RESUMO
OBJECTIVE: To investigate the clinical efficacy of haploid hematopoietic stem cells (haplo-HSC) combined with third-party umbilical cord blood (tpCB) transplantation in the treatment of X-linked chronic granulomatous disease (X-CGD). METHODS: The clinical data of 26 boys with X-CGD were retrospectively analyzed who were admitted to the Sixth Medical Center of PLA General Hospital between April 2014 and March 2018. All the patients were treated with haplo-HSC combined with tpCB transplantation. The median age of the patients was 3.5 years. The donor was the father in 25 cases and an aunt in 1 case. Transplantation was 5/6 HLA-matched in 9 cases, 4/6 in 12 cases, and 3/6 in 5 cases. The patients received busulfan, cyclophosphamide, fludarabine, or anti-thymocyte globulin for myeloablative preconditioning. Cyclosporine A and mycophenolate mofetil were used for prevention of acute graft-versus-host disease (aGVHD). Then the patients were treated with haploid bone marrow hematopoietic stem cells combined with tpCB transplantation on day 1 and haploid peripheral hematopoietic stem cells on day 2. The counts of median donor total nucleated cells, CD34+ cells, and CD3+ cells were 14.6×108/kg, 5.86×106/kg, and 2.13×108/kg respectively. RESULTS: The median time to neutrophil and platelet engraftment was 12 and 23 days after transplantation respectively. Full donor hematopoietic chimerism was observed on day 30. Twenty-five cases were from haplo-HSC and 1 was from cord blood. No primary implant failure and implant dysfunction occurred, and secondary implant failure occurred in one case. The NADPH oxidase activity returned to normal one month after transplantation. The incidence of grade I-II aGVHD and grade III-IV aGVHD was 35% and 15% respectively. Chronic GVHD (cGVHD) of the skin occurred in one case, and no progression was observed after steroid administration. During the follow-up period of 6-51 months, 25 patients survived, of whom 24 were disease-free (23 patients without cGVHD and 1 with cGVHD of the skin) and NADPH oxidase activity returned to normal; one patient developed secondary implant failure but survived; one patient died of viral interstitial pneumonia 16 months after transplantation. The 5-year event-free survival rate and overall survival rate were 81%±12% and 89%±10% respectively. CONCLUSIONS: Haplo-HSC combined with tpCB transplantation is one of the effective methods for the treatment of X-CGD in children.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Doença Enxerto-Hospedeiro , Doença Granulomatosa Crônica , Transplante de Células-Tronco Hematopoéticas , Pré-Escolar , Haploidia , Células-Tronco Hematopoéticas , Humanos , Masculino , Estudos Retrospectivos , Condicionamento Pré-TransplanteRESUMO
OBJECTIVE: To evaluate long-term cryopreserved human bone marrow cells (BMCs) as a source of functional mesenchymal stem cells (MSCs). METHODS: Samples of human BMCs that were cryopreserved for 23-25 years (n=20) were thawed to obtain an initial culture and a primary culture (P(0)) that was propagated through five passages (P(1)-P(5)) to obtain MSCs. Freshly collected human bone marrow samples (n=20) were used as controls for comparison of efficiency of recovery and growth characteristics of MSCs. P(3) cultures were tested for their capacity to differentiate into osteoblasts, adipocytes, and neuronal cells. Appropriate staining, immunohistochemical and biochemical methods were employed to ascertain cell type identities at different stages of culturing. RESULTS: In the initial culture, the cell adherence rate of the cryopreserved cells was significantly lower than that of controls (19.7% vs. 38.2%, p<0.05) while the relative rate of recovery of MSCs was only 48.5±8.6% in P(0). At the end of P(3), fibroblast-like cells accounted for about 95% of cells in both cryopreserved and control groups (p>0.05). These cells were positive for essential MSC surface molecules (CD90, CD105, CD166, CD44, CD29, CD71, CD73) and negative for haematopoietic and endothelial cell markers (CD45, CD34, HLA-DR). The cell growth and cell cycle patterns were similar for both groups. MSCs at P(3) from both groups had similar capacities to differentiate in vitro into osteoblasts, adipocytes, and neuronal cells. CONCLUSION: Using the methods described here, long-term (23-25 years) cryopreserved human BMCs can be successfully cultivated to obtain MSCs that have good differentiation capabilities.
