus-Mediated siRNA Inhibits Wear Debris-Induced Inflammation in Murine Air Pouch Model. Biotechnology Letters, 30, 1923-1929. http://dx.doi.org/10.1007/s10529-008-9788-0

[20]	Gordon, D., Glover, C.P., Merrison, A.M., Uney, J.B. and Scolding, N.J. (2008) Enhanced Green Fluorescent Protein-Expressing Human Mesenchymal Stem Cells Retain Neural Marker Expression. Journal of Neuroimmunology, 193, 59-67. http://dx.doi.org/10.1016/j.jneuroim.2007.10.019

[21]	Ren, G., Li, T., Lan, J.Q., Wilz, A., Simon, R.P. and Boison, D. (2007) Lentiviral RNA-Induced Downregulation of Adenosine Kinase in Human Mesenchymal Stem Cell Grafts: A Novel Perspective for Seizure Control. Experimental Neurology, 208, 26-37. http://dx.doi.org/10.1016/j.expneurol.2007.07.016

[22]	Goomer, R.S., Deftos, L.J., Terkeltaub, R., Maris, T., Lee, M.C., Harwood, F.L., et al. (2001) High-Efficiency Non-Viral Trasfection of Primary Chondrocytes and Perichondrial Cell for ex-Vivo Gene Therapy to Repair Articular Cartilage Defects. Osteoarthritis and Cartilage, 9, 248-256. http://dx.doi.org/10.1053/joca.2000.0382

[23]	Blanc, M.R., Anouassi, A., Abed, M.A., Canépa, S., Labas, V. and Bruneau, G. (2009) A New Method to Discriminate Immunogen-Specific Heavy-Chain Homodimer from Heterotetramer Immunoglobulin G Directly in Immunized Dromedary Whole Plasma Proteins: Western Ligand Blotting. Veterinary Immunology and Immunopathology, 127, 340-349. http://dx.doi.org/10.1016/j.vetimm.2008.10.334

[24]	Chassaigne, H., Trégoat, V., Norgaard, J.V., Maleki, S.J., van Hengel, A.J. (2009) Resolution and Identification of Major Peanut Allergens Using a Combination of Fluorescence Two-Dimensional Differential Gel Electrophoresis, Western Blotting and Q-TOF Mass Spectrometry. Journal of Proteomics, 72, 511-526. http://dx.doi.org/10.1016/j.jprot.2009.02.002

[25]	Li, S.L., Liu, Y. and Hui, L. (2013) Construction of Engineering Adipose-Like Tissue in Vivo Utilizing Insulin Gene-Modified Umbilical Cord Mesechymal Stromal Cells with Silk Fibroin Scaffolds with Silk Fibroin 3D Scaffolds. Journal of Tissue Engineering and Regenerative Medicine, Early View. http://dx.doi.org/10.1002/term.1695

[26]	Hofmann, S., Hagenmüller, H., Koch, A.M., Müller, R., Vunjak-Novakovic, G., Kaplan, D.L., et al. (2007) Control of in Vitro Tissue-Engineered Bone-Like Structures Using Human Mesenchymal Stem Cells and Porous Silk Scaffolds. Biomaterials, 28, 1152-1162. http://dx.doi.org/10.1016/j.biomaterials.2006.10.019

[27]	Wang, Y., Rudym, D.D., Walsh, A., Abrahamsen, L., Kim, H.J., Kim, H.S., et al. (2008) In Vivo Degradation of Three-Dimensional Silk Fibroin Scaffolds. Biomaterials, 29, 3415-3428. http://dx.doi.org/10.1016/j.biomaterials.2008.05.002

[28]	Liu, Y., Xiao, H.T. and Xue, M.S. (2010) The Optimal Aperture Screening: A Suitable Scaffold for the Construction of Tissue Engineered Adipose. Journal of Clinical Rehabilitative Tissue Engineering Research, 14, 1361-1364. (In Chinese)