Biological characteristics of JAK2 transduced CD34+ cells from cord blood during ex vivo expansion

AIM: To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34⁺ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer,dimerization (AP20187),was cloned (designated MGI-F₂JAK2).CD34⁺cells were enriched from cord blood with a MiniMACS system.The purified CD34⁺ cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2.Following transduction,cells were expanded into four groups: AP20187 alone,FL alone,TPO,alone,AP20187+FL+TPO,respectively.The expanded cells were monitored by GFP expression,immunophenotyping,progenitor colony assay,karyotype analysis as well as tumorigenesis in nude mice.RESULTS: The purity of selected CD34⁺ cells was over 91% and gene transfer rate was 49.32%±6.21%.Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34⁺ cord blood cells.The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture.Flow cytometry analysis showed that the phenotype of the expanded cells was CD33⁺,CD61⁺ and Gly-A⁺ partial positive;CD38⁺ and HLA-DR⁺ strong positive,while CD2,CD7 and CD19 were almost negative.Colony assays performed in methycelluos,which can give rise to BFU-E,CFU-GM and CFU-Mix,the CFU-GM was predominantly in all colonies.The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34⁺ cells in combination with FL and TPO.This system may have applications for studies in signaling transduction,hematopoiesis,and for gene and cell therapy.     

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.     

Role of NK cells in mouse allogeneic bone marrow transplantation

AIM: To study the effect of natural killer(NK)-cells on graft-versus-host disease(GVHD), graft rejection, engraftment and reconstituting of hematopoiesis in mouse allogeneic bone marrow transplantation. METHODS: Lethally irradiated BALB/c(H-2^d) mice were transplanted with C57/6j(H-2^b) bone marrow containing donor peripheral T cells and/or NK cells. Recipients CD₃₄⁺ cell counts and the expression of H-2K^b＋ cell were detected by flow cytometry, peripheral white blood cell(WBC) was detected by auto-cytometry, and the recipients survival rates, GVHD, engraftment and hematopoiesis recovery were then observed. RESULTS: In the group of transplanted with NK cells infusion, the incidence of GVHD was evidently lessened, the survival rates, WBC and CD₃₄⁺ cell counts and the expression of H-2K^b＋ cell were significantly high than that without NK cells infusion. CONCLUSION: In mouse allogeneic bone marrow transplantation, alloreactivity NK cells prevents GVHD, reduces graft rejection, and promotes engraftment and reconstituting of hematopoiesis.     

Mesenchymal stem cells contribute to the formation of liver fibrosis

AIM: Myofibroblasts play key roles in the formation of liver fibrosis. It has been reported that bone marrow mesenchymal stem cells can differentiate into myofibroblasts, and migrate to the damaged organs to participate the fibrotic process. Therefore, this study was designed to investigate the possible function and mechanism of bone marrow mesenchymal stem cells in developing liver fibrosis. METHODS: A mesenchymal stem cell line Ap8c3 was tagged with enhanced green fluorescent protein(eGFP)(Ap8c3-eGFP). The rat model of liver fibrosis was established by bile duct ligation(BDL) or injection of pig serum(IPS). Ap8c3-eGFP was intravenously injected into BDL or IPS-induced liver fibrotic rats. The eGFP positive(eGFP⁺) cells and expression of α-smooth muscle actin(α-SMA) in these eGFP⁺ cells in rat livers and bone marrow(BM) were detected. RESULTS: Intravenous engraftment of Ap8c3- eGFP resulted in homing to fibrotic livers as well as BM. Co-expression of α-SMA by eGFP⁺ cells was found in liver sections, and eGFP⁺ cells were found mainly in fibrotic septum in fibrotic livers. Ap8c3-eGFP was observed to differentiate into myofibroblasts, which specifically expressed α-SMA after homing to bone marrow.CONCLUSION: Differentiation of mesenchymal stem cells into myofibroblasts plays important roles in the formation of liver fibrosis. BM-derived myofibroblasts can migrate to damaged livers and participate in the formation of liver fibrosis.     