Assuntos
Bancos de Espécimes Biológicos/estatística & dados numéricos , Células da Medula Óssea/citologia , Criopreservação , Células-Tronco Mesenquimais/citologia , Adipócitos/citologia , Antígenos CD/análise , Células da Medula Óssea/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Contagem de Células , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Humanos , Imuno-Histoquímica , Imunofenotipagem , Células-Tronco Mesenquimais/efeitos dos fármacos , Neurônios/citologia , Osteoblastos/citologia , Fatores de TempoRESUMO
OBJECTIVE: To explore the effects of Yifei Huoxue Granule (YFHXG) on the proliferation and the transforming growth factor-beta (TGF-beta) activity of rat pulmonary artery smooth muscle cells (PASMCs) induced by platelet-derived growth factor BB (PDGF-BB). METHODS: Using tissue block adhering wall method, the primary rat PASMCs were cultured. PASMCs at the log phase growth were randomly divided into the control group, the PDGF-BB group, the PDGF-BB + high YFHXG group (at the final concentration of 7.5 mg/mL), the PDGF-BB + middle YFHXG group (at the final concentration of 1.5 mg/mL), and the PDGF-BB + low YFHXG group (at the final concentration of 0.3 mg/mL), respectively. MTT assay were employed to determine the cell proliferation rate of each group. Flow cytometric analyses were used to detect the cell cycle constituent ratio and the proliferation index (PI). In addition, TGF-beta protein's expression was determined by immunocytochemical assay (SP method). RESULTS: Compared with the control group, the proliferation of PASMCs in the PDGF-BB group was obviously active (P<0.01). But when compared with the PDGF-BB group, along with the increased concentration of YFHXG, the growth of PASMCs was obviously inhibited, the cell ratio of G0/G1, phase obviously increased, the cell ratio of S + G2/M phase significantly decreased, and PI significantly decreased. Besides, the expression of TGF-beta protein decreased in a dose-dependent manner (P<0.05, P<0.01). CONCLUSIONS: PDGF-BB could directly stimulate the proliferation of PASMCs. YFHXG had a significant inhibition on the proliferation of rat PASMCs induced by PDGF-BB and could regulate the expression of TGF-beta.
Assuntos
Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-sis/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Becaplermina , Células Cultivadas , Feminino , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas c-sis/administração & dosagem , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To explore the effects of hyperosmotic fluid and low temperature on hemorrhage and coagulation system after immersion of dogs with open abdominal wounds in seawater. METHODS: Twenty healthy dogs were subjected to open abdominal injury, then dogs were randomized equally into two groups: the control group (n=10) (without seawater immersion) and seawater immersion group (n=10). The variables of prothrombin time (PT), partial thromboplastin time (APTT), D-dimer and factor II, granule membrane protein-140 (GMP-140), endothelin-1 (ET-1) were determined. RESULTS: PT and APTT were significantly prolonged. D-dimer, GMP-140, and ET-1 were increased, while factor II was decreased after the dog with open abdominal wound was immersed in seawater (all P<0.05). Compared with the variables of control group, PT, APTT, D-dimer and factor II, ET-1 in seawater immersion group had markedly changed (all P<0.05) except GMP-140 at different time points (all P>0.05). CONCLUSION: Obvious vascular endothelial cell dysfunction, platelet activation, inhibition of coagulation factor activity, coagulopathy, and disorders in thrombosis and fibrinolysis system activation occur after dogs with open abdominal wounds are immersed in seawater.
Assuntos
Traumatismos Abdominais/sangue , Coagulação Sanguínea/fisiologia , Ferimentos Penetrantes/sangue , Animais , Modelos Animais de Doenças , Cães , Feminino , Imersão/fisiopatologia , Masculino , Distribuição Aleatória , Água do MarRESUMO
OBJECTIVE: Unrelated umbilical cord blood has the clear benefits of rapid availability and a reduced stringency of requirement for HLA match. The aim of this study was to investigate the efficacy of unrelated umbilical cord blood transplantation (UCBT) in the treatment of malignant leukemia in children. METHODS: Six children with malignant leukemia, including three cases of acute lymphocyte leukemia [two high-risk patients and one standard-risk patient in complete remission (CR)], two juvenile myelomonocytic leukemia (one in CR and one in the accelerating stage), and one acute myeloblastic leukaemia (in CR), received a UCBT. The umbilical cord blood grafts were HLA-matched (n=1) or HLA-mismatched at 1 (n=1) or 2 (n=1) or 3 (n=3) loci. Busulfan/cyclophosphamide/antithymocyte globulin (ATG) or total body irradiation (TBI)/cyclophosphamide/ATG was involved in the myeloablative pretreatment regimen. The median infused donor nucleated cell was 8.51 x 10(7)/kg of recipient weight, and the CD34+ cell was 1.81 x 10(5)/kg of recipient weight. Cyclosporin, corticoid, mycophenolate mofetil and daclizumab were used for prophylaxis of acute graft versus host disease (GVHD). RESULTS: The time to reach an absolute neutrophil count of 0.5 x 10(9)/L ranged from 11 to 35 days (median: 13 days) and the time to reach a platelet count of 20 x 10(9)/L ranged from 27 to 68 days (median: 30 days) after transplantation, and the donors' hematopoietic stem cells were shown in these patients. Four patients developed grade I to III acute GVHD but responded to steroids and daclizumab. Chronic GVHD was not found during a 3-16-month follow-up. Four patients survived and did not relapse during the follow-up. CONCLUSIONS: Unrelated umbilical cord blood is an alternative source of hematopoietic stem cells for patients with leukemia. UCBT can tolerate 1-2 HLA mismatches. The incidence of acute GVHD is high in UCBT recipients.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Leucemia/terapia , Criança , Pré-Escolar , Transplante de Células-Tronco de Sangue do Cordão Umbilical/efeitos adversos , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/etiologia , Hematopoese , Humanos , Lactente , MasculinoRESUMO
