To Induce Human Umbilical Cord Mesenchymal Stem Cells Transfected By HXOA4Gene To Differentiate Into Epidermal Cells In Vitro

Objective:The purpose of this study was to construct recombinant lentivirus vector-contain ning human HOXA4gene and transfect into human umbilicalcordmesenchymal stem cells(hUCMSCs) in vitro, to observre whether the biomedicalbehaviours of hUC-MSCs modified with HOXA4under the condition of monolayerculture. It will facilitated the following development of gene and cell therapy intreating disease of skin injure.Methods:1. HOXA4gene was amplified from plasmid HOXA4－MSCV by PCRtechnique and subcloned into the expression plasmid of lentiviral-GFP-CTB vectorThe lentiviral-GFP-HOXA4expression vector was constucted The corret HOXA4gene was confirmed by endoenzyme digestion and sequencing RecombinantLentiviral-GFP-HOXA4was produced by293T cells following the co-transfectionof lentiviral-GFP-HOXA4and packaging plasmids pRsv-REV pMDlg-pRRE andPMD2G The recombinant lentiviral-GFP-HOXA4were then used to infecthUCMSCs.2. The packaging lentivirus vectors transfected hUCMSCs in vitro,morphologicchange and expression of green fluorescent protein (GFP) was observed by fluores-cence phase contrast microscope.The efficiency of transfection was invest-igatedby fluorescence microscopy48h after transduction. Reverse TranscripatasePolymerase Chain Reaction (RT-PCR) was used to confirm expression of HOXA4inhUCMSCs.3. hUCMSCs modified with HOXA4grown on the high density monolayerculture without addition of any exogenous growth factors for21days. The epidermalcell specific molecular markers Cytokeratin14, Cytokeratin18,Cytokeratin19andBroad-spectrum keratin were detected by immunohistochemistry assay and Flowcytomerty． Results:The lentiviruse vector with human HOXA4cDNA was established successfully,which can effectively transfect hUCMSCs.The intensely expression of GFP wasobserved via fluorescence microscope and the efficiency of transfection of cellstransfected with Lenti-GFP—HOXA4or Lenti-GFP was more than90％respectively.RT-PC have detected HOXA4gene was overexpressed in hUC-MSCs. High densitymonolayer culture of these HOXA4transfected hUC-MSCs demonstrated thatspontaneous cell aggregation were formedat day21of cultureand subsequentlygenerate long shuttle changes. However,there was no the phenomenon of cellaggregation which occured in the cells trasducted by Lenti-GFP vectors or untreated.The expression of Cytokeratin14,Cytokeratin18Cytokeratin19and Broad-spectrum keratin in HOXA4transducted cells Was dectected by immunohist-ochemistry assay and Flow cytomerty．Conclusion．1．Lentiviral vector carrying HOXA4gene has been successfully constructed．2．HUC-MSCs display multilineage potential and they are amenable to modifyby exogenous gene；3．hUC-MSCs modified with HOXA4grown on thehigh density monolayer culture without the stimulation of exogenous growthfactors could also been induced differentiation into epidermal cell. These resultsindicated that HOXA4gene plays a important role in regulating differentiation ofMSCs．...