Effects of intravenous immunoglobulin on IL-4 levels and CD20+ cells in peripheral blood and recurrent rate of infants with wheezing

AIM: To investigate the effects of intravenous immunoglobulin on IL-4 levels and CD20⁺ cells in peripheral blood and the recurrence rate of infants with wheezing. METHODS: IL-4 levels and CD20⁺ cells in peripheral blood of 30 normal infants and 66 infants with wheezing were tested by flow cytometry and ELISA, respectively. The relief time of wheezing and recurrent rate were also recorded. RESULTS: The IL-4 levels and CD20⁺ cells in the wheezing infants were higher than those in controls(P<0.01). The IL-4 levels and CD20⁺ cells in the wheezing infants were decreased after routine treatment but were still higher than those in control infants after treatment. The IL-4 levels and CD20⁺ cells in the wheezing infants were decreased after immunoglobulin treatment and were almost the same as controls after treatment. The relief time of wheezing in the infants with immunoglobulin treatment was shorter than that in the infants with routine treatment(P<0.01), and recurrent rate of immunoglobulin treatment was lower than that of routine treatment(P<0.05). CONCLUSION: The IL-4 levels and CD20⁺ cells in peripheral blood are increased more significantly in infants with wheezing than those in control infants. The mechanisms of wheezing relief and decreasing the recurrent rate by intravenous immunoglobulin are associated with the down-regulation of IL-4 levels and CD20⁺ cells.     

Role of Kv1.3 channel of CD4+T lymphocytes in the formation of atherosclerosis in rat spleen

AIM: To investigate the function of voltage-gated potassium channel Kv1.3 and its possible role in CD4⁺ T lymphocytes in the formation of atherosclerosis (AS) in rat spleen. METHODS: The rat atherosclerosis model was established by feeding high-fat diet. The proportion of lymphocytes was determined by flow cytometry. The CD4⁺ T lymphocytes were separated using immunomagnetic bead. The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was detected. The concentrations of intracellular calcium and cytokines were also measured. RESULTS: (1) The proportion of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (74.93%±2.15% vs 67.80%±2.54%, P<0.05). (2) After stimulated with concanavalin A (ConA), the proliferation of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (1.1321±0.1750 vs 0.7971±0.0955, P<0.05). (3) After stimulated with ConA, the concentration of intracellular calcium in AS group was higher than that in control group. (4) In AS group, the releases of cytokines of IL-2 and TNF-α in AS group were significantly higher when stimulated with ConA for 48 h than that for 24 h. (5) The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was greatly higher in AS group than that in control group (3.670±1.579 vs 1). CONCLUSION: In AS rats, the increase in CD4⁺ T lymphocytes as well as the augmentation of Kv1.3 mRNA expression in the cells suggest that up-regulation of Kv1.3 mRNA expression in CD4⁺ T lymphocytes may be involved in the mechanism of atherosclerotic formation in rat spleen.     

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.     

Suppression of tumor growth by mRNA DC vaccine in severe combined immune deficiency mouse

AIM: To establish HCC Hu-PBL-SCID(severe combined immune deficiency) chimeric model,and to observe the antitumor effect of mRNA-dendritic cell vaccine.METHODS: Hu-PBL-SCID chimeric model was established by intraperitoneal injection of human peripheral blood lymphocytes.Human IgG in mouse serum was detected by ELISA to identify the model.After the model was established,the mice were divided into four groups,and were inoculated with mRNA DC vaccine,anti CD4+,CD8++mRNA DC,naive DC,and PBS,respectively.Then the animals were injected subcutaneously with 2×106 HepG-2 cells.Tumorigenecity,latent period,and tumor volume were observed,and antitumor efficacy of CTL was measured.RESULTS: The concentration of human IgG in mouse serum in Hu-PBL- SCID model was detected,indicating that the Hu-PBL-SCID model was established successfully.No significant difference of tumorigenecity among the four groups was observed.However,tumors in mRNA DC vaccine group grew slowly,tumor volumes were significantly smaller than those in anti CD⁴⁺,CD⁸⁺+mRNA DC,PBS and naive DC groups.The spleen cells in mRNA DC vaccine group specifically killed the HepG-2 cells but not SGC-7901 cells.CONCLUSION: mRNA DC vaccine shows anti-tumor immune response in vivo by inducing CD⁴⁺,CD⁸⁺T lymphocyte immune responses.     

