Dendritic cells induced from culture of cryopreservated G-CSF-mobilized peripheral blood stem cell harvests

AIM: To find the way of culturing the dendritic cells(DC) from the cryopreservated G-CSF-mobilized peripheral blood stem cell(PBSC) harvests. METHODS: The non-adherent or adherent cells from the fresh or refreshed PBSC harvests were induced to DCs separately. The amount of CD34+ cells, CD14+PI+ cells in the PBSC harvests and the characters of DCs were analyzed by flow cytometry. RESULTS: The DCs cultured from the non-adherent cells were similar to those from the fresh groups in the number and the characters. The amount of CD34+ cells was also similar between them, but CD14+PI+ cells were higher in the refreshed groups than that in the fresh groups. The adherent cells from the refreshed group were too few to be induced to DCs. CONCLUSION: The refreshed PBSC harvests could be induced to the matured activated myloid DCs cultured by the routine of CD34+ cells rather than by the way of monocytes.     

Effect of G-CSF on dendritic cell subsets in donors mobilized peripheral blood cells

AIM: To explore the effect of granulocyte colony stimulating factor (G-CSF) on donor’s dendritic cell （DC）subsets in peripheral blood cells (PBC). METHODS: The subsets of dendritic cells in PBC were analyzed by CD34^-Lin^- HLA-DR⁺ cells and the levels of IL-12p40, IL-10, IFN-γ, IL-4 in the serum were tested by ELISA before (25 cases) or after G-CSF mobiling. The relationship between the ratio of DC₁/DC₂ (CD11c⁺CD123^-/CD11c^-CD123⁺) and CD34⁺/MNC was explored. RESULTS: CD34⁺/MNC in PBC harvests was above 0.4% in 23 cases, and the ratio of DC₁/DC₂ was lower, the HLA-DR expression of DC₂ was enhanced after G-CSF mobiling than before, but the levels of IL-12p40, IL-10, IFN-γ, IL-4 in donors serum and CD83 expression of DC₂ were not changed by G-CSF. CONCLUSION: DC₂ increased accompanied by the increase in CD34⁺ cells in the PBC harvests. Although the expression of HLA-DR in DC₂ was up-regulated by G-CSF, these DC₂ did not regulate Th2 cells to excrete inhibitor cell factors and kept on the precursor characters.     

Hemangioblastic characteristics of human adipose tissue-derived adult stem cells in vivo

AIM: To investigate whether Flk1+CD31-CD34- cells isolated from human adult adipose tissue have characteristics of hemangioblasts in vivo. METHODS: After sublethally irradiated (300cGy) with a caesium source, the female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were injected with human adipose tissue-derived Flk1+CD31-CD34- cells (105 cells per mouse) via tail vain with 0.4 mL Roswell Park Memorial Institute medium (RPMI-1640). The control mice received the same volume of RPMI-1640 medium. All mice were killed 2 months after transplantation for further study. The differentiation potential of Flk1+CD31-CD34- cells was assessed in bone marrow and gastrointestinal tract by the methods of flow cytometry, RT-PCR, FISH, and triple-color immunofluorescence. RESULTS: Flk1+CD31-CD34- human adipose tissue-derived adult stem cells differentiated into endothelial cells and hematopoietic cells at the single-cell level in vivo. CONCLUSION: Human adult adipose tissue-derived Flk1+CD31-CD34- cells bear characteristics of hemangioblast in vivo and may have potential application for the treatment of hematopoietic and vascular diseases.     

Human mesenchymal stem cells differentiate into osteoblasts

AIM:To investigate the differentiation from human mesenchymal stem cells (hMSC) into osteoblasts. METHODS:MSC were separated from human marrow with Ficoll-Paque reagent and expanded in cuture medium. To detect the surface antigens, The labeled cells were analysed on a FACScan flow cytometer. hMSC were induced to differentiate from mesenchymal stem cells into osteoblasts with dexamethasone, vitamin C, β-GP. Cell morphology、AP activity、calcium deposition and osteopontin were detected. P10 MSC were compared to P3 MSC in the tendency of osteoblastic differentiation. RESULTS:The cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. hMSC showed a strong self-renewal capacity. After primary culture, approximately (5-6)×10⁵ cells were obtained. These expanded attached MSC were uniformaly positive for CD29,CD44,CD59,CD105,CD166 and didn’t express CD11a, CD14, CD33, CD34, CD45, CD38, CD80, CD86, CD117. After osteoblasts induction, the cells changed from spindle-shape to cuboidal and polygonal in cell morphology. The AP activity increased gradually and many scattered calcium nodes were observed. The expression of osteopontin was positive. CONCLUSION:hMSC can be induced to differentiate into osteoblasts.     