OBJECTIVE: To explore the maintaining measures for the vitality of hematopoietic stem cells (HSC) in vitro, so as provide technical support for ultra long distance transport of HSC collected from unrelated donors. METHODS: Peripheral blood hematopoietic stem cells (PBHSC) were treated by different methods according to various groups, then stored at 4 â in the refrigerator. The percentage of CD34+ cells, relative cell activity, relative cell proliferation rate, relative colony-forming rate, oxygen fraction and intracellular reactive oxygen species (ROS) were detected at 0, 24, 48 and 72 h after storage of PBHSC respectively. RESULTS: The percentage of CD34+ cells during 72 h storage did not altered. Along with the prolonging of storage time, the relative cell activity, relative cell proliferation rate and relative colony-forming rate gradually decreased in untreated PBHSC(control group), the related coefficients were -0.796, -0.883 and -0.815 respectively. Plasma dilution, antioxidants and oxygenation could improve the relative cell activity and relative cell proliferation rate, but oxygenation could decrease the relative colony-forming rate of PBHSC. The combination of 2 or 3 factors showed stronger protection effects on PBHSC. The intracellular level of ROS decreased gradually with the prolonging of storage time. Oxygenation of PBHSC could increase oxygen fraction, and also increase the intracellular level of ROS at the same time. The addition of antioxidants could reduce the level of ROS. CONCLUSION: The percentage of CD34+ cells can not serve as the indicator of PBHSC vitality. Plasma dilution, oxygenation and antioxidants can increase the survival and viability of PBHSC, but oxygenation can increase the intracellular ROS level and impair colony-forming ability of PBHSC. The combination of multiple factors can maintain the vitality of PBHSC better.
Assuntos
Células-Tronco Hematopoéticas , Antígenos CD34 , Antioxidantes , Espécies Reativas de OxigênioRESUMO
Aim of this study was to explore the effects of cryopreservation on biological characteristics of wharton's jelly derived mesenchymal stem cells (WJ-MSC), and to provide experimental evidence for clinical applications and the establishment of WJ-MSC bank. Primary WJ-MSC were produced by umbilical cord tissue culture in vitro. Fifth passage of WJ-MSC acquired by continuous cell culture were mixed with cryoprotectants, frozen in -80°C refrigerator and stored in liquid nitrogen. After the cryopreserved WJ-MSC were thawed, the first passage of WJ-MSC was obtained through cell culture and was taken as the 1st preserved passage (PP1). Thus, PP2-PP15 WJ-MSC were obtained by continuous cell subculture. The 1st control passage (CP1) to 15th passage (CP15) represented the 6th passage to 21st passage WJ-MSC acquired by subculturing in non-cryopreserved group. The biological characteristics of WJ-MSC from cryopreserved and control group, including the recovery rate of nucleated cells, trypan blue exclusion, CCK-8 activity, cell apoptosis, cell adherence, proliferation index, cell surface antigen, cell cycle and the capacities of induced differentiation into adipocyte, osteoblast and neuron, were detected and compared. The results indicated that the recovery rate of nucleated cells of cryopreserved WJ-MSC was 98.2%, trypan blue exclusion rate was 94.3%, CCK-8 activity was 91.4%, apoptotic rate was 3.9%, and the adherence rate was 92.6%. There was a statistically significant difference in proliferation index between PP1 and CP1 (P < 0.05), but there were no statistically significant differences between PP2-PP15 and their corresponding controls. The subculture cells highly expressed CD29, CD44, CD71, CD73, CD90, CD105, CD166 and HLA-ABC, and lowly expressed CD34, CD45 and HLA-DR. The expressions of above-mentioned surface antigens were not different statistically between two groups. The proliferation latency and logarithm proliferation of the subculture cells between two groups were also not different. After induced differentiation into adipocyte, osteoblast and neuron, the staining with oil red O, alkaline phosphatase and neuron-specific enolase was performed respectively, and the positive degrees were not clearly different macroscopically between two groups. Relatively high levels of triglyceride, alkaline phosphatase, and neuron-specific enolase in relevant cells could be detected, but had no significant differences between two groups. It is concluded that some WJ-MSC (< 10%) are damaged after cryopreservation, and the biological characteristics of WJ-MSC in cryopreservation group keep constant, as compared with that in non-cryopreservation group.