Correlation between CD4+CD28-T cells and lymphocytic apoptosis in rheumatoid arthritis patients

AIM: To investigate the fraction of CD4⁺CD28^-T cells and its correlation with lymphocytic apoptosis in peripheral blood of rheumatoid arthritis (RA) patients.METHODS: The RA patients and age-matched health controls were selected in the study. The lymphocytes were isolated from peripheral blood. CD4⁺ T cells without CD28 expression (CD4⁺ CD28^-) were analyzed by flow cytometry. The incidence of apoptosis in the cells cultured with or without PHA for 24 h was determined by flow cytometry with Annexin V-FITC/PI di-staining. The correlation between the fraction of CD4⁺CD28^-T cells and lymphocytic apoptosis was also observed.RESULTS: The fraction of CD4⁺CD28^-T cells was significantly higher in RA group than that in healthy control group (7.79%±3.52% vs 1.89%±1.78%, P<0.05). The apoptotic level in PHA cultured lymphocytes was significantly lower in RA group than that in healthy controls (11.38%±5.73% vs 19.46%±6.32％, P<0.05). Spearman analysis showed that there was a negative correlation between the fraction of CD4⁺CD28^-T cells and apoptotic level of activated lymphocytes (r=-0.433,P<0.01).CONCLUSION: The increased CD4⁺CD28^-T cells contribute to prolong the lifespan of activated lymphocytes in peripheral blood of RA patients, and the persistence of activated lymphocytes may contribute to the pathogenesis of RA.     

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.     

Supportive effects of human AGM region and fetal liver stromal cells on differentiation of murine embryonic stem cells into hematopoietic stem cells and in vivo hematopoietic reconstitution

AIM: To investigate the differentiation of murine embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) by the supportive effects of human aorta-gonad-mesonephros (AGM) region and fetal liver (FL) stromal cells.METHODS: E14 ESCs were induced into embryoid body (EB) first. Then the cells from EB were further co-cultured with human AGM region and FL stromal cells in non-contact system. On day 6, the cells derived from EB were collected for Sca-1⁺c-Kit⁺ cell analysis by flow cytometry, colony forming unit (CFU) assay and teratoma formation checking. BALB/c female mice conditioned with lethal dose of γ-ray irradiation were transplanted with EB cells from different culture systems. The survival rates, engraftment of donor cells, reconstitution of hematopoietic were monitored.RESULTS: Sca-1⁺c-Kit⁺ cells in EB cells co-cultured with human AGM region and FL stromal cells had the value of (21.96±2.54)%, and the total CFU was as (520±52)/10⁵ cells, which were statistically greater than those in EB cells only cultured with human AGM region stromal cells (P＜0.05). No teratoma was found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region and FL stromal cells. In BALB/c female mice transplanted of EB cells co-cultured with human AGM region and FL stromal cells, the survival rate was 77.8%, and the peripheral blood cell count was obviously improved on day 14. PCR results showed the recipients all had sry gene copies from donor in bone marrow. The recipient mice transplanted with EB cells only cultured with human AGM region stromal cells all died within 15 days.CONCLUSION: Stromal cells from human AGM region and FL enhance the directed differentiation of ESCs into HSCs which can reconstruct hematopoiesis in vivo.     

The influence of anesthesia and means of postoperative pain control on T lymphocyte subtypes of blood in patients with rectal cancer

AIM: To evaluate the influence of anesthesia and different means of postoperative pain control on the T-lymphocyte during the perioperative period in patients with rectal cancer.METHODS: 40 adult patients, aged 65 or older, of American Society of Anesthesiologists (ASA) class 2-3 were divided into two groups according to the type and means of postoperative pain managements. Group Ⅰ (n=20) received intravenous anesthesia and patient controlled analgesia(PCA), fentanyl (13 μg/kg) for post pain; group Ⅱ (n=20) received intravenous anesthesia plus lumber epidural anesthesia and epidural PCA of morphine 5 mg plus ropivacaine 100 mg for post operative pain. Blood samples from internal jugular vein were obtained before surgery, at the completion of surgery and 24, 48, and 120 h post surgery for detecting CD3⁺, CD4⁺, CD4/CD8 counts of peripheral T-lymphocytes. In addition, blood cortisol level and pain intensity were assessed by visual analogue score (VAS)at each time point. RESULTS: Baseline(before anesthesia) values of CD3⁺,CD4⁺, CD4/CD8 in patients were messured and there was a significant decrease of all these values from completion of surgery to 48 h after surgery in both groups (P<0.01). However, group Ⅱ showed a higher CD4⁺ at 48 h, higher CD3⁺,CD4⁺, CD4/CD8 at 120 h post surgery than group Ⅰ (P<0.05). Patients in both groups obtained good pain relief post surgery,but VAS in group Ⅱ were significantly lower than those in group Ⅰ at 24 and 48 h post surgery (P<0.01). Compared with baseline, blood cortisol levels in both groups increased markedly at completion of surgery, and at 24, 48 h after surgery (P<0.01),while the increased cortisol level in group Ⅱ at completion of surgery and 24 h after surgery was less than that in group Ⅰ (P<0.05).CONCLUSION: Combined intravenous anesthesia with lumber epidural anesthesia appears to reduce the perioperative stress response and exerts less negative effects on the T-lymphocytes, suggesting that such a means of anesthesia might be more suitable to the elderly patients with rectal cancer.     

