The transdifferentiation of Sca-1+ cells from murine fetal liver

AIM:To explore transdifferentiation potential of Sca-1⁺ cells from murine fetal liver. METHODS:2×10³ of Sca-1⁺ cells from male murine fetal liver were transfused into female mouse irradiated lethally with γ ray from ⁶⁰ Co source (10 Gy) via tail vein. Two months later, FISH and immunohistochemistry were used to detect the situation for transdifferentiating of the donor cells (male cells) in tissues of female recipient mouse. RESULTS:The renal tubular epitheliocyte-like and neurocyte-like cells with Y chromosome were found on the sections of renal and brain tissues from female recipient mice. These cells have phenotype characteristics of RCA⁺/CD₄₅^-F_4/80^- and NueN⁺/CD₄₅^-F_4/80^-, respectively. CONCLUSION:The evidence is provided for Sca-1⁺ cells from murine fetal liver to transdifferentiate into both renal and brain tissue cells.     

Sca-1+ cells from mouse fetal liver can differentiate into neurons in vitro

AIM: To study whether Sca-1⁺ cells from fetal liver can be induced to differentiate into neuronal cells in vitro. METHODS:Sca-1⁺cells from 14 5-days-old murine fetal liver were isolated with a magnetic cell sorting kit, and were cultured in Dulbecco s modif ied Eagle s medium(DMEM)/F12 supplemented with 10%fetal bovine serum(FBS), and passaged at a rat io of 1 3 when cells reached more than 80%confluence.The 5 passage cells were induced by 10^-3mol/Lβ-mercaptoethanol(β-ME)and 5×10^-7 mol/L all-trans-retinoic acid(RA)for 24 hours, and then incubated in serum-free medium for 5 hours to 5 days.The characteristics of treated cel s were assayed by immunocytochemistry staining analysis at 5 hours, or 5 days.RESULTS: Cells treated with β-ME and RA exhibited neuronal phenotype and expressed neuron-specific protein such as neuron-specific nuclear protein (NeuN), neuronfilament-M, and neuron-specific tubulin-1 (TuJ-1) but not tau, MAP-2, or the astrocyte-specific marker glial fibrillary acidic protein (GFAP).CONCLUSION: Sca-1⁺ cells from fetal liver, of which most are regarded as hematopoietic stem cells, could differentiate into early immature neuronal cells in vitro. These findings suggest that Sca-1⁺ cells from fetal liver may be an alternative source in cell therapy and gene therapy of neural dysfunction.     

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.     

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.     

Differentiation of Sca-1+ cells from murine fetal liver into renal cells in mice with acute renal failure

AIM: To study the effect of acute renal failure (ARF) on the differentiated frequency of Sca-1+ cells from murine fetal liver in irradiated mice. METHODS: The Sca-1+ cells from murine fetal liver were isolated with magnetic cell sorting (MACS) technique, the sex of which was identified by PCR. The 2×104 Sca-1+ cells were transplanted into a lethally irradiated (［60Co］, 8 Gy) inbred female mouse. After 8 weeks, these recipient mice were divided to A, B, and C groups at random (A group: irradiated; B group: ARF; C group: ARF and Sca-1+). The mice in B and C groups were induced to ARF with 50% (V/V) glycerin (11.6 mL/kg). 72 hours later, the mice in C group were injected with the fresh prepared Sca-1+ cells again. 8 weeks later, mice were sacrificed, and their kidneys were taken out, fixed and slices were prepared. Fluorescence in situ hybridization (FISH) of renal slices was performed and the pictures of them were taken and analyzed. RESULTS: The cells containing Y chromosome were found in renal slices from the mice in A, B and C groups, which located in epithelial cells of renal tubules, interstitium, glomeruli, and glomerular margin and increased gradually. The double and encircle zone of Y chromosome cells were found in the slices from the mice in B and C groups separately, which was consist of new renal tubules. The differentiation frequency of Sca-1+ cells in kidney in A, B and C groups were (1.65±0.18)%， (8.58±1.34)% and (18.13±1.91)%， respectively, which showed significant difference between former group and later group (P<0.01). CONCLUSION: The differentiation of Sca-1+ cells from murine fetal liver into renal cells and tissue is promoted by physiological microenvironment of acute damage and regeneration.     