Assuntos
Criopreservação/métodos , Células-Tronco Mesenquimais/citologia , Geleia de Wharton/citologia , Diferenciação Celular , Sobrevivência Celular , Humanos , Sincalida/metabolismo , Cordão Umbilical/citologia , Cordão Umbilical/metabolismoAssuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Osteopetrose/terapia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/efeitos adversos , Doença Enxerto-Hospedeiro/etiologia , Teste de Histocompatibilidade , Humanos , Lactente , Masculino , Osteopetrose/etiologia , Transplante HomólogoRESUMO
OBJECTIVE: To investigate the inhibitory effect of Yifei Huoxue Granule (, YFHXG) on the hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and its mechanism of decreasing pulmonary arterial pressure. METHODS: Twenty male Sprague-Dawley (SD) rats were randomly divided into four groups: saline, and 0.66, 3.30 and 16.50 g/kg of YFHXG groups, the saline and different concentrations of YFHXG were given twice daily for 7 days, respectively. Serum-pharmacology method was used in the preparation of YFHXG serum. Tissue block anchorage was employed in the primary culture of rat PASMCs. The PASMCs were randomly divided into normoxia group, hypoxia group, and hypoxia+YFHXG group (0.66, 3.30 and 16.50 g/kg doses of YFHXG-treated serum groups, exposed to hypoxic condition). PASMCs in normoxia and hypoxia group were cultured with saline serum, hypoxia+YFHXG groups were cultured with different concentrations of YFHXG serum. Cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle was analyzed using flow cytometry. In addition, hypoxia inducible factor-1-alpha (HIF-1α) protein expression was evaluated by immunocytochemistry analysis, the concentration of intracellular reactive oxygen species (ROS) and Ca(2+) were determined by laser scanning confocal microscopy (LSCM). RESULTS: MTT assay and flow cytometry showed that hypoxia could directly activate the proliferation of PASMCs, while YFHXG dose-dependently inhibited hypoxia-induced proliferation of rat PASMCs. Immunocytochemistry showed that hypoxia enhanced HIF-1α protein expression, and LSCM showed that hypoxia significantly increased intracellular ROS and Ca(2+), while YFHXG decreased the expression of HIF- 1α and attenuated the hypoxia-induced increase in intracellular concentration of ROS and Ca(2+). CONCLUSIONS: YFHXG could inhibit hypoxia-induced proliferation of rat PASMCs, which may decrease pulmonary arterial pressure and vascular remodeling. The anti-hypoxia effect of YFHXG may be explained by its regulation of HIF-1α expression and of the levels of intracellular ROS and Ca(2+).
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Artéria Pulmonar/citologia , Animais , Cálcio/metabolismo , Ciclo Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
The objective of this study was to explore the effect of mesenchymal stem cells (MSC) on HL-60 proliferation and its molecular mechanism. HL-60 cells co-cultivated with MSC were used as experiment group, and the cells cultivated solely were taken as control group. HL-60 cells in the two groups were counted at different time. The time-quantity curve was drawn. The cell cycle and apoptosis ratio of HL-60 cells were compared between the two groups and expressions of Survivin and Bcl-2 protein were detected by flow cytometry. The results showed that HL-60 cells cultivated with MSC were obviously inhibited, especially on day 5 and 7 (P < 0.01). HL-60 cells were distributed on the phase of G(0)/G(1) [control group (47.0 ± 9.0)% vs experiment group (70.0 ± 16.0)%, P = 0.003], and apoptotic peak appeared. Both of Survivin and Bcl-2 protein expressions in HL-60 cells decreased [Bcl-2 protein in control group (63.0 ± 9.1)% vs experiment group (50.0 ± 14.1)%, P = 0.045; Survivin in control group (94.0 ± 9.3)% vs experiment group (77.0 ± 11.8)%, P = 0.006]. It is concluded that the MSC can inhibit HL-60 cell proliferation, and promote HL-60 cell apoptosis.
