RESUMO
OBJECTIVE: To establish a novel method to isolate endothelial progenitor cellsï¼EPCï¼ from cryopreserved umbilical cord blood (cryoUCB), to investigate the biological characteristics of EPC and to improve the rate of EPC obtained from cryoUCB. METHODS: Twelve cryoUCB samples during 2000 to 2001 years were collected from allogeneic cord blood bank, cryoUCB was thawed rapidly in a water bath at 37 â, total nucleated cells (TNCs) were washed by phosphate-buffered saline (PBS). TNCs were seeded onto fibronectin-coated dishes to isolate EPC. Flow cytometry and immunofluorescence were used to identify EPC. The function of EPC was identified in vitro, such as the incorporation of Dil-Ac-LDL and FITC-UEA-I, the formation of capillary-like structure in matrigel, and the release of VEGF by ELISA. RESULTS: One to five cluster of cobble stone-like cells appeared at 2-3 weeks after seeding. Flow cytometric analysis showed that positive rates of CD31, CD34, CD144, and VEGFR (CD309) wereï¼92.91±5.20ï¼%, ï¼30.0±23.27ï¼%, ï¼88.55±3.83ï¼% and ï¼67.21±12.12ï¼% in passage 1 to passage 3 of EPC. EPC could uptake Dil-Ac-LDL and FITC-UEA-I, form capillary-like network on Matriget and release VEGF. CONCLUSION: EPC had been successfully isolated from cryopreserved umbilical cord blood by this method with high stability and reproducibility. EPC can be obtained in 85% frozen umbilical cord blood. This method may lay a foundation to supply abundant EPC for clinical application.
Assuntos
Células Progenitoras Endoteliais , Sangue Fetal , Diferenciação Celular , Células Cultivadas , Reprodutibilidade dos Testes , Células-TroncoRESUMO
The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat's weight (260-330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa's score, a larger infarct volume and a greater model success ratio were screened using the Longa's score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.
Assuntos
Isquemia Encefálica/fisiopatologia , Modelos Animais de Doenças , Reabilitação do Acidente Vascular Cerebral , Acidente Vascular Cerebral/patologia , Animais , Encéfalo/fisiopatologia , Isquemia Encefálica/reabilitação , Infarto da Artéria Cerebral Média/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Reperfusão , Traumatismo por ReperfusãoRESUMO
OBJECTIVE: This study was to expand the cytotoxic T lymphocytes (CTL) through inducing the differentiation of umbilical blood monomuclear cells (UBMNC) by using various combination of cytokines, and to investigate the functions of expanded CTL. METHODS: The MNC were isolated by ficoll density gradient centrifugation. Then, the PHA-P, IFN-γ combined with IL-2, IL-15 and other cytokines were used for induction and expansion of the cord blood-derived CTL. The biological function of CTL was examined by phenotype analysis, cytotoxic tests and real-time fluorescence quantitative PCR. RESULTS: After expansion for 15 days, the cell number increased by 1522% ± 137%. The content of CD3(-)CD8(-) cells in uncultured cord blood MNC was 95%, and the CD3(+)CD8(+) CTL cells reached 82.77% in cultured cord blood MNC after expansion for 15 days. The expanded CTL cell showed the cytotoxic activity against K562 and HeLa cell line. The killing rate of MNC was 61.88 ± 1.08%. After expansion, the killing rate could reach to 90% with the average value of 90.33 ± 2.02%. The expanded CTL cells highly expressed some key cytokines, such as granzyme A, granzyme B, GM-CSF, granulysin, IFN-γ, TGF-ß, TNF-α and perforin. Compared with the control group, the expression of IFN-γ and TGF-ß significantly increased (P < 0.05), and the other factors dramatically increased (P < 0.01). CONCLUSION: The cord blood-derived CTL can be expanded by different combinations of cytokines. These protocols may provide alternative choices for CTL cell expansion in tumor adoptive immunotherapy.
Assuntos
Sangue Fetal , Linfócitos T Citotóxicos , Citocinas , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Granzimas , Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Classe II , Humanos , Imunoterapia Adotiva , Perforina , Fito-HemaglutininasRESUMO
This study was objective to explore the effect of IFN-γ on immunosuppressive capability of mesenchymal stem cells (MSC) derived from umbilical cord. The immunomodulating capability of MSC was changed by stimulating cell surface receptors like Toll-like receptors (TLR). The inhibition of T-lymphocyte proliferation by MSC was tested via cell co-cultures. Further RT-PCR and ELISA were performed to examine the expression changes in gene and protein level. The results showed that the IFN-γ could promote the immunosuppressive effect of umbilical cord derived MSC. IFN-γ-stimulated MSC could suppress the proliferation of T cells more effectively. IFN-γ stimulation up-regulated the expression of immunosuppressive genes like IDO1, COX2, HLA-G, and soluble suppressive proteins such as HLA-G, KYN, IL10, PGE2 of MSC. And the immuno suppression capability of IFN-γ-stimulated MSC was 2-7 folds higher than control in MSC and lymphocyte co-culture tests. It is concluded that IFN-γ can effectively enhance the immunosuppressive capability of MSC.