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The Experimental Study on MSCs Transfected by EGF Gene for Wounding Healing

Author: HeBin
Tutor: ZhaoBaiCheng
School: Central South University
Course: Burns and Plastic Surgery
Keywords: Epidermal growth factor Adeno-associated virus Mesenchymal stem cells Wound healing Burn
CLC: R644
Type: Master's thesis
Year: 2010
Downloads: 72
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Abstract

Objective: The main reason for the lack of repair cells and various biologically active factors promote wound repair reduce wounds difficult to heal skin tissue defects. How adequate autologous skin transplantation restrictions, timely and effective repair impaired wound healing possible to restore the normal function of the skin healing wound clinical difficulty. Therefore, this study proposed by AAV-mediated EGF gene modified MSCs, to explore the possibility of promoting wound repair, and the source of the skin tissue engineering seed cells and transgenic technology is used to promote wound healing to provide the theoretical basis and practical basis. Methods and Results: 1. Construct recombinant adeno-associated virus vector carrying the EGF gene was amplified by RT-PCR from placental tissue 175bp size of the EGF gene, cloned into pMD18-T vector and transformed into competent cells. Extracted for recombinant plasmid DNA with restriction enzymes EcoRI and Sal I digestion, the ends were the EGF gene fragment with EcoRI and Sal I restriction sites, and ultimately cloned into pAAV-IRES-hrGFP plasmid proved successfully constructed pAAV-EGF-GFP plasmid by restriction enzyme digestion and sequencing. AAV-EGF virus packaging, purification and detection pAAV-EGF-GFP, pAAV-RC, pHelper 3 plasmids were co-transfected into HEK293 cells, the packaging containing EGF gene viral vector AAV-EGF; chloroform-PEG / NaCl precipitate - chloroform extraction \Ultimately prove to obtain high purity and high titer AAV-EGF virus. Bone marrow mesenchymal stem cells cultured by density gradient centrifugation combined with adherent method vitro cultured to get mouse bone marrow of MSCs and induce MSCs to osteogenic differentiation of three directions into the cartilage and into fat, respectively Von Kossa , Alcian blue Ponceau staining and Oil Red O staining method to detect. The results show that the primary and passaged in vitro culture of purified mouse MSCs have the ability to differentiate to multiple directions, we cultured cells for bone marrow mesenchymal stem cells. Transfected MSCs cells 4.AAV-EGF the AAV-EGF to obtain viral transfection of MSCs, respectively in cultured 3d, 14d, 27d after total RNA was extracted from the cells by RT-PCR was used to detect the EGF gene in mouse MSCs cells secretion expression. The results showed that the AAV transfected MSCs cell mediated in vitro expression of the EGF gene, and the expression of the EGF gene is increased with time. Conclusion: In this study, in vitro successfully isolated and cultured mouse bone marrow differentiation potential of MSCs; build pAAV-EGF-GFP plasmid carrying human EGF gene recombinant; obtained AAV-EGF virus referral guide EGF gene in a small expression in the mouse bone marrow MSCs.

The Experimental Study on MSCs Transfected by EGF Gene for Wounding Healing

Abstract

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