Human mesenchymal stem cells differentiate into neuron-like cells with Tanshinone II A

AIM:To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells with Tanshinone II A.METHODS:hMSC were separated from rib marrow with Ficoll-Paque reagent and expanded in culture medium. To detect the surface antigens, the labeled cells were analysed on a FACScan flow cytometer to determine the effect of the capacity of proliferation and differentiation of the mesenchymal stem cells with FGF-2. hMSC were induced to differentiate into neurons with DMEM Tanshinone II A. Neuron-specific enolase(NSE), neurofilament(NF), Nestin, glial fibrillary acaidic protein(GFAP) were detected by immunohistochemistry.RESULTS:hMSC were expanded as undifferentiated cells in culture for more than 15 passages. The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. These expanded attached MSC were uniformly positive for CD29, CD44, CD90, CD105, CD166 and didn't express CD11a, CD14, CD34, CD38, CD45, CD80, CD86. FGF-2 have special effect on low denisity MSCs. Simple methods with Tanshinone II A induced hMSC to exhibit a neuronal phenotype, expressing NSE, NF-M, Nestin at 5 hours. But the neuron-like cells didn't express the glial astrocyte marker GFAP.CONCLUSION:hMSC can be induced to differentiate into neurons with Tanshinone II A.     

Protective effect of atrial natriuretic peptide on alveolar type II cells

AIM: To study the protective effect of atrial natriuretic peptide (ANP) on alveolar type II cells (AT-Ⅱ) damaged by lipopolysaccharide (LPS).METHODS: AT-Ⅱ were placed in a 6 well cell culture cluster (0.5×106 cells/cm2) and divided into 3 groups: (1) control group (n=6), the medium consisted of RPMI-1640 without FBS. (2) LPS group (n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS (1 mg/L). (3) ANP group (n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS (1 mg/L) and ANP (10-8, 10-7, 10-6 mol/L). After 4, 12 and 24 h, the cell culture mediums of control group, LPS group and ANP (10-7 mol/L) group were collected, and those of the ANP (10-6, 10-8 mol/L) group were collected after 12 h. Alkaline phosphatase(AKP), lactate dehydrogenase(LDH), malondialdehyde(MDA), total phospholipids (TPL) and surface tension (ST) in the medium of every group were examined. RESULTS: AT-Ⅱ were characterized by AKP staining. The contents of LDH, AKP and MDA in the medium of every ANP group were lower than those in the corresponding LPS group. The TPL content in the medium of every ANP group was higher than that in the corresponding LPS group, and the change of ST of the medium was opposite to that of TPL. The effect at 12 h was the most significant, for example, at 12 h, the activities of AKP in the mediums were: control (43.5±10.4) U/L, LPS (98.1±16.4) U/L, LPS+ANP (10-6) (46.4±10.5) U/L, LPS+ANP(10-7) (60.7±9.5) U/L, LPS+ANP(10-8) (91.3±13.9) U/L.CONCLUSION: ANP protects the AT-Ⅱ from being damaged by LPS and promotes the secretion of pulmonary surfactants.     