Deficiency in Na-K-2Cl co-transporter impaired hearing and balance in mice

AIM: We generated transgenic mice of NKCC1^-/- (homozygous mutant),NKCC1^+/- (heterozygous) and NKCC1^+/+ (wild-type) that have a targeted disruption in the NKCC1 gene to investigate the role of Na-K-2Cl (NKCC1) channel in auditory function of the inner ear.METHODS: Hearing threshold and endocochlear potential (EP) were measured in the NKCC1^-/-,NKCC1^+/- and NKCC1^+/+ mice by auditory brainstem response (ABR) and EP recordings,respectively.The inner ears of the mice were removed and examined morphologically with the light microscope.RESULTS: The auditory function of NKCC1^+/+ mice was normal,the mean value for ABR thresholds in response to click sound was ［（23.13±3.78）dB,SPL］,EP was （98±16）mV.The mean value for ABR thresholds to click sound was elevated in NKCC1^+/- mice ［（38.49±12.29） dB,SPL］,relative to that significantly increased in NKCC1^+/+ mice (P<0.01).EP in NKCC1^+/- mice was about （78±7） mV,significantly decreased than that in NKCC1^+/+ mice (P<0.05).NKCC1^-/- mice were completely deaf,the ABR waveform was not observed for even 100 dB SPL sound stimuli used and EP was nearly disappeared (EP,4 mV±6 mV).NKCC1^-/- mice were deaf and demonstrated difficulties in maintaining their balance.NKCC1^-/- mice exhibited a marked atrophy of the stria vascularis,contraction of the endolymphatic compartments and collapse.CONCLUSION: NKCC1 channel plays a critical role in potassium homeostasis of endolymph in the inner ear.Mice lacking of NKCC1 can lead to changing K⁺ concentration in endolymph and influence auditory function subsequently.NKCC1 knockout mice exhibit marked structural abnormalities of the cochlea as well.     

Harmful factors affect glycine receptor α1 subunit mRNA expression in the neonatal rat cardiomyocytes

AIM: To investigate the effects of lipopolysaccharide, hypoxia/reoxygenation,isoproterenol and high concentration of glucose on glycine receptor α₁ subunit mRNA expression in the neonatal rat cardiomyocytes. METHODS: Isolation of cardiomyocytes from Sprague-Dawley rats aging 1～3 d were performed. Cardiomyocytes （1×10⁵～5×10⁵ cells·L^-1）were cultured in DMEM medium containing 15% fetal bovine serum at 37 ℃ in 5%CO₂ atmosphere for 72 h. Then, cultured rat cardiomyocytes were treated with lipopolysaccharide, isoproterenol or high concentration of glucose for 24 h, respectively, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell survival rate was measured using CCK-8 reactant and RT-PCR was applied to monitor the expression of glycine receptor α₁ subunit mRNA. RESULTS: Compared with the control group, no significant difference in the cell survival rate was observed （P＞0.05）. The expression of glycine receptor α₁ subunit mRNA was increased （P＜0.01） in lipopolysaccharide（5,10,20,40,80 mg/L）,isoproterenol（20,100,500 μmol/L） or hypoxia/reoxygenation, hypoxia groups, but decreased（P＜0.01）in the group treated with high concentration of glucose（25, 50 mmol/L）. CONCLUSION: Lipopolysaccharide, isoproterenol, hypoxia/reoxygenation or hypoxia upregulates the expression of glycine receptor α₁ subunit mRNA,but high concentration of glucose down-regulates the expression of glycine receptor α₁ subunit mRNA in cultured neonatal rat cardiomyocytes.     