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 adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

Mesenchymal stem cell-induced CD8α+ Jagged2high CD11bhigh regulatory dendritic cells prevent and treat mouse aGVHD

AIM: To investigate the role of mesenchymal stem cell-induced regulatory dendritic cells (MSC-DCregs) in mouse acute graft-versus-host disease(aGVHD) model. METHODS: Bone marrow cells from BALB/c (H-2^d) mice were isolated and were induced to differentiate into DCs. The DCs were selected by flow cytometry, and after 10 d co-culture with MSCs, they were induced to be MSC-DCregs. Male 8-week-old C57BL/6 (H-2^b) mice were used as donor mice. The female 8-week-old BALB/c (H-2^d) mice, who had received 100 cm source-skin distance, 30 cm×30 cm radiation field, 700 cGy total body irradiation (TBI) pretreatment were used as recipient mice. The recipients were divided into 5 groups: control group, TBI group (injected with medium only), bone marrow transplantation group (injected with 1×10⁷ bone marrow cells), aGVHD group (injected with 1×10⁷ bone marrow cells and 1×10⁷ spleen cells), and MSC-DCregs group (injected with 1×10⁷ bone marrow cells, 1×10⁷ spleen cells and 1×10⁶ MSC-DCregs). The white blood cell count, recipients' chimerism, clinical evaluation of aGVHD, survival analysis and pathological changes were determined. RESULTS: Hematopoieic recovery was seen at 10 d after transplantation. The recipients' chimerism was parallel to the donors' at 30 d. The median survival time of the mice in aGVHD group and MSC-DCregs group was 27 d and 33 d, and the survival rates at 30 d were 20% and 100% (P<0.01), respectively. The clinical scores of the mice in MSC-DCregs group were lower than those in aGVHD group (P<0.01). Moreover, the pathological changes in the skin and liver of the mice in MSC-DCregs group were less serious than those in aGVHD group. CONCLUSION: The MSC-DCregs induce an aGVHD tolerance in vivo, and further research of its mechanism is still in great necessary.     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.     

Association between development of CD4+CD25+ regulatory T cells and thymus CD4-CD25+ cells

AIM: To explore the correlation between development of CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺ Tr) and thymus CD4^-CD25⁺ cells. METHODS: The ratios of CD4⁺CD25⁺ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4^-CD25⁺ cells to CD4^- T cells in thymus were measured by flow cytometry. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4⁺CD25⁺ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4^-CD25⁺ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells showed no proliferation in response to ConA, while CD4⁺CD25⁺ Tr showed a transient enlargement of cell size. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells underwent proliferation in response to PDB plus ionomycin. CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4⁺CD25⁺ Tr showed significant proliferation and CD4⁺CD25^- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4^-CD25⁺ cells are probably the precursor of CD4⁺CD25⁺ Tr during cell development.     

Establishment of method for PD-1 immunophenotype detection in TCR Vβ repertoire of CD3+, CD4+ and CD8+ T-cell subsets

AIM:To establish the method for detecting the immunophenotype of immunosuppressive receptor programmed cell death protein 1 (PD-1) in T-cell receptor (TCR) Vβ repertoire of CD3⁺, CD4⁺ and CD8⁺ T-cell subsets, therefore to evaluate the distribution of PD-1 in T-cell repertoire from human peripheral blood (PB). METHODS:The PB samples from 10 cases of healthy individuals (HI) were collected. Using multi-colored fluorescence flow cytometry, the distribution frequency of PD-1 in TCR Vβ repertoire was detected with a wide panel of anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-PD-1 and 24 anti-TCR Vβ repertoire (IOTest^® Beta Mark TCR Vβ Repertoire Kit, containing 8 tubes which labeled A~H, each tube is a composite antibody of FITC and PE coupling, each cocktail contains antibodies direc-ted to 3 different Vβ subfamilies) monoclonal antibodies. RESULTS:The total number of the 24 TCR Vβ repertoire detected in CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells from 10 cases of HI was consistent with the Quick Reference Card data provided by the kit. The preliminary results showed that the frequency of Vβ usage in CD3⁺, CD4⁺ and CD8⁺ T cells was different. High usage of Vβ2, Vβ3, Vβ8, Vβ9, Vβ5.1, Vβ13.1 and Vβ13.2 was found in CD3⁺ T cells, while high usage of Vβ2, Vβ3, Vβ8, Vβ5.1, Vβ9 and Vβ13.1 in CD3⁺CD4⁺ T cells, and high usage of Vβ1, Vβ2, Vβ3, Vβ9, Vβ13.1 and Vβ13.2 in CD3⁺CD8⁺ T cells were also observed. Further analysis showed that the expression of PD-1 was detected in all 24 TCR Vβ subfamilies of CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. The higher frequency of PD-1⁺ T cells was CD4⁺Vβ5.2⁺ T cells, whereas the higher frequency of PD1⁺ T cells in CD8⁺Vβ11⁺ and CD8⁺Vβ13.6⁺ T cells was detected. CONCLUSION:The method for detection of the immunosuppressive receptor PD-1 in TCR Vβ repertoire of T-cell subsets is successfully established, which provides a new method for further analysis of immunosuppressive characteristics of TCR Vβ repertoire in the patients with leukemia.     