Biological characterics of human fetal cord blood mesenchymal stem cells

AIM: To investigate the biological characterics of human second-trimester fetal cord blood mesenchymal stem cells (MSC) and its application prospects in utero gene transfer/therapy (IUGT). METHODS: Nuclear cells separated from cord blood were cultured in DMEM medium. Surface antigens of the MSC were analyzed by the FACScan flow cytometry. Adipogenic and osteogenic mediums were used to assess the differentiation ability of the cells. Adenovirus vector deliver green fluorescent protein gene (Ad-GFP) was used to transfected the MSC and the expressing of GFP was detected by fluorescent microscope. The MSC were injected into the liver of newborn rat. The immunofluorescence analysis was conducted to determine the presence of double-positive CD105⁺/CD166⁺ cells in different organs of rats. MSC were subcutaneous injected into the human-nonobese diabetes/severe combined immunodeficiency disease (NOD/SCID) mice and carcinogenesises of the MSC in vivo were detected by pathological diagnosis. RESULTS: MSC could be separated from fetal cord blood. These cells were uniformly positive for CD29, CD44, CD59, CD105, CD166 and negative for CD34, CD45, CD80, CD86, HLA-DR. The cells had the abilities to differentiate into adipogenic and osteogenic cells in vitro, expressed the GFP at high levels (56.32%±3.28%). The MSC were located at different organs after injected into the newborn rats and didn't have carcinogenicity in vivo. CONCLUSION: Human second-trimester fetal cord blood MSC is an promising target cells in fetal IUGT.     

Effect of bone marrow mesenchymal stem cells on engraftment of hematopoietic stem/progenitor cells in sensitized mice

AIM: To evaluate the effects of bone marrow-derived mesenchymal stem cells (MSCs) on engraftment of hematopoietic stem/progenitor cells in sensitized mice. METHODS: Mouse bone marrow-derived MSCs were cultured by adherent culture method. MSCs combined with or without hematopoietic stem/progenitor cells were implanted into the sensitized mouse model, which was established by allogeneic splenocyte transfusion, and were divided into 6 groups: MSC intervention groups, including sensitized mice with MSCs on day 11, sensitized mice with MSCs on day 0 and sensitized-mice with MSCs both on day 11 and day 0; control groups, including sensitized mice without MSC intervention, non-sensitized mice without MSC intervention and non-sensitized mice without MSCs or transplantation of hematopoietic stem/progenitor cells. The survivors were assessed after transplantation and hematopoietic recovery was monitored weekly including hematological change, immune function reconstruction, bone marrow cell recovery, chimera analysis and graft-versus-host disease development. RESULTS: Compared with different control groups, MSC intervention did not prolong the survival rates of the sensitized model mice after lethal irradiation. CONCLUSION: Under the experimental conditions, MSC combined with C57BL/6 bone marrow hematopoietic stem/progenitor cells fail to promote the growth of engraftment in C57BL/6 allogeneic splenocyte-sensitized BALB/c mice in vivo.     

Relationship between Th1/Th2 balance and CD28/CTLA-4 expression on peripheral blood T cells in patients with systemic lupus erythematosus

AIM: To investigate the pattern of Th1/Th2 balance in systemic lupus erythematosus(SLE) patients and the relationship between CD28/CTLA-4(cytotoxic T-lymphocyte antigen-4) molecule expression and Th1/Th2 balance.METHODS: Eighteen SLE patients met the ARA 1997 updated SLE criteria were selected in the study. According to Bombardier's SLEDAI criteria, all patients were classified into two groups: active group(12 cases) and static group(6 cases). Fourteen normal individuals, matched for age and sex of the patients, served as controls. The peripheral blood mononuclear cells(PBMCs) were isolated by density gradient centrifugation and cultured in RPMI-1640 culture medium. After treated with PMA(5 μg/L) and ionomycin(500 μg/L) for 72 h, the PBMCs were collected, the contents of IFN-γ and IL-10 in the supernatant of cultured PBMCs were detected using enzyme linked immunosorbent assay(ELISA). The expression of CD28 and CTLA-4 molecules on T cells were detected by flow cytometric technique with double staining by FITC or PE labeled monoclonal antibodies. RESULTS: The level of IL-10 was higher in the PBMCs of active and static SLE patients(351.29 ng/L±153.31 ng/L and 319.37 ng/L±153.39 ng/L) than that in controls(254.48 ng/L±120.69 ng/L), but the difference did not reach statistical significance(P>0.05). The level of IFN-γ was significantly lower in the PBMCs of active SLE patients(25.76 ng/L±16.09 ng/L) than that in controls(50.71 ng/L±27.92 ng/L, P＜0.05). The ratio of IL-10/IFN-γ was significantly higher in active SLE patients(18.74±13.77) than that in controls(6.66±4.95, P<0.05). Either before or after culture, the expression of CD28 molecule on CD3⁺and CD8⁺ T cells from all SLE patients was not remarkably different from that in the cells of controls. Before culture, the expression of CTLA-4 molecule on CD3⁺T cells of active SLE patients(0.79%+0.37%) was significantly lower than that in the cells of controls(1.31%+0.61%, P<0.05). After culture, the expression of CTLA-4 molecule on CD3⁺ T cells of SLE patients was still lower than that in the cells of normal controls without statistical significance(P＞0.05).The expression level of CD28 molecule on CD3⁺ or CD8⁺ T cells in active SLE patients and controls was not correlated with the levels of IFN-γ and IL-10 in the supernatants(P＞0.05). The level of CTLA-4 molecule expression on CD3⁺ T cells of active SLE patients was positively correlated with IFN-γ level(r=0.681, P<0.05), while was negatively correlated with IL-10 levels(r=-0.624,P＜0.05) and the ratio of IL-10/IFN-γ(r=-0.738, P<0.01). The level of CTLA-4 molecule expression on CD3⁺ or CD8⁺ T cells of controls showed no correlation with IFN-γ levels, while showed negative correlations with IL-10 level(r=-0.587, P<0.05; r=-0.563, P<0.05, respectively).CONCLUSION: There is a bias in the differentiation of Th0 cells towards Th2 in SLE patients. CTLA-4 probably plays an important role in this mechanism through suppressing the signal transmitted by CD28.     