The ex vivo expansion characteristic of endothelial progenitor cells

AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34⁺ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34⁺ and CD34^- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34⁺ and CD34^-,total MNC led to a significant increase in the expansion of CD34⁺ cells compared with CD34 enrichment (P＜0.05). There was a trend toward decreased apoptosis in cultures when early passage was performed once the linear cord like structures appeared. There was no significant effect on apoptosis between with VEGF and without VEGF group (P＞0.05). These differentiated EPCs were stained positive for CD34⁺, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34⁺ and AC133⁺cells accounted for 68.2%±6.3% (n=6) and 57.2%±9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34⁺ and CD34^- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.     

Effects of dexamethasone on intracellular expression of TH1/TH2 cytokines in experimental asthma mice

AIM: To study the effects of dexamethasone (DXM) on intracellular expression of T_H1/T_H2 cytokines and the mechanism of that during the development of asthma. METHODS: Eighteen BALB/c mice were divided into 3 groups at random: control group, asthma group, and DXM treated group, with 6 mice in each. The expressions of T-box expressed in T cells (T-bet) and GATA-binding protein-3 (GATA-3) in lung tissue were detected by Western blotting. The expressions of intracellular cytokines interleukin-4 and interferon-γ in CD4⁺ T cell were measured by flowcytometry.RESULTS: The results of flow cytometry indicated that the ratio of intracellular cytokines IL-4/IFN-γ in CD4⁺ T cells in asthma group was much higher than that in control group (P<0.01), the ratio of intracellular IL-4/IFN-γ in T cells in DXM group was lower than that in asthma group significantly (P<0.01). The expression of T-bet in lung tissue in asthma group was lower than that in control group significantly (P<0.01), while GATA-3 was higher than that in control group significantly (P<0.01). The expressions of T-bet and GATA-3 in DXM group were much lower than those in asthma group (P<0.01), but the decreased degree of GATA-3 was more than that of T-bet. CONCLUSION: With pathological process of asthma, to reverse the ratio of IL-4/IFN-γ in CD4⁺ T cell by regulating T-bet and GATA-3 expression can improve the inflammatory reaction and may be one of the mechanisms of DXM in treating asthma.     

Effects of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells

AIM: To observe the expression of hypoxia-inducible factor-1alpha (HIF-1α) and stromal cell-derived factor 1 (SDF-1) in pulmonary artery endothelial cells (PAECs) of SD rats and to investigate the role of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells to PAECs. METHODS: Immunomagnetic beads were used to separate and purify the CD34⁺/CXCR4⁺ progenitor cells derived from the peripheral circulation of SD rats. The expression of HIF-1α and SDF-1 in PAECs exposed to hypoxia (1% O₂, 5% CO₂ and 94% N₂) was detected by immunofluorescence, Western blotting and ELISA. The migration index and adhesion rate were measured in the progenitor cells, which were subjected to the following different treatments: (1) normoxia (21% O₂); (2) hypoxia 12 h; (3) hypoxia 12 h +HIF-1α inhibitor (2ME2); (4) hypoxia 12 h+SDF-1 neutralizing antibody; (5) hypoxia 12 h+2ME2+SDF-1 neutralizing antibody.RESULTS: The expression of HIF-1α and SDF-1 in PAECs was effectively induced by the hypoxic exposure, and both of them reached the peak levels after 12 h of hypoxic treatment (P<0.01), while administration of 2ME2 decreased the hypoxia-induced SDF-1 expression (P<0.05). Treatment of the PAECs with 2ME2 or SDF-1 neutralizing antibody attenuated the migration index and adhesion rate of progenitor cells to the PAECs (P<0.05). CONCLUSION: There is a HIF-1α/SDF-1 signaling axis in hypoxia-exposed PAECs, which may play a crucial role in the migration and adhesion of progenitor cells to PAECs.     