Assuntos
Células da Medula Óssea/citologia , Proliferação de Células , Células-Tronco Mesenquimais/citologia , Adulto , Apoptose , Técnicas de Cocultura , Citometria de Fluxo , Células HL-60 , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , SurvivinaRESUMO
The objective of this study was to evaluate the value of morphologic diagnosis for acute leukemia (AL), to explore the relation of morphologic diagnosis with immunology, cytogenetics and molecular biology diagnosis of AL and to analyze the onset characteristics of AL in 10 years. The samples of bone marrow and peripheral blood from 233 newly diagnosed cases of AL were collected during 2001-2011 years; the morphologic examination and immunologic, cytogenetic and molecular biologic examination (ICM) were carried out, the consistency of morphologic diagnosis with ICM diagnosis was compared, the onset characteristics of AL was analyzed. The results showed that: (1) the consistent rate of immunology, cytogenetics, molecular biology diagnosis with morphologic diagnosis was 84.3%. The order of consistent rat was AUL, M0 < M1 < HAL < M4 < M2 < M3 < M5 < ALL < M6, M7, AP; (2) Misdiagnosis always occurred among AUL, M0, M1, ALL and HAL or among M2a, M3v, M4 and M5. (3) In 233 cases, the highest ratio of blast was observed in M1 (92.5%), while the lowest ratio of blast was observed in M2 (49.5%). (4) AL occurred more frequently in males than that in female (147:86). (5) AL occurred in patients aged from 1 to 88 years. The median age was 41.5 for AUL, 40.8 for M0, 43.4 for M1, 46.3 for M2, 33.8 for M3, 42.6 for M4, 48.8 for M5, 77.3 for M6, 2.5 for M7, 65.0 for AP, 29.1 for ALL and 40.3 for HAL. (6) The number of patients in the later five years (139 cases) was significantly greater than that in the first five years (94 cases), especially the patients with M1, M2, M3, M4, and M5. It is concluded that morphologic diagnosis has important clinical value in the MICM diagnosis of AL. Attaching importance to the confusing cell morphology and onset characteristics of AL can improve the diagnostic accuracy.
Assuntos
Leucemia/diagnóstico , Leucemia/patologia , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Análise Citogenética , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Biologia Molecular , Estudos Retrospectivos , Adulto JovemRESUMO
OBJECTIVE: In order to investigate whether or not thrombopoietin (TPO) could promote the fibrogenesis of bone marrow stromal cells in absence of megakaryocytes (MKs). METHODS: Improved dexter culture system with various TPO concentrations was used for ex vivo culture of bone marrow stromal cells. Relative proliferation index, the expressions of fibronectin, laminin and type IV collagen, and the systhesis of type III procollagen were detected at different time points during culture process. RESULTS: TPO stimulated the proliferation of bone marrow stromal cells. Relative proliferation index of the stromal cells increased with the TPO concentration increasing, and was not related to the exposure time. The expressions of fibronectin, laminin, and type IV collagen appeared stronger in the TPO groups than those in the control group. But the expressions of these molecules were not dependent upon the culture time. TPO could accelerate the synthesis of type III procollagen in bone marrow stromal cells, and this acceleration was unrelated to the TPO concentration. CONCLUSION: These findings suggested that TPO could stimulate the stromal cells with a consequence of increased syntheses and secretions of the extracellular matrix and collagen in absence of MKs. In other words, TPO could promote the fibrogenesis of bone marrow stromal cells without the existence of MKs.
Assuntos
Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/patologia , Trombopoetina/farmacologia , Células Cultivadas , Colágeno Tipo III/metabolismo , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Fibrose/patologia , Humanos , Laminina/metabolismo , Megacariócitos/citologia , Células-Tronco Mesenquimais/metabolismoRESUMO
The aim of this study was to investigate the best method to preserve human bone marrow cells and the effectiveness of long term cryopreservation at -80 degrees C. The human bone marrow cells in 20 samples were firstly frozen by a programmed freezer or -80 degrees C refrigerator, and then were preserved in liquid nitrogen with DMSO-AuP (10% dimethylsulfonamide, 10% autologous plasma) or DMSO-HES-HuA (5% dimethylsulfonamide, 6% hydroxyethyl starch, 4% human serum albumin) as cryoprotectant for 21 to 25 years. They were thawed in 38 degrees C. The cell sample frozen in -80 degrees C refrigerator was frozen at a low frozen speed of 1 degrees C/min which was the same as the programmed freezer before -30 degrees C. Before detection the bone marrow cells were taken from liquid nitrogen and were thawed in 38 degrees C, then the suspension of bone marrow cells was prepared for detection. The cell morphology and recovery rate of erythrocytes, nucleocytes and platelets; the recovery rate of hematopoietic stem progenitors cells, as well as mesenchymal stem cells were determined. The results showed that the protective effectiveness of DMSO-HES-HuA was better than DMSO-AuP. The mature erythrocytes were destroyed lightly [(3.5 +/- 1.5)% versus (12.6 +/- 4.8)%], the hemolysis rate was lower [(3.3 +/- 1.6)% versus (23.1 +/- 5.1)%]. Osmotic fragility of erythrocytes in the former was not changed, but was dropped in the latter. The recovery rates of red cell, platelet, granulocyte-macrophage colony forming units and