The Study About The Support Effect Comparison Of Extraorgan Amplification Of Human Placenta Mesenchymal Stem Cell To Haemopoietic Stem Cell Resource From Bone Marrow, Cord Blood, Placenta

Background and objectiveMesenehymal stem cell is a kind of tissue stem cell that be to follow with interest and to possess multi-differentiate potency.Fridenestein etc adherence cell culture with bone marrow form fibroblast and other nterstitial cells in 1976.Whereafter other scholars describe the non-hematopoiesis dherent cells source from bone marrow and they may differentiate to be full-grown mesenchymalis.They consider that these cells may form bone marrow and involucrum mesenchymalis;construct haematopoietic microenvironment;emerge connective tissue backbone and produce cell factors, chemokines and extracellular matrix protein adjust homing and proliferation of hematopoietic cell.MSC in vitro culture secrete many kinds of haematogenesis fators.And can express many kinds of surface adhesion molecules,that possess determinate effective in haematogenesis regulation.Therefore explantation MSC to make recover bone marrow microenvironment, support autoallergic and allogene haemopoietic stem cell transplantation and reconstruction, to cause a good deal of researchers to pay close attention to.Amplification and generation of hematopoietie Stem/progenitor Cell,haematogenesis factors are indispensable.Hyanesworht ect Cultivated primitive mesenchymal cell that can differentiate direction to osteoblast and chonseoblast,they express SH-2,SH-3 and SH-4 with monoclonal antibody detection.In the condition of without hematopoietic cell and differential stimulus,they can produce many kinds of haematogenesis correlation factors,comprehend granule array colony stimulus factors(G-CSF),stem cell factor(CSF),leukaemia inhibitory factor(LIF),M-CSF,IL-6,IL-11.But (MG-CSF),IL-3,TGF-β2 didn't be detected.To use the factor of IL-Ia that possesses enhance marrow stroma cell haemopoiesis capability;to stimulate the kind of primitive mesenchymal cells,it can elevate the level of production G-CFS,M-CFS,LIF, IL-6 and LI-11.Furthermore,IL-Ia even more can induce it product GM-CSE Majumdar etc have compared the kind of MSC source from marrow to tradition stroma cell;have detected mRNA expression of haematopoiesis correlation factor consequences indicated MSC express stably multiple haematopoiesis correlation factor:IL-6,IL-7,IL-8,IL-11,-12,IL-14,IL-15,M-CSF,SCF,Flt-3,FL,as well the expression level of IL-11,IL-12 obviously super-stroma-cell,the two kind of cells altogether expess G-CFSandGM-CFS at the tradition of IL-Ia induction.But the MRNA of LIF and IL-Ia only be inducted and express in MSC.Now out-of-body amplification methods of hematopoietic stem cell CD34⁺major depend on adding different component factors.But the method has many disadvantages,these factors of extraorgan admoveantur very difficult to analogue haematopoietic microenvironment,furthermore it need uninterrupt admoveantur in the whole cultivation process,cost is very expensive,it is inconcinnity long-term cultivation.Recently,some researchers cultivate CD34⁺ cells source from bone marrow with bone marrow groundmass to support,it gets commendable effective with mesenchyme stem cells source from placenta to support CD34⁺ cells source from cord blood.Placenta as accompaniment of fetus,is dealed to take as offal.Recent years there are many researchers acquire hematopoietic stem cells and mesenchyme stem cells in the human placenta tissue and blood,moreover,investigations confirm placenta mesenchyme stem cells may secrete many kinds of haematogenesis correlation factors. Accordingly,we segregate placenta mesenchyme stem cells from the human placenta, conduct and actions feeder layer of CD34⁺ cells source from different tissues,observe the distinction of its support and amplificate effect for distinct source hematopoietic stem cells. materialPlacentas and cord blood samples:normal healthy and fresh placenta tissue and cord blood come from department of gynaecology and obstetrics of The first affiliated hospital of Zhengzhou University.Bone marrow samples come from pediatrics of The first affiliated hospital of Zhengzhou University.Magnetism cell sorter and CD34⁺ cell detachment case come from Miltenyi Biotec company.Cell factors and antibodies:FGF-b Human come from Gibco company anti-CD29, CD19,CD34,CD44,CD45,HLA-DR,HLA-ABC,CD106 and UEA-1monoclonal antibodies come from PharMingen company.Major reagents:bovine serum albumin,methyl cellulose,mercaptoethanol,cellulose, mercaptoethanol,]glutamine,hydrocortisone,collagenaseⅣ,Hyaluronidase,DNase1 all come from Sigma company.DMEM culture medium,folium ilicis chinensis fetal calf serum come from Hyclone company.Method1.Isolated culture and identification of the human placenta mesenchymal stem cells: to digest placenta tissue with collagenase,colat postdigestive tissue with stainless steel meshwork of 100 mesh,wash twice with PBS liquid,inoculate these cells in the DMEM culture medium contained 20 percent FBS.Cellcultures were maintained at 37℃with a water-saturated atmosphere and 5%CO₂.after 48-72 hours,removal nulli- attached cells.Medium was replaced one time every 3-4 days.When cells were more than 80%confluence,they were recovered with 0.25%trypsinase,serial subcultivation.2.To detect surface antigens with flow cytometry:to get the sixth,the ninth,the twelfth generation cells with 0.25%trypsin digestion.After these cells were washed with PBS and were counted.To part with anti-CD19,CD29,CD34,CD44, CD45,CD105,CD106,HLA-DR,HLA-ABC,UEA-1monocl-onal antibodies react 30 minutes at ambient temperature and protection from light.After these cells were washed with PBS,detected their surface markers.3.CD34⁺ cells extraction:extract rulely mononuclear cells of bone marrow,cord blood and placenta,segregate CD34⁺ cells with immunomagnetic beads.4.Amplification group of haemopoietic stem cell:experiment separate to A:without feeder layer group B:have feeder layer group A group separate toA1:(bone marrow group ) A2:(cord blood group)A3:(placenta group) B group separatet B1(bone marrow group) B2(cord blood group)B3(placenta group),every group 6 ambiholes. To take different sources CD34⁺ cells put in the above-mentioned medium, carry out nucleated cells count,to detect CD34⁺ percentage and to calculate amplification multiples of CD34⁺ every week.5.Statistical treatment.Enumeration data apply duplication metered analysis of variance.Result.1.It can separate out placenta mesenchymal cells form human placenta,moreover certificate they were mesenchymal stem cells.2.It have more apparente amplification effect with HPDMSCS for feeder layer than without feeder layer.The support effect of human placenta mesenchymal stem cell to haemopoietic stem cell,the group resource form human placenta has an obviously advantage other groups,amplificate(2.6±0.17)-fold(8.7±1.2)-fold (16.8±2.2) -fold(11.6±1.8) -fold.Cord blood is worse,amplificate(2.0±0.2) -fold(4.3±1.2) -fold(11.2±2.4) -fold(6.7±1.9) -fold Bone marrow is worst; amplificate(1.8±0.3) -fold(4.6±1.1) -fold((8.7±1.4) -fold(4.2±0.4) -fold.Conclusion1.it can separate out mesenchymal stem cells from placenta.2.Human placenta mesenchymal stem cells have hematopoiesis support effect,provid hemopoietic microenvironment for haemopoietic stem cell,They can become feeder layer of different haemopoietic stem cell extraorgan amplification,especially suitable to haemopoietic stem cell resource human placenta.3.These haemopoietic stem cells source from placenta grow best.Amplification multiples were max.It indicated the support effect is most strong congelleric mesenchymal stem cell to congelleric haemopoietic stem cell.