Tissue irradiation injury and past-transplantation distributing diversity of Sca-1 positive cells from murine fetal liver

AIM: To study the relationship between tissue irradiation injury and past-transplantation distributing diversity of Sca-1 positive cells from murine fetal liver and their offspring cells in multi-organs after syngeneic sex mismatch hematopoietic remodeling.METHODS: The Sca-1 positive cells from the livers of C57BL/ 6j male mouse fetus aged 14.5 day were identified and separated by quick PCR and magnetic cell sorting (MACS) techniques. In order to reconstruct hematopoiesis of the adult female mice, which were lethally irradiated with 8G of ［Co⁶⁰］, the 2×10⁴ of Sca-1+ cells were transplanted through caudal vein into each of them. After 6 months, these recipient mice were sacrificed, and their kidneys, livers, lungs, stomachs, and small intestines were taken out, fixed and slices were made. Fluorescence in situ hybridization was performed and observed by fluorescent microscope. Images were captured and analyzed through appropriative cool CCD and software. RESULTS: After 2×10⁴ of Sca-1+ cells were transfused, the hematopoietic function in the lethally irradiative female mice was completely restored. The cells containing Y chromosome were observed 6 months latter in multi-organs, including kidney, liver, lung, stomach, and small intestine. The frequency of the cells containing Y chromosome respectively was kidney (1.65±0.18)%, liver (0.90±0.10)%, lung (1.90±0.60)%, stomach (6.10±0.50)%, and small intestine (7.61±2.30)%, presented the trend of small intestine>stomach>lung>kidneys>liver. Combined informational analysis showed that the presenting frequency of the cells containing Y chromosome was consistent with the irradiative sensitivity of the organ. CONCLUSION: These findings suggest that the capability of differentiation of Sca-1 positive cells from murine fetal liver was potentially connected to the extent of damage in these organs when transferred in vivo.     

Supportive and expansive effects of aorta-gonad-mesonephros (AGM)-derived stromal cells on hematopoietic stem in vitro

AIM: To explore the supportive and expansive effects of aorta-gonad-mesonephros(AGM) region derived stromal cells on hematopoietic stem cells (HSC) in vitro.METHODS: The murine stromal cells were separated and cultured from AGM region of a 11 day postcoitum (dpc) mouse embryo and 6 week mouse. After identification by Wrights staining and flow cytometry, the stromal cells were co-cultured with the embryonic stem cell(ESC)-derived, cytokine-induced HSCs, and the maintenance and expansion of HSCs were evaluated by detecting CD34+，CD34+Sca-1+cells with flow cytometry.Blast colony-forming cell (BL-CFC) and high proliferative potential colony-forming cells(HPP-CFC) were determined by semi-solid medium clonal culture.RESULTS: AGM-derived and bone marrow(BM)-derived stromal cells were similar in morphology and phenotype, and had common character of stromal cells. Supported by AGM stromal cells or by BM stromal cells, more primitive progenitor cells HPP-CFC were expanded, but BL-CFC expansion was only detected in AGM-derived stromal cells. In the supporting of BM stromal cells CD34+ hematopoietic stem/progenitor cells were expanded 3-4 times, but no significant expansion in CD34+Sca-1+ cells was observed. While in the supporting of AGM stromal cells, both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded significantly from 4 to 5 times, respectively (P<0.05).CONCLUSION: AGM-derived stromal cells significantly support the expansion of HSC, and also maintain the self-renewal activity and multi-lineage differentiation of HSC in vitro.     

Intracellular expression of cytokines in placenta cells from NOD/SCID mice and its relationship with pregnancy outcomes

AIM: To investigate the relationship between the intracellular expression of Th1 and Th2 type cytokines in placental lymphocytes and the pregnancy outcomes in NOD/SCID mice. METHODS: The resorption rate of embryos (RR) was compared between BALB/c and NOD/SCID mice. In addition, the expression rates of Th1 type (TNF-α and IL-2) and Th2 type (IL-10) cytokines were detected intracellularlly in placental lymphocytes by four-color flow cytometry. RESULTS: Although multiple-immunodeficiency was confirmed in the NOD/SCID, no significant difference was observed in RR between BALB/c and NOD/SCID mice. At the same time, a dramatically elevated level of CD8+IL-10+/CD8+ cell percentage was found at the feto-maternal interface in NOD/SCID×NOD/SCID, as compared with BALB/c×BALB/c mice, while no significant difference was observed for TNF-α and IL-2 expression in CD4+ and CD8+ cell subsets isolated from these mice. CONCLUSION: The spontaneous elevation of CD8+IL-10+/CD8+ cell percentage at the feto-maternal interface may be related to the roughly normal fertility in NOD/SCID mice.