long term culture-initiating cells were higher in the former than that in the latter [(96.1 +/- 1.8)%, (70.0 +/- 9.5)%, (49.2 +/- 10.9)%, (54.2 +/- 13.8)% versus (76.3 +/- 5.6)%, (52.7 +/- 8.1)%, (43.5 +/- 12.3)%, (47.2 +/- 13.6)% respectively]. With each kind of cryoprotectant or frozen method, the frozen MSC could keep the original growth properties. With the same cryoprotectant and different frozen method, the cryopreservative effectiveness was not different. The influence of the cryoprotectant prescriptions and the frozen methods on the cryopreservative effectiveness was little. It is concluded that the human bone marrow cells with DMSO-AuP or DMSO-HES-HuA as cryoprotectant, frozen by a programmed freezer or -80 degrees C refrigerator, could be then preserved in liquid nitrogen for long time. When the preserving time was as long as 21 to 25 years, the morphology, the recovery rate and the activity of various kinds of cells were still good. The method of freezing by -80 degrees C refrigerator with 5% DMSO-6% HES-4% HuA and preserving in liquid nitrogen would be convenient, cheap and easily-manipulated for preservation of the human bone marrow cells.
Assuntos
Células da Medula Óssea/citologia , Criopreservação/métodos , Crioprotetores , Nitrogênio , Sobrevivência Celular , Ensaio de Unidades Formadoras de Colônias , Humanos , Fatores de TempoRESUMO
This study was aimed to compare the influences of bone marrow mesenchymal stem cells (BMMSCs) from patients with acute myeloid leukemia (AML), AML patients with complete remission (CR) and non-leukemia patients on HL-60 cells. The HL-60 cells were divided into three groups: group of co-cultivation with BMMSCs of AML patients, group of co-cultivation with BMMSCs of AML patients with CR and group of co-cultivation with BMMSCs of non-leukemia patients. The count of HL-60 cells, the CD11b and survivin expression of HL-60 cells, the cell cycle distribution of the HL-60 cells in 3 groups were compared by flow cytometry, the morphology and differentiation rate of HL-60 cells in 3 groups were observed and compared by microscopy. The results showed that there were no differences in HL-60 cell count at five and seven days, in HL-60 distribution at the G(0)/G(1) phase, in survivin and CD 11b expressions in 3 groups. All cells of 3 groups began to mature, and the differentiation rates in 3 groups were 18.0 +/- 3, 17.0 +/- 1.3 and 19.0 +/- 2.0 respectively, therefore there were no significant differences between the 3 groups (p = 0.23). It is concluded that there is no influence of BMMSCs in 3 groups on the proliferation and differentiation of HL-60 cells.
Assuntos
Células da Medula Óssea/citologia , Leucemia/patologia , Células-Tronco Mesenquimais/citologia , Diferenciação Celular , Proliferação de Células , Técnicas de Cocultura , Células HL-60 , HumanosRESUMO
This study was aimed to investigate whether the thrombopoietin (rhTPO) may facilitate myelofibrosis or not. The modified Dexter culture system with various concentrations of rhTPO was used to culture the stromal cells in vitro; the proliferative activity of cells was detected by MTT method; the morphologic changes were observed by light and scanning electron microscopy; the staining changes of ALP, PAS, AS-D NCE and IV type collagen were observed by cytochemistry method; the changes of fibronectin, laminin and IV type collagen were assayed by immunohistochemistry method; the cell surface antigens were assayed by flow cytometry. The results indicated that rhTPO could promote the proliferation of stromal cells which was related to the concentrations of rhTPO. Proliferative activity of stromal cells increased with increasing of rhTPO concentration, and was not related to the exposure time. On day 3 stromal cells adhered to the wall, and became oval. On day 7 stromal cells turned to fusiform and scattered dispersively. On day 12 to 14 these cells ranged cyclically and became long fusiform. Cells covered 70%-80% area of bottle bottom at that time. By day 16 to 18 these cells covered more than 90% area of bottom and ranged cyclically. They displayed the same shape as fibroblasts. By light microscopy with Wrights-Giemsa staining, fibroblasts predominated morphologically, few macrophages, endothelial cells and adipose cells were found. There were no significant differences between experimental group and control group. On day 14 to 42 the adherent cells were positive with PAS staining, poorly positive with ALP and naphthol AS-D chloroacetate esterase (AS-D NCE) staining, and the difference in cytochemistry was not significant between two groups. When these cells were dyed with Masson's trichrome and Gomori's staining, neither collagen fibers nor reticular fibers were positive, but fibronectin, laminin, and collagen type IV appeared positive stronger in experimental group than those in control. The expressions of these molecules were not dependent on culture time. By scanning electron microscopy microvilli and fibers on cell surface appeared more and more, monolayer cells evolved into multilayer cells, and newly-formed fibroblasts appeared gradually as culture time prolonged. These alterations were not different among various groups. The expressions of CD34, CD45, CD105, CD106, and CD166 were not affected obviously by rhTPO. It is concluded that rhTPO had no effects on histochemical properties of stromal cells. Fiber staining and scanning electron microscopic examinations revealed that rhTPO can not facilitate fiber formation of stromal cells. But rhTPO may be able to augment the expressions of fibronectin, laminin and collagen type IV of stromal cells. Therefore it is still necessary to follow up the patients for a long time, who have received rhTPO therapy clinically.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Trombopoetina/farmacologia , Células da Medula Óssea/citologia , Proliferação de Células/efeitos dos fármacos , Fibroblastos , Humanos , Células Estromais/citologiaRESUMO
