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1.
Zhonghua Yi Xue Za Zhi ; 92(36): 2574-7, 2012 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-23158802

RESUMO

OBJECTIVE: To validate a novel method of expanding late endothelial progenitor cells (EPC) in vitro. METHODS: We cultured mononuclear cells (MNC) from human peripheral blood on the plate with the feeder layer cells, i.e. irradiated late EPC or human umbilical vein endothelial cells. After 21 days, the numbers of late EPC colonies were counted separately. And the surface antigen of late EPC was detected by fluorescence-activated cell sorter (FACS) and their in vitro ability of forming vascular structure examined by matrigel. RESULTS: Both colony numbers of late EPC with feeder layer cell culturing were over 20 times than those without (40.0 ± 3.9, 39.3 ± 3.1 vs 2.0 ± 1.3, both P < 0.05). And the former's late EPC colony appeared earlier. The late EPC expressed CD31, CD34, eNOS, Flt-1, P1H12, Sendo, VE cadherin and CD117. And vascular structures were discerned. CONCLUSIONS: The method of feeder layer cells may vastly expand the quantity of late EPC. And microenvironment plays an important role in its expansion.


Assuntos
Técnicas de Cultura de Células , Células Endoteliais/citologia , Células-Tronco/citologia , Adulto , Diferenciação Celular , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Masculino , Trofoblastos/citologia , Adulto Jovem
2.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 34(3): 202-6, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22776649

RESUMO

OBJECTIVE: To study the role of the feeder layer cells as niche in the process of expansion of late endothelial progenitor cell in vitro. METHODS: We cultured mononuclear cells (MNC)from human peripheral blood (PB)on the plate with the feeder layer cells which were irradiated late endothelial progenitor cells(EPC)or human umbilical vein endothelial cells (HUVEC) by EGM-2. After 21 days, the numbers of obtained late EPC colonies were counted separately, and their surface antigen of the late EPC was verified by fluorescence-activated cell sorter (FACS) analysis, and their ability of forming vessel structure with Matrigel in vitro. The differentiation of single stem cell on the feeder layer cell was traced by video-microscopy. RESULTS: After 21 days of culture,(40.0±3.9)and(39.3±3.1)late EPC colonies that MNC of a hundred milliliter PB were cultured, respectively, on the feeder layer cells of EPC and HUVEC were much more than (2.0±1.3) colonies cultured on without the feeder layer cells (all P <0.05). These cells also expressed CD31,CD34,eNOS,FLt-1,P1H12,Sendo,VE cadherin,and CD117, as shown by FACS analysis. Furthermore, they formed vessel structure with Matrigel in vitro. The video-microscopy showed the asymmetric cell division was participated by the feeder layer cell during the expansion of single stem cell. CONCLUSION: The massive expansion of late EPC can be achieved by the provision of the feeder layer cells, which may be involved in the stem cell asymmetric cell division.


Assuntos
Microambiente Celular , Células Endoteliais/citologia , Células Alimentadoras , Células-Tronco/citologia , Comunicação Celular , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Sangue Fetal/citologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Trofoblastos/citologia
3.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 26(4): 405-9, 2004 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-15379265

RESUMO

OBJECTIVE: To isolate single chain antibody fragments (scFv) against cell surface molecules by pathfinder selection from an anti-KG1a cell scFv phage library. METHODS: The anti-KG1a scFv library was enriched by KGla cell panning for three rounds, or unenriched, then processed for pathfinder selection respectively using anti-CD34 monoclonal antibody as pathfinder molecule. ScFv phage clones were randomly picked and identified by binding KG1a cells using immunofluorescein and flow cytometry. The KG1a+ clones were further identified by KG1a, HL60, U937, and CEM cell lines and ELISA. Their antigenic molecules on cell surface were digested by chymopapain and analyzed by flow cytometry. DNAs from ten positive clones were sequenced. The scFv clones with different primary structure were used to analyze the molecular weight of their antigens by Western blot. RESULTS: One hundred and two KG1a+ scFv phage clones were isolated from 144 enriched and 96 unenriched scFv phage library respectively, among which 47 bound KG1a, HL60, U937, and CEM cells, 55 bound KG1a cells exclusively. None of 28 KG1a+, HL60-, U937-, and CEM- scFv clones bound to the CD34 antigen, as confirmed by ELISA, although most of their antigens were sensitive to chymopapain digestion. DNA sequences from ten positive clones showed that they were from four different clones. They bound antigens with different molecular weight. CONCLUSIONS: One hundred and two scFv phage clones specific for hematopoietic stem and progenitor cells have been isolated from an anti-KG1a cell scFv phage library. The pathfinder selection has showed advantages to improve the screening efficacy of scFv phage clones against antigens, which present at very low densities on the cell surface.


Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/genética , Células-Tronco Hematopoéticas/imunologia , Fragmentos Fc das Imunoglobulinas/biossíntese , Especificidade de Anticorpos , Bacteriófagos/genética , Clonagem Molecular , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Biblioteca de Peptídeos , Anticorpos de Cadeia Única
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