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.     

Effect of granulocyte colony-stimulating-factor on acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in a murine model

AIM: To explore the impact of granulocyte colony-stimulating factor (G-CSF) on acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in a murine model and its possible mechanisms. METHODS: Male C57BL/6 (H-2^b) and BALB/c (H-2^d) mice were used as the allogeneic and syngeneic donor mice, respectively. Moreover, female BALB/c mice were used as recipient mice. The recipient mice were conditioned by a single dose (8 Gy) of total body irradiation (TBI). The recipient mice were randomly divided into 7 groups: TBI group, Syn-BMST control group, post-Syn-BMST G-CSF administration (Syn-BMST+G-CSF)group, allo-BMT control group, post-allo-BMT G-CSF administration (allo-BMT+G-CSF)group, allo-BMST control group and post-allo-BMST G-CSF administration (allo-BMST+G-CSF) group. The mice in control groups and G-CSF administration groups were subcutaneous injected with 0.1 mL normal saline (NS) and 0.1 mL NS containing 2 μg G-CSF per day from 1st day, respectively. The effect of G-CSF on aGVHD was evaluated by clinical manifestations and pathological changes, as well as survival time of the mice in different groups. The serum levels of IL-2, IL-4, IFN-γ and TNF-α in allo-BMST and allo-BMST+G-CSF groups were detected by ELISA at 10th day. Flow cytometry was used to analyze the immunophenotypes of splenocytes at 10th day. RESULTS: The mice in TBI group were all died for hematologic failure on 9~15 d after TBI. No effect of G-CSF on the survival of the mice underwent Syn-BMST and transplantation of single allogeneic marrow cells was observed. The mean survival days in allo-BMST group and allo-BMST+G-CSF group were (34.8±4.5) d and (19.8±6.1) d'respectively (P<0.01). Moreover, post-transplant administration of G-CSF increased the spleen total nucleated cells count (SpTNC), NK cells subset, and DC1/DC2 ratio in the spleen with over 99% of donor chimerism rate at 10th day. No difference in the levels of serum IL-2, IL-4, IFN-γ and TNF-α between the 2 group at 10th day was found. CONCLUSION: The administration of G-CSF after allo-BMST significantly aggravates mouse aGVHD. The expansion of NK cells stimulated by G-CSF may be involved in the mechanism of generating alloreactivity against host cells. These results imply there may be potential risk of evoking or aggravating acute GVHD if G-CSF is administered in the early stage of clinical allo-HSCT.     

Establishment of an acute GVHD animal model in EL9611 erythroleukemia mice

AIM: To establish an acute graft-versus-host disease (GVHD) model in EL9611 erythroleukemia mice. METHODS: Using C57BL/6 (H-2^b) mice as the donor and BALB/c (H-2^d) mice as the recipient in allogeneic bone marrow transplantation (allo-BMT), the acute GVHD model was established. The mice were divided into leukemia group (n=10), radiation control group (leukemic mice given radiation without allo-BMT, n=4), GVHD group (leukemic mice given radiation+allo-BMT, n=10) and normal control group (n=4). In leukemia group, 2×10⁶/mouse EL9611 erythroleukemic cells were transfused via tail vein into BALB/c mice to build the erythroleukemia model. In GVHD group, 7 days after leukemic cell transfusion, the mice received total dose of 8.0 Gy γ of total body irradiation(TBI), and within 5 h, 2×10⁶ C57BL/6 bone marrow cells and 1×10⁷ C57BL/6 spleen cells per mouse were transfused via tail vein to build the acute GVHD model in EL9611 erythroleukemia mice. The clinical manifestations of posture, fur, stool and so on were observed. Pathological examination was conducted to examine the changes of liver, spleen, skin, small intestine and peripheral blood. The survival rate was also calculated. RESULTS: (1) In leukemia group, the mean survival time (MST) was (14.5±2.1) days,or (7.5±0.7) days when irradiation day was as day 0(P<0.01 compared with GVHD group). The death rate was 100% with no spontaneous remission. The dead mice showed splenohepatomegalia and high WBC count . Pathological examination showed disorganization of normal tissues and leukemic cell infiltration. (2) In radiation control group, MST was (9.0±0.7) d, with significant difference as compared with GVHD group and normal group (P<0.01). The death rate was 100%. Pathological examination showed hematopoiesis exhaustion. (3) In GVHD group, MST was (32.0±3.2) d (P<0.01 compared with other groups). The death rate was 100%, the symptoms were observed on day 10-13 after allo-BMT. Clinical manifestations and pathological examination corresponded to those of I degree to II degree of GVHD. CONCLUSION: Intravenous infusion of 2×10⁶/mouse EL9611 leukemic cell successfully establishes the EL9611 erythroleukemia animal model. Seven days after EL9611 leukemic cell transfusion, lethal dose of TBI and allo-BMT can successfully build the acute GVHD model of EL9611 leukemic mice.     

