The Expression Of MUC2in Umbilical Cord Mesenchymal Stem Cells Over Expressing CDX2

Background and Purpose: caudal type homeobox2(CDX2) is specifically expressedin the intestinal mucosa. it is also a critical gene that promote definitive endodermdifferentiating into hindgut. Esophageal epithelial cells modified by CDX2can express theintestinal mucin (MUC2) and promote intestinal metaplasia in esophagus. Internalribosome entry site(IRES) can link CDX2and Enhanced Green FluorescentPortein,(EGFP), so we can make a coexpression of CDX2and EGFP. The first part ofexperiment intends to build, amplify, extract CDX2-IRES-EGFP eukaryotic expressionvector, and lay the foundation for the gene overexpression in umbilical cord mesenchymalstem cells. UC-MSCs which is the member of adult stem cells mainly exist in themesenchyme surrounding blood vessel of umbilical cord. They have the ability toself-renew and to differentiate into various tissues or cells.In the second part, we isolatethe cells from mesenchyme of human umbilical cord,then the morphology,proliferativecapacity,immunophenotype will be studied for confirming that these cells are UC-MSCs inorder to lay the foundation for further research to study the expression of MUC2inUC-MSC overpressing CDX2. Ulcer colitis can make mucosal mucus layer thinner andMUC2is the main part of the mucosal mucus layer.so here we research the expression ofMUC2after the overexpression of CDX2in UC-MSCs, explore the method of makingMUC2overexpress in MSCs by genetical modification. Study the necessity of inducingUC-MSCs differentiating into endodermal cell for overexpressing MUC2. Lay thefoundation for studying UC-MSCs differentiate into goblet cells in intestinal mucosa inorder to cure ulcer colitis..Methods: amplify CDS area of CDX2gene sequences from cDNA library by RT-PCRand clone the CDS area into the IRES-EGFP plasmid backbone through in-fusion method.Then transform CDX-IRES-EGFP into competent bacteria by heat shock method andculture these bacteria with selection culture containing antibiotic. Positive clones werepreliminarily identificated by PCR, and then extracted by alkaline lysis method.Finallydetect the concentration and purity of CDX2-IRES-EGFP by spectrophotometry forsubsequent experiments. In the second part, mesenchyme is separated from the umbilicalcord taking from a healthy full-term newborns and cut them into shreds.Put these shredsinto flask with DMEM/F12medium and FBS in order to make these tissues adhere flaskand MSCs grow from them. we start the subculture when a number of cells grow from tisstue.Then we observe the morphology, cell proliferation of each generation and detectthe immunophenotype(CD34/CD45/CD105/CD73/CD90/HLA-DR) of third generationcells with flow cytometry. In the research of expression of MUC2, there are five groups:they are Single-transfection Group: CDX2is transfected into UC-MSCs; Single-inductionGroup: induce UC-MSCs differentiate into endodermal cells; Induction and transfectiongroup: CDX2is transfected into UC-MSCs after induction. Single-induction Group:justtransfect vector without CDX2;Control group: UC-MSCs without any treatment.Single-induction Group should be treated by transfection reagent in order to exclude theeffect of transfection reagen. Finally, the mRNA of MUC2and CDX2are detected in eachgroup.Endodermal cells is induced by recombinant human Activin A, recombinant humanWnt3a.Results:The length of PCR product of target gene is962bp, gel electrophoresis bandposition is consistent with expectations; sequencing result is consistent with publishedGenebank sequence; the expected length of PCR product of positive transformants was823bp, gel electrophoresis band position is consistent with expectations. The extractedeukaryotic expression vector CDX2-IRES-EGFP concentration is843.6ng/ul, OD260/280=1.74, OD260/230=2.23. We can see a few cells which are spindle and polygonal shapecrawled out from tissue and adhere the flask after7-8days of primary culture.After10-14days, intensive cells grow surrounding the tissue. the time interval of subculture isabout5-6days. The cell after subculture have consistent and spindle shape, high ability ofproliferation which grow trend is swirly until the16th generation.The result of flowcytometric analysis show, that CD90/CD105/CD73is positive, CD34/CD45/HLA-DR isnegative. In the research of expression of MUC2,green fluorescence can be seen byfluorescence microscope in Single-transfection Group, Induction and transfection Groupand Single-induction Group after48h. GATA4and sox17increase by27.0±7.0-fold and54±6.4-fold which both are marker of endoderm until the5th day when UC-MSCs isinduced in Single-transfection Group and Induction and transfection Group. Five group isdetected by RT-PCR,the result show that CDX2increase by113.2±13.5-fold, MUC2increase by30.2±9.2-fold in Single-transfection Group; CDX2increase by196.7±58.8-fold, MUC2increase by98.8±17.8-fold in Induction and transfection Group. Theexpression of CDX2and MUC2do not change significantly in the other three groups.Conclusions: Eukaryotic expression vector CDX2-IRES-EGFP was constructedcorrectly, its concentration, purity meets transfection requirements. Human umbilical cord is rich in UC-MSCs, UC-MSCs are spindle shaped and have strong proliferative capacity.we can get UC-MSCs from tissue adherent to flask which have MSCs phenotype and donot have hematopoietic stem cell phenotype.Generally our study here get enoughUC-MSCs with high proliferative activity,high purity.Finally, overexpression of CDX2canincrease the expression of MUC2that is a marker of goblet cell in UC-MSCs. Theexpression of MUC2can be increased if UC-MSCs is induced to endodermal cells inadvace.But more MUC2enough for curing ulcer colitis would be got until betterexperiment condition is found.