Cell Differentiation ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; Fetal Blood ; cytology ; Hepatocytes ; cytology ; drug effects ; Humans ; Stem Cells ; cytology ; drug effects

The study was aimed to investigate the hematopoietic function of placenta tissue and clarify the effect of human placental chorionic tissue in different periods on proliferation of hematopoietic stem cells in vitro, in order to further understand the changes of the hematopoietic function of placenta with the time prolonging. The experiments were divided into four groups: early placenta (group B), mid-term placenta (group C), full-term placenta (group D), and blank group (group A). The hematopoietic stem cells were amplified in co-culture way, and the colony formation ability after the expansion was observed. The results showed that compared to initial concentration, the CD34(+) cells cultured with full-term placenta were amplified by (2.60 ± 0.20) times, which was significantly higher than those CD34(+) cells cultured with mid-term placenta (1.74 ± 0.24) and early placenta (1.14 ± 0.12), but that in blank group was reduced without amplification. After culture for 14 days, the colony number of group C and group D were significantly higher than that of group A and group B. Among them the number of CFU-GM, CFU-GEMM, BFU-E of group C all were a little higher than that of group D. It is concluded that human placental extract in different period without any exogenous cell factors all can support the proliferation of hematopoietic stem cells, this ability is getting stronger with time increasing. The colony formation ability of the amplified cells shows weakened after the first increase, this colony formation ability of the amplified cells in group C is strongest, slightly stronger than that of group D.
Antigens, CD34 ; Cell Proliferation ; drug effects ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; drug effects ; Humans ; Placental Extracts ; pharmacology

To investigate the effect of S-nitrosoglutathione (GSNO), a nitric oxide donor, on platelet production from megakaryocytes differentiated from cord blood CD34(+) cells in vitro, the CD34 (+) cells from eight fresh umbilical cord blood samples by a high-gradient magnetic cell sorting (MACS) system were cultured in serum-free medium for 14 d with thrombopoietin (TPO) 50 ng/ml, IL-3 10 ng/ml, stem cell factor (SCF) 50 ng/ml and rHuGM-CSF 20 ng/ml. Then, CD61 (+) cells were purified by MACS system from these CD34 (+) cells, and were cultured in serum-free medium supplemented with TPO 50 ng/ml, IL-3 10 ng/ml and SCF 50 ng/ml in the presence (treatment group) and absence (control group) of GSNO for 30 min or 2 h. Platelet-sized particles were counted by flow cytometry; megakaryocyte structure was detected by scanning electron microscope. Aggregation of the thrombin-induced platelet particle was observed under inversion microscope. cGMP was assessed by commercial ELISA kit. The results showed that, compared with the control group, the number of platelet-sized particles significantly increased (P<0.05) in the treatment group, in which megakaryocytes presented significant pseudopod formation and extensive membrane blebbing. The platelet particle aggregation could be observed under microscope after thrombin induction. cGMP activity was significantly increased after treatment with GSNO (P<0.05). These results propose that GSNO can facilitate platelet production from megakaryocyte, and it may be partly through cGMP pathway.
Antigens, CD34 ; analysis ; Blood Platelets ; cytology ; Cell Differentiation ; drug effects ; Cyclic GMP ; blood ; Female ; Fetal Blood ; cytology ; Hematopoiesis ; drug effects ; Hematopoietic Stem Cells ; cytology ; Humans ; Megakaryocytes ; cytology ; Nitric Oxide ; physiology ; Platelet Aggregation ; drug effects ; Pregnancy ; S-Nitrosoglutathione ; pharmacology

This study was aimed to investigate whether aspirin has effect on function of late endothelial progenitor cells (EPC). Cord blood CD34(+) cells were purified using the ficoll density gradient centrifugation and human CD34 positive selection kit, then the cells were inoculated on fibronectin-coated culture plate. After culture for 2 weeks, adherent cells were identified as EPC by flow cytometry, immunofluorescence, RT-PCR, uptake of Dil-Ac-LDL and matrigel tube formation assay. EPC were treated with different concentrations of aspirin (0.1, 1, 10, 100, 1 000, 10 000 µmol/L) for 24 h, then the proliferation, adhesion and migration ability of these cells were analyzed by CCK-8 assay and transwell methods. The results indicated that the low concentrations of aspirin (0.1 and 1 000 µmol/L) promoted late EPC adhesive and migratory capacity, but no obvious effect on proliferation of late EPC were observed. On the other hand, the high concentrations of aspirin (10 000 µmol/L) inhibited proliferation and migratory capacity of EPC, but had no obvious effect on adhesive ability of EPC. It is concluded that low concentration of aspirin promotes migration and adhesion of late EPC, while the high concentration of aspirin decreases EPC proliferation and migratory capacity of EPC.
Aspirin ; pharmacology ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endothelial Cells ; cytology ; drug effects ; Fetal Blood ; cytology ; Humans ; Stem Cells ; cytology ; drug effects

