Study On Effect Of Down-regulation CD151on Biological Characteristics Of Umbilical Cord Mesenchymal Stem Cell

Background：The mesenchymal stem cell (Mesenchymal stem cell, MSC) is a kind ofpluripotent stem cell which is originated from mesoderm with the capacity ofself-renewal and differentiation potential. The presence of MSCs is demonstrated insome tissues and organs, such as bone marrow, umbilical cord, cord blood, placenta,chorionic. They can amplify and also can be induced into osteocyte, adipocyte,chondrocyte, neurocyte. The unique biological characteristics of MSCs are aprerequisite for a broad experimental research and clinical applications. Thefour-transmembrane protein super family is a kind of small molecule glycoprotein,crossing the cell membrane four times. In the tetraspanin-enriched microdomainsbetween the different four-transmembrane proteins or with the other transmembraneprotein can form four transmembrane protein complex which is the structural basis ofthe important biological functions by mutual coupling. CD151is one of the importantmembers of the four-transmembrane protein family, first be discovered in the plateletendothelial cells, playing a key role in cell adhesion, migration, proliferation,activation and tumor cell growth, metastasis and into the blood vessels.Objective：Our previous study found that the umbilical cord-derived MSCs almost100percent express CD151but so far whether CD151has effect on the biologicalcharacteristics of the umbilical cord-derived MSCs without no-depth study. Theexperiments carried out on this result. siRNA interfere with the CD151on the surfaceof UC-MSCs in order to decrease CD151expression. To compare the biologicalcharacteristics of UC-MSCs such as stem cell surface markers, the capacity ofadipogenic, osteogenic differentiation. And preliminarily examine the effects onimmune regulation and hematopoietic support.Method：(1) Primary cells which were cultured with purified adherent culture derivedfrom umbilical cord tissue were isolated by collagenase digestion. Some stem cellsurface markers were detected by Flow cytometry，the capacity of adipogenic,osteogenic differentiation in vitro.(2) siRNA interfere CD151on the surface ofUC-MSC. The cells were divided into three groups, interfering with siRNA-CD151 group、negative control group (interfering with siRNA-NC)、blank group(withoutinterference). In order to confirm the best time the interferential time was lasted24、48and72hours. The efficiency of interference after24、48、72hours and the changesof other surface markers were detected by flow cytometry. Their ability ofdifferentiation was assessed by oil red O and von Kossa staining.(3) PHA-stimulatedhuman peripheral blood mononuclear cells were established co-culture system withthree groups of cells. The inflammatory factor interferon-γ and HGF were measuredby ELISA. The mRNA expression of CD151、HGF、TGF-β1、COX-2and IDO inhUC-MSC were detected by Real-time quantitative PCR.(4) The CD34+cells inumbilical cord blood were sorted by immunomagnetic beads. The sorting efficiencywas detected by flow cytometry. Collecting conditioned medium of the groups ofinterfered cells that co-culture with CD34+cells，The co-culture were divided intothree groups: experimental group, negative control group, positive control group,observing the morphology of hematopoietic colonies at the7th、14th、21st dayrespectively and calculating the amount of the colonies.Result：1. Separation of the umbilical cord tissues, cultured UC-MSCsPrimary cells were obtained by collagenase digestion which were cultured in linewith stem cell classical morphology、expressed the stem cell phenotypes and had thecapacity of differentiation into adipocytes, osteoblasts in vitro;2. siRNA interfere CD-151on the cell surface of UC-MSCs(1) Interfered UC-MSCs72hours was the optical time by siRNA-CD151.comparingto the control group (95.66±1.56%) and blank group (95.48±4.04%) CD151expression on the surface of experimental group (11.97±2.63%)cells was significantlydecreased (P <0.05, P <0.05) as the same as CD151mRNA (P <0.01, P <0.01);(2) The cell of three groups was all adherently grew with typical fibroblastmorphology. The stem cell surface markers were detected by flow cytometry allexpress CD73、CD90、CD105、HLA-ABC whereas do not express CD34、CD45.CD105expression of the experimental group (83.37±0.71%) decreased than control group (93.66±0.21%) and blank group (91.87±0.75%)(P <0.05, P <0.05).The restof surface markers had on significant differences;(3) In the different differentiation medium state three types of cells had the capacity ofadipogenic and osteogenic differentiation which had no significant difference in lipiddroplets but in osteogenic differentiation the volume of calcium deposition decreasedand its number increased;3. The effect on immunomodulatory property of UC-MSCs after interference with theCD-151(1) UC-MSCs were co-culture with PBMC+PHA, Compared to control group andblank group the secretion of IFN-γ in the supernant with interfering siRNA-CD151was significantly increased (P <0.05, P <0.05);(2) Compared to control group and blank group the experimental group UC-MSCHGF, TGFβ-1mRNA expression decreased significantly (P <0.01, P <0.01) COX-2mRNA expression increased (P <0.05) and IDO mRNA expression had no difference.The experimental group UC-MSC HGF secretion also similarly decreased by ELISA(P <0.01);4. The effect on hematopoietic supportive capacity of UC-MSCs after interferencewith the CD-151The cells of experimental group and negative control group are able to formhematopoietic colonies including CFU-G、CFU-GM、CFU-M with small size, diffuseddistribution, small number, later appearance while the CFU-E is absent. There is nodifference in total number of colonies between two group cells. All kinds of coloniesare observed in the positive group. The conditioned medium of UC-MSC after siRNAinterfering72hours still can promote CD34+cells differentiation and hashematopoietic supportive capacity. It makes CD34+cells differentiation into myeloidcells except erythroid cells.Conclusion:1. siRNA-CD151can successfully down-regulate CD151mRNA expression ofUC-MSC while after interfering siRNA-CD151don’t change other surface markersexcept CD105as well as maintain the capacity of adipogenic differentiation. But it may affect osteogenic differentiation capacity;2. siRNA-CD151has decreased the ability that US-MSC inhibit IFN-γ secretion ofPBMC with changing some related soluble immunomodulatory factors to participatein immunomodulatory property;3. The conditioned medium still can promote CD34+cells differentiation and keephematopoietic supportive capacity while there is no difference in total number ofcolonies between cells of two groups indicate CD151may not be the main moleculeparticipates in hematopoietic supportive capacity of UC-MSC.