Effect of lung cancer cells transfected with interleukin 18 gene on the immunological activity of dendritic cells

AIM: To study the effect of interleukin 18 gene transfected lung cancer cells on the phenotype and immunological activity of dendritic cells (DC). METHODS: A secretive IL-18 expression vector containing IL-12 P40 signal sequence was constructed and transfected into NCI-H460 lung cancer cells. DC induced from human peripheral blood were divided into 4 groups (NT, PV, GT and PD). DC were stimulated by non-transfected NCI-H460 cells, pure vector transfected NCI-H460 cells and IL-18 transfected NCI-H460 cells respectively for group NT, PV, GT, and non-stimulated DC for group PD. CD54, CD80, CD83 and CD86 on DC in the 4 groups were detected with flow cytometry. T cell proliferation stimulated by DC in the 4 groups was assayed with MTT method. IL-12 release in cultured DC supernatant was measured by ELISA. RESULTS: Sequencing result of the secretive IL-18 was correct. The transfected cells expressed IL-18 fusion gene and 18 kD IL-18 protein. DC in GT group expressed more surface molecules than those in other 3 groups. T cell proliferation and IL-12 secretion in GT group were higher than those in other 3 groups. CONCLUSION: IL-18 gene transfected NCI-H460 cell increases surface molecule expressions on DC. It also enhances immunological activity and IL-12 secretion in DC.     

Isolation of endothelial progenitor cells from cord blood with CD133 immunomagnetic sorting

AIM: To isolate, purify and differentiate endothelial progenitor cells from cord blood in vitro and to study their biological characteristics. METHODS: CD133+ cells were selected from fresh cord blood mononuclear cells (MNC) by magnetic activated cell-sorting system (MACS). EPC was studied by flow cytometry, immunocytochemistry and immunofluorescence staining. Isolated cells were cultured in IMDM medium supplemented with or without VEGF, bFGF, SCF. RESULTS: The percentage of CD133+ cells of cord blood MNC was (1.41±1.14)%, and purity was 75%-85% (FACS method). CD133+ cells were grown on fibronectin-coated chamber slides in the presence of VEGF, bFGF, SCF. Within 1-2 hours of culture cells became adherent. On day 7-10, the adherent cells displayed a typical “cobblestone” morphology. After 14 days of culture, the adherent cells revealed a heterogeneous cell population, comprising small-sized round cells, spindle-like cells and formed tube-like structure. Weibel-Palade bodies were shown on the transmission electron microscopy photomicrographs. Compared with the original, cell markers CD133 and CD34 decreased significantly (77.0%±3.3% to 1.6%±2.2% and 93.1%±4.7% to 37.4%±4.9%, P<0.05), while Flk-1 increased significantly (from 22.3%±3.3% to 94.3%±4.1%, P<0.05) after 14 days of culture with VEGF, bFGF, SCF. The vWF was strongly expressed (77.9%±3.3%) on the 14th day later. CONCLUSION: Vascular endothelial progenitor cells were isolated from cord blood with specific expression of CD133/CD34/Flk-1. With the stimulation of the growth factors, seven-ten days after culture EPCs could be turned to endothelial cells.     