OBJECTIVETo observe the effect of Bushen Yiqi Huoxue Recipe (BYHR, a Chinese medical prescription for reinforcing Shen, replenishing qi and promoting blood circulation) on placental trophoblast apoptosis in fetal growth restricted (FGR) pregnant rat for the sake of explore its mechanism of action in treating FGR.

METHODSFGR animal model was established by passive smoking, 32 pregnant rats were divided into four groups at random: the normal group, the model group, the Chinese medicine (CM) group (model rats treated by BYHR) and the Western medicine (WM) group (model rats treated by arginine). The histological change of placenta was examined with HE stain, the trophoblast apoptosis was detected by TUNEL and RT-PCR, and the expressions of Bcl-2 and Bax in the placenta were detected by immunohistochemical method.

RESULTSBlood stasis and villous ischemia were seen in placenta of FGR model rats. Placental microcirculation was significantly improved in the CM group, but in the WM group only partially improved. The median apoptotic index of syncytial trophoblast cells in the four groups, in normal, model, CM, and WM order, was 45%, 75%, 57% and 70%, as compared with the model group, it was much lower in the normal group and the CM group (P < 0.01), but a similar level was shown in the WM group. No significant difference in mRNA and protein expressions of Bcl-2 and Bax was found among the four groups.

CONCLUSIONBYHR can improve the placental microcirculation in FGR rats to prevent excessive apoptosis through a mechanism other than the classical Bax/Bcl-2 apoptotic pathway, which needs further exploring.

Animals ; Apoptosis ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Fetal Growth Retardation ; prevention & control ; Microcirculation ; drug effects ; Placenta ; blood supply ; cytology ; Pregnancy ; Rats ; Trophoblasts ; cytology

OBJECTIVETo investigate the effects of Compound Salvia injection (CSI) on the number and activity of endothelial progenitor cells (EPCs).

METHODMononuclear fraction of human umbilical cord blood was obtained by density gradient centrifugation and plated on fibronectin coated culture dishes. Cells were divided in to five groups: group control, group VEGF, group CSI 50, group CSI 10 and group CSI 2 (supplemented with none cytokine, VEGF 10 ng x mL(-1), CSI 50, 10, 2 microg x mL(-1), respectively). After six days in culture, cell clusters were viewed with an inverted microscope, fluorescence-activated cell sorting (FACS) analysis of PE-CD34 and FITC-VE-Cadherin was performed to detect number of EPCs, adhesion assay was performed by replating cells on fibronectin coated dishes, and then counting adherent cells.

RESULTNumbers of EPCs of group VEGF, group CSI 10 and group CSI 2 were significantly increased as compared with those of group control ( P < 0.01, P < 0.05, P < 0.01, respectively), and numbers of EPCs of group CSI 2 were more than those of group CSI 10 and group V (P < 0.01). Compared with group control, number of EPCs of group CSI 50 was significantly decreased (P < 0.01). Compared with group control, numbers of clusters and adhesive EPCs of group CSI 10 and group CSI 2 were significantly increased, while those of group CSI 50 were significantly decreased.

CONCLUSIONLow concentration CSI can significantly promote EPCs augmentation and enhance its functional activity, while high concentration CSI significantly restrains it.

Blood Cell Count ; Cell Adhesion ; drug effects ; Dose-Response Relationship, Drug ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Endothelium, Vascular ; cytology ; Fetal Blood ; cytology ; Humans ; Injections ; Plants, Medicinal ; chemistry ; Salvia miltiorrhiza ; chemistry ; Stem Cells ; cytology ; drug effects

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.
Cell Differentiation ; drug effects ; Cells, Cultured ; Endothelial Cells ; cytology ; enzymology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Panax notoginseng ; chemistry ; Saponins ; isolation & purification ; pharmacology

