] MSCs. MSCs, ,,, PHA. Immune regulatory effects of mesenchymal stem cells derived from human umbilical blood
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1 IMMUNOLOGICAL JOURNAL Vol. 23 No. 3 May [ ] (2007) MSCs 1, 2, 1, 1 1 3, (1., ;2., ) [ ] (MSCs) MSCs ; MSCs (PHA),H 3 2TdR MSCs,CD29 CD105 CD166,CD14 CD34 CD45 ; MSCs PHA MSCs [ ] ; ; [ ] R392 [ ] A Immune regulatory effects of mesenchymal stem cells derived from human umbilical blood WANG Meng, YANG Yuan, YANG Dong2ming, WANG Xu2quan, XU Jian2zhong ( Orthopedic Center of PLA, Southwest Hospital, Third Military Medical University, Chongqing ) [ Abstract] Objective To study the isolation, expansion, and purification of mesenchymal stem cells (MSCs) from human umbilical cord blood, and investigate its immune regulatory effects on homologous lymphocyte.methods Human umbilical cord blood samples were ob2 tained sterilely. The cord blood mononuclear cells were isolated by lymphocyte separation medium and individual colonies were selected and cul2 tured. The phenotype of MSCs derived from umbilical blood was analyzed by flow cytometry. The MSCs derived from umbilical blood were added to mixed lymphocyte cultures and PHA2induced transformation cultures with various concentrations. The proliferation of lymphocyte was mea2 sured by [ 3 H]2thymidine incorporation using a liquid scintillation counter. Effects of MSCs derived from umbilical blood on mixed lymphocyte response and PHA2induced lymphocyte transformation were investigated. Results The adherent cells isolated from the cord blood mononuclear cells displayed a fibroblast2like morphology and expressed several MSCs2related antigens CD29, CD44, and CD105, not CD34, CD45, or CD106. The MSCs derived from umbilical blood showed negative immunomodulatory effects and could inhibit evidently the mixed lymphocyte re2 sponse and PHA induced transformation. Conclusion The MSCs derived from umbilical blood with the immune regulatory effect could be an important and novel source of allogenic seed cells for bone tissue engineering. [ Key words] Umbilical cord blood ; Mesenchymal stem cells ; Immunomodulation ; Bone tissue engineering,, ( mesenchymal stem cells, [1 MSCs), ] MSCs [ ] ;[ ] [ ] ( AA02A122) [ ] ( ),, DNA 306, ( Tel) ; ( E2 mail) com 3, 1980,, [2 ] MSCs, MSCs
2 288 23,35 1 2, MSCs 34 1, 80 %90 %,, (1 mmolπl EDTA), Mesencult TM,4872 h, 3 6 7,, ; 1. 3,, 2025 d MSCs, 20 gπl BSA PBS, Π PE,, FITC CD14 CD29 CD34 CD45 CD105 CD166, %90 % (EB ) 10 L,, 4 30 min,pbs 2,10 gπl 4, %, 3 30 min,facscalibur (BD ),, CellQuest (1) 812 d, MSCs, gπl : 13 d,36 d (MNC), 200 mlπlfbs RPMI 1640,7 9 d ( Gibco ), 37, 50 mlπl CO 2 24 h C(sigma ) 25 gπml,37 50 mlπl CO 2 30 min 3 C MSCs C Π Π, Π 1 MSCs ( 40), 5,37 50 mlπl kbq 3 H2 cpm 1. 5 MSCs 1 C Π 24 ;, Π, ml, 12 h PHA(Sigma ), 30gΠmL, molπl ph 7. 4 PBS 1 1, PHA gπl,, 5,37,50 mlπl CO rπmin 20 min,, PBS 2 3 d 96,pH Mesencult TM ( Stem Cell, 3 H2TdR ) 4 gπl ml - 1 (Sigma ) 90 %, SPSS10. 0, t 50 mlπl CO 2, Fig 1 Mesenchymal stem cells derived from umbilical blood( 40) CO 2 4 d 2. 2 MSCs : MSCs TdR, 18 h CD29 CD105 CD166, CD14
3 3,1 MSCs 289 CD34 CD45 Π (2) 2 MSCs Fig 2 Identification of mesenchymal stem cells derived from umbilical blood by flow cytometry 2. 3 MSCs Π, ( cpm) 3 H2TdR cpm ( ( ) 10 4 MSCs ) 10 4 ( ) 10 4 ( ), 10 4 ( ) 10 4 ( ) 10 4, MSCs MSCs Π cpm ( (4) 01297) 10 4 ( ) 10 4 ( ) 10 4 ( ) 10 4 ( ) 10 4 MSCs ( P < 0. 01) MSCs, (3) 3 MSCs Fig 3 Effects of MSCs derived from umbilical blood on mixed lymphocyte reaction 2. 4 MSCs [3 ] (mesenchymal stem cell, MSCs PHA MSCs) ( P < 0. 01), MSCs, MSCs,, 3 H2TdR cpm ( MSCs Fig 4 Effects of MSCs derived from umbilical blood on 3 PHA2induced lymphocyte reaction MSCs ) 10 4, MSCs 1
4 [4 ], HLA 20 d MSCs MSCs [7,8 ] MSCs MSCs, : MSCs, MSCs MSCs : MSCs PHA, MSCs MSCs MSCs, 3 H2TdR cpm ( ) , MSCs [ Π, 3 H2TdR ] 2000, cpm ( ) 10 4 Erices ( ) 10 4 ( ) 10 4 ( ) 10 4 ( ) 10 4, [6 MSCs ] MSCs MSCs ph Mes2 MSCs encult TM MSCs MNC,3,45 80 %90 %, 10 3 [9 ],23, [10 80 %, 3 ] :,,,,, 812 d, 15 MSCs MSCs MSCs MSCs, MSCs MSCs MSCs MSCs MSCs,, 3, 15 [ ] MSCs [1 ] Pittenger MF, Mackay AM, Beck SC,et al. Multilineage po2 : tential of adult human mesenchymal stem cells [J ]. Science, CD29 CD105 CD166, CD ,284(5 411) : CD34 CD45 [2 ] Tse W, Laughlin MJ. Umbilical cord blood transplantation : a new alternative option [ J ]. Hematology Am Soc Hematol Educ Program, 2005 : Π ( 294 )
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