AIM: To explore proper cryopreservative systems for hematopoietic stem cells. METHODS: Peripheral blood mononuclear cells from 20 persons were mixed with different cryopreservative agent, dimethyl suflfoxide (DMSO) or combination of DMSO and hydroxyethyl starch (HES), then cooled in -80 degrees C low temperature refrigerator (Refr) or autocontrolled programmed cryogenic system (PCS), preserved in Refr or in liquid nitrogen. GM-CFU, LTC-IC, CD34+ cells and typeran blue resistance (TBR) were assayed after different period of cryopreservation. RESULTS: The recovery rates of CFU-GM, LTC-IC, CD34+ cells and TBR in peripheral blood mononuclear cells which were cooled and preserved in Refr with 5% DMSO-6% HES were 82.2% +/- 14.7%, 83.0% +/- 12.2%, 94.2% +/- 4.3% and 97.7% +/- 3.9% respectively, significantly higher than that in Refr with 10% DMSO (P < 0.05). When cells were cryopreservated with the same cryopreservatives, there was no significantly difference of recovery rate in group of Refr and group of Refr with PCS. Meanwhile, there was not significantly difference of recovery rate among all three groups, preserved in Refr ahead of liquid nitrogen, in Refr merely, in liquid nitrogen with PCS within one year (p > 0.05). However, the recovery rate of CFU-GM, LTC- IC, CD34+ cells and TBR decreased dramatically if cells were cooled and preserved in Refr for two years. After cells were thawed, the cell activity declined gradually at room temperature if the cryopreservatives were not removed or diluted. The cell activity of 10% DMSO group was affected more than that of 5% DMSO-6% HES group. CONCLUSION: 5% DMSO-6% HES is better than 10% DMSO as cryopreservatives for hematopoietic stem cells. Refr cryopreservation is a simple and effective method if cells would be cryopreserved for less than one year. If cells would be cryopreserved for more than one year, liquid nitrogen cryopreservation should be recommended. The cryopreservatives should be diluted or removed immediately after cells were thawed.
Assuntos
Preservação de Sangue/métodos , Criopreservação/métodos , Células-Tronco Hematopoéticas/citologia , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , HumanosRESUMO
To investigate the effect of total panax notoginseng saponins (tPNS) to induce the differentiation of mononuclear cells (MNC) in cord blood into endothelial cells, the DMEM culture media containing tPNS were used to induce the MNC of cord blood. Then, the morphology of the adherent cells was observed by the light microscopy and the fluorescence microscopy, the changes of cell surface markers (UEA-1), function marker (vWF) and CD31 were detected by flow cytometry. The results showed that the number of adherent cells produced by 250 mg/L tPNS and the positive rate of cells expressing CD31 and UEA-1 were higher than those in the groups of other concentrations (P < 0.05). There was no significant difference in the number of adherent cells expressing CD31 and UEA-1 between 50 ng/ml VEGF + 250 mg/L tPNS and 50 ng/ml VEGF. It is concluded that the traditional Chinese drug tPNS can induce partial MNC in the cord blood to differentiate into endothelial cells. No synergistic effect has been found between tPNS and VEGF.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Endoteliais/citologia , Leucócitos Mononucleares/citologia , Panax notoginseng/química , Saponinas/farmacologia , Células Cultivadas , Células Endoteliais/enzimologia , Sangue Fetal/citologia , Humanos , Saponinas/isolamento & purificaçãoRESUMO
This study was aimed to investigate the influence of cryopreservation on biological properties and function of leukemic dendritic cells (L-DCs) derived from patients with acute or chronic leukemia. Some fresh leukemic cells were detected immediately; some were cultured immediately; some were cryopreserved in -80 degrees C with 5% DMSO-6% HES as cryopreservor. After being thawed, they were cultured. The combination of rhGM-CSF, rhIL-4, rhTNF-alpha and other cytokines were added into the culture system. 12 days later, L-DCs were assayed for morphology, immunophenotype, mixed lymphocytic reaction (MLR) and CTL cytotoxicity on autologous leukemic cells. The results showed that both fresh and cryopreserved leukemic cells obtained from patients with acute or chronic leukemia revealed typical DC morphologically by means of using combinations of cytokines in culture, but there was no significant difference between pre-or post cryopreservations. L-DCs also upregulated the expression of CD80, CD54, HLA-DR, CD1a, CD83 and CD86, and downregulated the expression of CD14, but there was also no difference as compared with L-DCs befor cryopreservation. L-DCs derived from leukemic cells were also capable of stimulating MLR and inducing CTL which could kill autologous leukemic cells obviously. It is concluded that leukemic cells, regardless of fresh or frozen, can induce L-DCs after culture with cytokine combination. The L-DCs can induce CTL targeting autologous leukemic cells, and may be used to treat MRD as immunotherapy. The induction and biological properties of L-DCs are not influenced by cryopreservation.