This study is to investigate whether the synthesized chiral compound Nordy has influence on the function of endothelial progenitor cells (EPCs) from human umbilical cord blood induced by vascular endothelial growth factor (VEGF). EPCs were isolated from human umbilical cord blood by density gradient centrifugation. After cultured for 7 -10 days, EPCs were prepared for detecting effect of Nordy on proliferation, migration and tubule-forming activity in Matrigel induced by VEGF. Incubation of EPCs with 100 micromol L(-1) Nordy for 24 h initially inhibited the proliferative capacity of EPCs induced by VEGF (P <0.05). Moreover, 25 -50 micromol L(-1) Nordy also exhibited inhibitory effect at 48 -72 h. In addition, 25 - 100 micromol L(-1) Nordy impaired EPCs migratory and tubule-forming capacity in vitro (P < 0.05). Nordy could inhibit in EPCs the functions of proliferation, migration and tubulogenesis induced by VEGF in vitro, which might be a possible mechanism of its anti-EPCs effects.
Antineoplastic Agents ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endothelial Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Masoprocol ; analogs & derivatives ; pharmacology ; Neovascularization, Physiologic ; drug effects ; Stem Cells ; cytology ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors

OBJECTIVETo investigate the effects of platelet factor 4 (PF4) on the adherence, and the expressions of adherent molecules CD(49d) and CXCR4 and the receptor of SDF-1 of fresh and expanded cord blood CD(34)(+) cells.

METHODSCD(34)(+) cells were isolated from cord blood using MACS immune magnetic beads. The adherent ability was assayed by using crystal violet staining and the expression of adherent molecule CD(49d) and CXCR4 by FACS.

RESULTS(1) PF4 could increase the adherent ability of the fresh cord blood CD(34)(+) cells, the effect being positively correlated with the dose of PF4. (2) SDF-1 at concentration of 100 ng/ml increased the adherent ability of the fresh cord blood CD(34)(+) cells. (3) The spontaneous and the SDF-1 induced adherent ability of the cord blood CD(34)(+) cells began to decrease after being cultured for 10 days without PF4, while in the presence of PF4 at 100 ng/ml, the ability of the cord blood CD(34)(+) cell adhering to the stroma layer still remained at higher level. At day 14, the adherent ability was (262.04 +/- 64.81)% and (64.35 +/- 8.29)% in PF4 group and control group, respectively, if it was defined as 100% at day 0. SDF-1 at concentration of 100 ng/ml induced adherent ability was (138.31 +/- 32.39)% and (67.66 +/- 12.44)% in PF4 group and control group, respectively. (4) The expression of CD(49d) and CXCR4 increased 13.02% and 17.33%, respectively, when incubated with PF4.

CONCLUSIONSPF4 could increase the adherent ability and promote the expression of CD(49d) and CXCR4 of the cord blood CD(34)(+) cells, suggesting that PF4 promote the circulating stem cells homing to the marrow in the process of stem cells transplantation.

Antigens, CD34 ; blood ; Cell Adhesion ; drug effects ; Fetal Blood ; cytology ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Integrin alpha4 ; blood ; Platelet Factor 4 ; pharmacology ; Receptors, CXCR4 ; blood

Purpose of this study was to establish an effective method in vitro to proliferate natural killer T (NKT) cells from umbilical cord blood (UCB) and peripheral blood (PB), and to study their different phenotype. Mononuclear cells (MNC) from UCB and PB were cultured in the presence of IL-2 (100 U/ml), with or without alpha-Galcer. TCR Valpha24 Vbeta11 double positive natural killer T-cells (NKT cells) and their other phenotypes were determined by flow cytometry. The results showed that after expansion for 7 days, TCRValphabeta(+) NKT cells from UCB-MNCs increased by (8.74 +/- 4.37) x 10(2) times as much, but most of them did not express NK1.1 and its TCR Vbeta11(+) was higher than TCR Valpha24(+). After expansion for 14 days, TCR Valphabeta(+) NKT cells from PB-MNCs increased by (3.72 +/- 2.01) x 10(2) times, the expression of NK1.1 was high and its TCR Vbeta11(+) was almost equal to TCR Valpha24(+). It is concluded that human TCR Valpha24 Vbeta11 double positive NKT cells can expand by addition of alpha-Galcer. The proliferation efficiency in UCB-MNCs is greater than that in PB-MNCs. Most of the UCB-NKT is NK1.1(-), while the PB-NKT is NK1.1(+), a new subset of NKT cells.
Cell Proliferation ; Cells, Cultured ; Fetal Blood ; cytology ; Galactosylceramides ; pharmacology ; Humans ; Interleukin-2 ; pharmacology ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; Leukocytes, Mononuclear ; cytology ; Phenotype ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; immunology