Generation of hematopoietic stem/progenitor cells with property of strengthened cell mediated immunity from an embryonic stem cell line

AIM:To induce lymphoid stem cells and/or T-cell precursors to differentiate into functional mature T lymphocyte, and to increase the surface marker of T lymphocytes such as CD₃⁺, while embryonic stem(ES) cells differentiated into hematopoietic stem/progenitor cells(HSPCs) in vitro. When they were injected into lethally irradiated mice, these differentiated cells had the advantage in immune reconstitution. METHODS:Embryonic stem cells formed embryoid bodies(EBs) in the medium containing methycellulose, hematopoietic growth factors(HGFs) was added to the culture system on the 6th day, thymopeptide was added at the same time. Flow cytometry were performed to detect the surface marker CD₃₄⁺ and CD₃⁺ of the differentiated cells. Finally the differentiated cells were injected into lethally irradiated mice, 60 days later, the incidence rate of graft versus host disease(GVHD) was taken as the mark of cell mediated immunity, PCR was performed to detect the sex determining region of the Y-chromosome(Sry) in bone marrow cells and spleen cells of the survival host female mice. RESULTS:The percentage of CD₃⁺ T lymphocytes was 10.52% and the incidence rate of GVHD was 0% on the 13th day, but they respectively rose up to 22.93% and 100% if thymopeptide was added in the procedure of inducing ES cells to differentiate into HSPC in vitro. CONCLUSION:The quantity of CD₃⁺ T lymphocytes increased in medium containing thymopeptide when ES cells differentiated into CD₃₄⁺ HSPC.     

EP2A promotes human CD34+ cell homing in vitro but not proliferation

AIM: To investigate the role of prostaglandin E₂ receptor 2 agonist (EP₂A) in proliferation and homing of human CD34⁺ cells. METHODS: Bone marrow fluid and peripheral blood containing stem cells were collected from healthy donors mobilized by granulocyte colony-stimulating factor in our department. Human CD34⁺ cells were isolated by the method of magnetic-activated cell sorting microbeads. Bone marrow mononuclear cells were isolated by Ficoll-Paque centrifugation, and the bone marrow mesenchymal stem cells (BMMSC) were cultured with L-DMEM. Human CD34⁺ cells and BMMSC were divided into 4 groups, and treated with PGE₂ (as positive control), DMSO (as negative control), EP₂A and EP₂A+prostaglandin E₂ receptor 2 antagonist (EP₂AA), respectively. After exposed to the reagents, human CD34⁺ cell viability was measured by CCK-8 assay, the number of colonies was evaluated by colony-formation assay, the cell cycle distribution was analyzed by flow cytometry, and the protein expression of survivin, β-catenin and CXC chemokine receptor 4 (CXCR4) was detrmined by Western blot. Moreover, the concentration of stromal cell-derived factor-1α (SDF-1α) in the BMMSC was detected by ELISA. RESULTS: The cell viability and the colony number of human CD34⁺ cells in EP₂A group were not higher than those in negative control group. Furthermore, the proportion of human CD34⁺ cells treated with EP₂A in G₂/M phase was not elevated compared with negative control group. The protein expression of survivin and β-catenin did not up-regulated in human CD34⁺ cells exposed to EP₂A, but the protein expression of CXCR4 in human CD34⁺ cells and the concentration of SDF-1α in BMMSC were elevated. CONCLUSION: EP₂A promotes human CD34⁺ cell homing in vitro but not proliferation.     