Effect of GYY4137 on cytosolic lipid decomposition in mouse primary hepatocytes

AIM: To evaluate the effect of GYY4137, a novel hydrogen sulfide (H₂S) donor, on cytosolic lipid decomposition in mouse primary steatosis hepatocytes. METHODS: Oleic acid (OA) was used to induce hepatic steatosis model in vitro. The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion were divided into 4 groups:the cells in control group were incubated with normal medium for 54 h; the cells in model group were incubated with OA at 1.2 mmol/L for 48 h followed by serum-free phenol red-free RPMI-1640 for 6 h; the cells in H₂S group or DL-propar-gylglycine (PAG; an inhibitor of cystathione γ-lysase, inhibiting H₂S synthesis) group were incubated with OA at 1.2 mmol/L for 48 h followed by serum-free phenol red-free RPMI-1640 which contained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h. The glycerin release and the protein expression of hormone-sensitive lipase (HSL) in the cells were mea-sured. RESULTS: Compared with model group, the glycerin release and the protein expression of phosphorylated HSL (p-HSL) in H₂S group decreased significantly, while those increased significantly in PAG group. CONCLUSION: In steatosis hepatocytes, exogenous H₂S possibly decreases cytosolic lipid decomposition by decreasing the protein level of p-HSL.     

Effect of ex vivo short time expansion with SCF and IL-6 on the adhesion and transmigration activities of hematopoietic stem/progenitor cells

AIM:To investigate the expression and function of homing related molecules and transmigration ability of human cord blood CD34+ hematopoietic stem/progenitor cells after short time stimulation with cytokine SCF and IL-6.METHODS:CD34+ cells were separated by Ficoll density gradient centrifugation and stimulated by SCF and IL-6 cytokines for 48 h. The changes of CD49d (VLA-4), CD11a (LFA-1), CD62L (L-selectin) and CD184 (CXCR4) were analyzed by flow cytometry. The adherent and migration activities of CD34+ cells were evaluated in human fibronectin (FN) coated microplates (96 wells) and transwell system.RESULTS:The numbers of CD34+ cell expanded to 3 folds and the percentages of CD34+ cells that were positive expressions for CD49d, CD11a, CD62L or CD184 increased 1 to 2 folds after the cytokine stimulation. The spontaneous adhesion between CD34+, FN and SDF-1 induced migration increased after SCF+IL-6 stimulated.CONCLUSION:SCF+IL-6 can improve the most of the homing related characteristics and activities in the short time expansion of CD34+ hematopoietic stem/progenitor cells, which may be partly related to the increased intrinsic homing potential.     

Effect of exogenous hydrogen sulfide on lipophagy in mouse primary hepatocytes

AIM: To evaluate the effect of exogenous hydrogen sulfide (H₂S) from GYY4137 on lipophagy in mouse primary hepatocytes. METHODS: The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion method were divided into 4 groups: the cells in control group were incubated with normal medium; the cells in model group were incubated with 1.2 mmol/L oleic acid (OA) for 48 h; the cells in H₂S group or propargylglycine (PAG) group were incubated with 1.2 mmol/L OA for 48 h followed by serum-free phenol red-free RPMI-1640 medium which contained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h. The cells were collected to conduct immunofluorescence staining of LC3 and photography under fluorescence microscope, phase-contrast microscope or transmission electron microscope. The protein expression of LC3-Ⅰ/Ⅱ in the hepatocytes was determined by Western blot. RESULTS: In contrast with the model group, the fluorescent particles of LC3, the protein expression of LC3, the number of autophagic lysosome and vacuoles in hepatocytes in H₂S group increased. CONCLUSION: In steatosis hepatocytes, exogenous H₂S promotes the lipophagy.     