Assuntos
Criopreservação , Células Dendríticas/citologia , Leucemia Mieloide/imunologia , Leucemia Mieloide/patologia , Linfócitos T Citotóxicos/imunologia , Células da Medula Óssea/citologia , Antígenos CD8/metabolismo , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-4/farmacologia , Receptores de Lipopolissacarídeos/metabolismo , Ativação Linfocitária , Proteínas Recombinantes , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Endothelial progenitor cell (EPC) is a subtype of progenitor cells that can circulate, proliferate and have the ability to differentiate into mature endothelial cells. Exploration of EPC have significance to understand the vascular forming process of adults physiologically and pathologically. Cytokines, especially haematopoiesis-related cytokines, cellular stimulating factors in the body also influence EPC. In this review, the advances of research on the relationship between EPC and some cytokines related with haematopoiesis (G-CSF, EPO, HAPO and IL-18) and the potential signal transduction pathway were summarised.
Assuntos
Citocinas/fisiologia , Células Endoteliais/citologia , Hematopoese/fisiologia , Células-Tronco/citologia , Eritropoetina/fisiologia , Fator Estimulador de Colônias de Granulócitos/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Humanos , Interleucina-18/fisiologia , Proteoglicanas/fisiologiaRESUMO
AIM: To observe the basical properties of adherent stromal cells in culture derived from cryopreserved bone marrow cells (BMCs), and to provide laboratory evidences for clinical application of cryopreserved stromal cells. METHODS: Fresh BMCs and adherent stromal cells cultured for 14 days in Dexter long-term culture system (fresh stromal cells) plus 5% DMSO-6% HES cryopreservatives were frozen in -80 degrees C refrigerator, and cryopreserved in - 196 degrees C liquid nitrogen for 2 weeks (the former is called cryopreserved BMCs, the latter called cryopreserved stromal cells). These cells were cultured in Dexter long-term culture system after they were thawed. We have examined the growth features, constituents and stimulating functions of the culture of these cells. RESULTS: Growth features: Cryopreserved BMCs produced adherent stromal cells, cell clusters and cell layer 1-2 days later than fresh BMCs. Cryopreserved stromal cells formed cell layer in 2nd day of culture, and were 12-18 h later than fresh stromal cells. Cryopreserved BMCs and stromal cells proliferated significantly less than fresh BMCs and stromal cells. Constituents: The ratio of fibrocytes and endothelial cells were lower, and the ratio of macrophages and fat cells were higher in culture of cryopreserved BMCs than that in fresh BMCs. The measurements in culture of cryopreserved stromal cells were much more significant compared with that in fresh stromal cells. Numbers of cells containing apoptic bodies in culture of cryopreserved BMCs and stromal cells were more than that in fresh BMCs and stromal cells. TBRR of cryopreserved BMCs and stromal cells were 92.5% and 89.5% respectively. The expression rates of CD14 and HLA-DR in culture of cryopreserved BMCs and stromal cells were higher than that in fresh BMCs and stromal cells, and just in contrast with the expression rates of CD45 and CD33. Stimulating functions: CAFA and LTC-IC on the stromal cell layer derived from cryopreserved BMCs, cryopreserved stromal cells, fresh BMCs and fresh stromal cells were all growing well, and there were no significant differences among these groups. CONCLUSION: Biological properties of adherent stromal cells derived from BMCs and stromal cells were injured slightly and still maintained completely after cryopreservation with 5% DMSO-6% HES cryopreservatives.