Morphological study of macrophages infected with constructed recombinant adenovirus carrying gp120 gene of Chinese HIV-1 strain

AIM: To construct a recombinant adenovirus carrying gp120 gene of Chinese HIV-1 strain,which can infect mouse bone marrow-derived macrophages (BMM). METHODS: Co-transfection of shuttle and backbone plasmids of AdMax system into 293Ad5⁺ cells was performed, followed by viral packaging, propagation and purification. These viruses were subject to Karber TCID₅₀ titration. The expression of gp120 protein in 293Ad5⁺ cells was determined by ELISA. The viral titration was validated by a multiplicity of infection (MOI) test with BMM. RESULTS: The titers of the outcome viruses, including AdMax-HIV-1 gp120 (Ad-gp120) and its vector control Ad-GFP, were 10^8.3 and 10^8.1 TCID₅₀/mL, respectively. Both recombinant adenoviruses infected BMM with similar capacity of 293Ad5⁺ cell infection, which validated the TCID₅₀ titration.The gp120 protein was positive in 293Ad5⁺ cell lysates. BMM activation was observed morphologically after Ad-gp120 infection as compared with Ad-GFP-infected cells. CONCLUSION: Functional adenovirus containing HIV-1 gp120 of prevalent strains in China was successfully constructed. Infection of Ad-gp120 causes BMM activation.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Butylated hydroxyanisole promotes expression of neural specific genes in mouse fetal liver cells through extracellular signal-regulated kinase signaling pathways

AIM: To study the mechanism of butylated hydroxyanisole-induced neural differentiation of fetal liver Sca-1+ cells in vitro. METHODS: Sca-1+ cells from 14.5-day-old mouse fetal liver were isolated with a magnetic cell sorting kit. Cultured cells pretreated with or without extracellular signal-regulated kinase (MEK1) inhibitor, PD98059, were induced by 200 μmol/L butylated hydroxyanisole (BHA) for 24 hours, and then incubated in serum-free medium. Expression of genes in treated or untreated cells were assayed by Western blotting and RT-PCR. RESULTS: There was low level of neuronfilament-L (NF-L) and brain factor-1 (BF-1) in fetal liver Sca-1+ cells, but no neuronfilament-H (NF-H) and tyrosine hydroxylase (TH) was observed. BHA significantly promoted the expression of neuron-specific NF-L, NF-H, BF-1, and TH in fetal liver Sca-1+ cells. NF-L, NF-H, BF-1 and TH increased by 6.32 fold, 2.73 fold, 3.37 fold and 2.68 fold, respectively (all P<0.01 vs untreated cells). However, the expression of BHA-induced genes were inhibited by ERK kinase inhibitor PD98059 (25 μmol/L). CONCLUSION: BHA induces neural differentiation of fetal liver cells through ERK.     

Construction of mouse ESC line stably expressing Pdx1 by Tet-On system

AIM To construct the mouse embryonic stem cell （ESC） line with stable pancreatic and duodenal homeobox 1 （Pdx1） expression by Tet-On system, which may lay a foundation for further research on the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells. METHODS The Pdx1-overexpressing lentiviral vector with green fluorescent protein marker and puromycin resistance was constructed by Tet-On system and was used to infect the mouse ESC. The cells were divided into 3 groups： blank control group （ESC group）, empty lentivirus control group （PDX1^- ESC group） and Pdx1 lentivirus transfection group （PDX1⁺ ESC group）. Flow cytometry was used to detect the transfected cells after screening by doxycycline （DOX）. The function of Tet-On system and the expression of Pdx1 gene were detected. The transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were sorted by flow cytometry, and constructed ESC line with stable expression of Pdx1 and negative control ESC line were verified. RESULTS （1） The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were 90.72% and 94.01% after screening by DOX, respectively. The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group was 97.84% and 98.13% after sorting by flow cytometry, respectively. （2） With DOX, green fluorescence was observed in PDX1^- ESC group and PDX1⁺ ESC group. The mRNA and protein expression of Pdx1 was significantly increased in PDX1⁺ ESC group （P<0.05）. Without DOX, no green fluorescence was observed in the cells of the 3 groups, and no significant difference in the mRNA and protein expression of Pdx1 was observed （P>0.05）. （3） After 3 months of cryopreservation, the cell lines still survived in resuscitation culture and were regulated by DOX. CONCLUSION Using Tet-On system, the mouse ESC line with inducible Pdx1 expression were successfully established and could be used as an effective cell model to research the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