Adult human mesenchymal stem cell differentiates into adipocytes

AIM:To investigate the differentiation from human mesenchymal stem cells (hMSC) to adipocytes.METHODS: hMSC were separated from rib marrow and expanded in culture medium. To detect the surface antigens, the labeled cells were analysed on a FACScan flow cytometer. hMSC were induced with dexamethasone, insulin, 1-methy1-3-isobutylxanthine and indomethacin which acted as adipocyte differentiation inducer. The cells were stained with Oil Red O. The number of adipocytes were counted on a phase-contrast microscope.RESULTS: hMSC were expanded as undifferentiated cells in culture for more than 5 passages. The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. These expanded, attached MSC were uniformly positive for CD29, CD44, CD90, CD105, CD166 and didn't express CD14, CD34, CD45, CD11a. After induced with induction medium, lipid vacuoles were first detectable within the cells at 48 hours. Two weeks later, more than 85% MSC differentiated into adipocytes which displayed a perinuclear accumlation of lipid vacuoles, as detected by Oil Red O. CONCLUSION:hMSC can be induced to differentiate into adipocytes.     

Effects of uremic serum on the proliferation and trans-differentiation of human renal tubular epithelial cells

AIM: To explore the effects of uremic serum on proliferation and trans-differentiation of human renal tubular epithelial cells. METHODS: Human renal tubular epithelial cell line (HK-2) was cultured in RPMI-1640 medium. The proliferation effects of uremic serum at different concentrations were evaluated by methylene blue assay (MTT method) and flow cytometry. The positive cells percentage of α-smooth muscle actin(α-SMA)in different concentration uremic serum medium was also measured by flow cytometry in vitro. RESULTS: Absorbance 490 (A₄₉₀) was increased in 5%-20% uremic serum groups compared with that in normal controls with the use of MTT. Cells in G₁ phase were decreased, but proliferation index (PI) was increased in 10%-20% uremic serum groups compared with that in normal controls with the use of flow cytometry. No significant difference of cell proliferation index was found among uremic serum groups. The positive percentage of α-SMA cells was increased significantly in uremic serum groups compared with that in normal controls, and increased in parallel with the increasing of uremic serum concentration. CONCLUSION:These data suggest that AGEs could induce a high expression of MIP-1αm RNA and protein in cultured HUVECs in a dose-dependent and time-dependent manner.     

Transfusion of mesenchymal stem cells improve the hematopoiesis in mice with aplastic anemia by regulating immune cells

AIM: To study the possible immunologic mechanism of mesenchymal stem cells (MSCs) by which the hematopoiesis of mice with aplastic anemia (AA) is improved. METHODS: The mice model of immuno-mediated aplastic anemia was established. Thirty BALB/c mice were divided into 3 groups: radiation group, AA model group and MSCs group. The mice in radiation group only had processing of 5 Gy ［60Co］γ radiation. The mice in AA model group were intravenously injected with the cell suspension of thymocyte and lymph-node cell mixture (prepared from DBA/2 mice, 1×106 cells) after 5 Gy ［60Co］γ radiation. The animals in MSCs group received the same treatment as the mice in AA model group did, and afterwards was injected with 1×106 MSCs intravenously at 3 days. All the mice in the 3 groups were observed and analyzed the following parameters: peripheral blood cells, pathological features of bone marrow, the relation between the changes of peripheral CD4+, CD8+, CD4+/CD8+, NK cells and the hematopoiesis. RESULTS: After 5 Gy ［60Co］γ radiation, the peripheral blood cells in all groups decreased at 7 days, while at 21 days those in MSCs group and radiation group restored to normal but those in AA model group was still at lower level. At 7 days, the cells of CD4+, CD8+ and CD4+/CD8+ in each group were similar without statistical difference, but the number of NK cells in MSCs group was quite lower than that in the other 2 groups (P<0.05). At 14 days, the CD4+ cells in all groups were obviously decreased, while the CD8+ cells in MSCs group and AA model group were significantly higher than those in radiation group. At 21 days, the CD8+ and NK cells in MSCs group were quite the same as those in radiation group while those in AA model group were obviously higher compared to the other 2 groups. The number of adipose cells observed in the pathological slice of femoral bone in MSCs group and radiation group was no difference while that in AA model group was much higher. CONCLUSION: MSCs transfusion improves the hematopoiesis in mice with aplastic anemia by regulating the functions of immune cells.     

Ovarian carcinoma cells inhibit ζ chain expression and the secretion of Tc1/Tc2 type cytokines in CD8+ T cells

AIM: To investigate the effect of ovarian carcinoma cells on ζ chain expression and the secretion of Tc1/Tc2 type cytokine in CD8+ T cells, and its role in the ovarian carcinoma induced immunosuppression.METHODS: The supernatants of human ovarian carcinoma cell lines of OVCAR3, CAOV3 and SKOV3 and RPMI-1640 were added into CD8+ T cells (groups I, II, III, and control), which were isolated from the peripheral venous blood of healthy persons. The expression of ζ chain was analyzed by Western blotting. Thiazolyl blue(MTT) method was used to detect the effects of those cell line supernatants on the growth of CD8+ T cells. The secretion of the Tc1 type cytokine interferon (IFN)-γ mRNA and the Tc2 type cytokine interferon (IL)-10 mRNA were detected by RT-PCR. RESULTS: The expression of ζ chain was significantly lower in groups I, II, and III in comparison with that in control group. The absorbance at the wavelength 570 nm of CD8+ T cells culture in the group I, II, and III was all significantly lower than that in the control group. The IFN-γ expression was significantly lower in groups I, II, and III in comparison with that in control group, while the expression of IL-10 was significantly higher. CONCLUSION: Ovarian carcinoma may suppress CD8+ T cell proliferation and secretion of the Tc1/Tc2 type cytokine through inhibition of ζ chain, which may play an important role in the ovarian carcinoma induced immunosuppression.     

Isolation and characterization of rabbit muscle-derived stem cells

AIM:To explore the feasibility of isolation and characterization of the muscle-derived stem cells (MDSCs) of rabbits.METHODS:Skeletal muscle specimens were harvested from rabbits of 1.5 months.Preplate technique was applied to isolate MDSCs,and flow cytometry (FCM),immunocytochemistry and Western blotting were performed to characterize MDSCs.RESULTS:The isolating process contained 6 consecutive days of differentiated attachment.From day 3,many round-shaped or short spindly-shaped cells began to appear,which were markedly smaller than the skeletal muscle cells and grew to confluence after about 10 d with obvious tendency to fuse.When passaged below confluence of 30%,these cells maintained their original shape well,and were shown >80% desmin+,>70% Bcl-2+,CD45- by FCM.Desmin+ by immunocytochemistry and decreasing expression of α-SMA by WB were also observed.When passaged at high confluence (>50%) or cultured with low concentration of serum,these cells tended to fuse to form myotubes,and were skeletal myosin+.CONCLUSION:Preplate technique can effectively isolate MDSCs,this provides tissue engineering with a new type of seed cells.     

The culture and differentiation of adult bone marrow-derived pluripotential mesenchymal stem cells into insulin-producing cells

AIM: To study the culture and characteristics of mouse adult bone marrow-derived pluripotential mesenchymal stem cells and its potential to differentiate into insulin secretion cells. METHODS: Cells were plated on 60% DMEM-LG and 40% MCDB-201 medium supplemented with 2% fetal calf serum and 10 μg/L PDGF-BB, 10 μg/L EGF and 1×106 U/L LIF. The proliferation rate, phenotype and oct-4 mRNA were tested. After it was plated on serum-free medium DMEM/F12 with GLP-1 and nicotinamide, the nkx2.2 ngn3, pdx-1 and insulin 2 mRNA were tested. RESULTS: The cells were round with large nucleus and scant cytoplasma. They were CD13+, CD44-, CD45- and MHCⅡ-. Oct-4 mRNA were present. The nkx2.2 pdx-1 and insulin 2 mRNA were presented in cells plated on the inducing medium at 14 days. CONCLUSION: The adult bone marrow-derived pluripotent stem cells were cultured and they has the possibilities to be induced into insulin-secreting cells.