Protective effect of HSF1 on mice with LPS-induced acute lung injury and screening of relevant differentially-expressed genes

AIM: To study the protective effect of heat shock factor1 (HSF1) on the mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI), and to screen the relevant differentially-expressed genes. METHODS: ALI mouse model was established by LPS intracheal instillation. The macroscopic and pathological changes of the lung tissue were observed, and the concentrations of total protein, TNF-α, IL-β, IL-6 and VEGF in the bronchoalveolar lavage fluid (BALF) were analyzed. Differentially-expressed genes in the lung tissues of HSF1^+/+ mice and HSF1^-/- mice with ALI induced by LPS were screened by gene chips. The key gene was verified by real-time qPCR. RESULTS: The macroscopic and pathological changes of the lung injury in HSF1^-/-+LPS mice were more serious than those in HSF1^+/++LPS mice. The concentrations of total protein, VEGF, TNF-α, IL-1β and IL-6 in the BALF of HSF1^-/-+LPS mice were significantly higher than those of HSF1^+/++LPS mice (P<0.05). Compared with the HSF1^+/+ mice, a total of 918 differentially-expressed genes were indentified in the HSF1^-/- mice, among which the expression levels of 65 genes had obvious diffe-rence, with 28 genes up-regulated, including Atg7, ccr1, cxcr2, Tbl1xr1, Mmp9, Pparg, Plcb2, Arrb2, Cntn1, Col4a6, etc, and 37 genes down-regulated, including Fgfr1, Fgfr2, Map4k4, Ddx58, Tfg, Stat3, Smad4, Lamc1, Sdc3, etc. The results of real-time qPCR showed that the mRNA level of CXCR2 in HSF1^-/-+ LPS mice was significantly higher than that in HSF1^+/++ LPS mice, which was consistent with the results of gene chips. CONCLUSION: HSF1 has protective effect on the mice with LPS-induced ALI. CXCR2 may be involved in the protective effect of HSF1 on this process.     

Chinese herbal preparation Qing Yi Tang Ⅱ granule protects against experimental acute pancreatitis in mice

AIM: To investigate the protect effect of Chinese herbal preparation, Qing Yi TangⅡgranule (QYT), on acute pancreatitis (AP) mice and its mechanism. METHODS: Adult male and female C57BL/6 mice (n=24) were randomly divided into control group, AP group and AP+QYT group. Severe AP was induced by combined intra-peritoneal injection of caerulein (50 μg/kg) and lipopolysaccharide (LPS; 10 mg/kg). Drinking water or 24% QYT solution was given to the mice in AP group or AP+QYT group by oral gavage. The mice in control group were intraperitoneally injected with equivalent volume of normal saline and gavaged with water. The mice were sacrificed 3 h after the last injection. Severity of AP was assessed by biochemical markers and histology. The plasma level of IL-6 and MCP-1, and lung myeloperoxidase (MPO) levels were determined for assessing the extent of systemic inflammatory response. The intestinal microflora, T lymphocytes and T-lymphocyte subgroups were examined for assessing the function of the intestinal barrier. RESULTS: Compared with control group, the mice in AP group presented significant increases in pathological histological scores, plasma amylase activity and IL-6 and MCP-1 levels, as well as the MPO activity in the lung and pancreatic tissues. QYT attenuated these changes to some extent. Furthermore, the increased intestinal microflora was significantly reversed by QYT. No difference of the numbers of Peyer's patches in small intestine in the 3 groups was observed, but the percentage of CD3⁺ T lymphocytes decreased significantly in AP group, and increased percentage of CD4⁺ and CD4⁺/CD8⁺ ratio were found in AP group and AP+QYT group. CONCLUSION: QYT protects against cearulein and LPS-induced acute pancreatitis in mice. The mechanisms may be related to the suppression of the inflammatory response, promoting intestinal bacteria removal, and regulating the functions of T lymphocytes in the intestinal barrier.