Effects of hyperbaric oxygen and cyclosporin A on thelevels of active oxygens and nitric oxide in spleens of skin transplanted mice

AIM: To study effects of hyperbaric oxygen (HBO) and cyclosporin A (CsA) on the contents of active oxygens and nitric oxide (NO) in spleens of skin transplanted mice. METHODS: The donor mice BALB/C and receptor mice C₅₇BL/6 were tested for skin transplantation. The HBO group mice were treated with 99.2% oxygen under 0.25 MPa for 1.5 hours, while CsA group mice were treated with CsA 0.5 mg·kg^-1·d^-1 by abdomen injection. After 14 days, the spleen were extracted the contents of malondialdehyde (MDA) and NO and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) and NO synthases (NOS) were determined. RESULTS: (1) Compared with the control group, the transplantation group, HBO group and CsA group have markedly increased the content of MDA and the activities of GSH-PX and CAT; Compared with the transplantation group, the CsA group have markedly increased activity of SOD and reduced activities of GSH-PX and CAT; the HBO group have markedly reduced the activity of GSH-PX and increased the activities of CAT and SOD (P<0.01). (2) Compared with the control group, the transplantation group have markedly increased the content of NO and the activity of NOS; Compared with the transplantation group, the HBO group have markedly increased the activity of NOS and reduced the content of NO (P<0.01); The content of NO and the activity of NOS in CsA group was not changed significantly. CONCLUSION: In the lymphocytes of the transplantation group, the peroxidation is intensified, and the content of NO and the activity of NOS increased. HBO and CsA may activate the systems of oxidation/antioxidation and NO/NOS in spleen, which may be related to their mechanism of inhibition rejection.     

The prevention of acute graft versus host disease by Rhodamine 123-mediated photodynamic therapy

AIM: To evaluate the effect and safety of Rhodamine 123 (Rh123)-mediated photodynamic treatment (PDT) on acute graft versus host disease. METHODS: An acute graft versus host disease (aGVHD) mice model was established using C57B/6 mice as donors and BALB/c mice as recipients. Mixed lymphocytic cells were cultured with Rh123 (50 nmol/L) and irradiated by argon laser 30 mW/cm2 for 3 min, then transplanted to BALB/c recipient mice mixed with donor bone marrow. Hepatopoietic recovery, aGVHD occurrence, survival time after transplantation and pathological changes were observed. In addition, CD3+CD69+ positive rates of MLC were examined by flowcytometry. RESULTS: Occurrence of aGVHD decreased, degree of pathological manifestation became milder, survival rates were higher than non PDT groups. CD3+CD69+ rates of MLC cells treated with photodynamic therapy (PDT) and cultured for 24 h significantly decreased. CONCLUSION: Rh123-mediated PDT can effectively prevent aGVHD of allogeneic bone marrow transplantation in mice.     

Study on efficacy of the decoction of invigorating kidney and activating blood circulation in BALB/C mice with immune mediated aplastic anemia

AIM and METHODS: To study the efficacy of the decoction of invigorating kidney and activating blood circulation(DIKABC) in BALB/C mice with immune mediated aplastic anemia. 50 female BALB/C mice at 8~12 weeks of age were divided into 5 groups:(1) control,(2) model,(3) model+DIKABC(H), (4) model+DIKABC(L), and (5) model+ciclosporin A(CsA). The model of immune mediated aplastic anemia in BALB/C mice, which received sublethal whole body irradiation, lymph node and thymus cells of DBA/2 mice, was given different liquid from groups and administered appropriately for 9 days, blood cells and bone marrow nucleated cells(BMNC) and CD₄₅⁺ cells were determined by flow cytomytry(FCM).RESULTS: RBC,WBC,Platelet,BMNC in DIKABC groups were higher than those in model group (P＜0.01/0.05), but those in CsA group were lower than those in DIKABC groups (P＜0.05).Compared with model group, CD₄₅⁺ cells in DIKABC groups increased (P＜0.05),but were lower than those in control groups (P＜0.05). CONCLUSION: The decoction of invigorating kidney and activating blood circulation could increase the percentages of CD₄₅⁺ cells in bone marrow and prevent immune mediated aplastic anemia in BALB/C mice.     

The role of chlamydia pneumoniae in the course of transformation of C57 BL/6J mouse peritoneal macrophages into foam cells

AIM: In order to understand the role of chlamydia pneumoniae in the course of macrophages transformation into foam cells experiments with chlamydia pneumoniae standard strain AR-39 wese made. METHOD: C57 BL/6J Mouse peritoneal macrophages C57 BL/6J wese incubated 24 h, and they were divided into 6 groups to be incubated continually: A₁~DMEM; A₂~DMEM+10 IFUs/L AR-39; B₁~DMEM+10mg/L LDL; B₂~DMEM+10mg/L LDL+10 IFUs/L AR-39; C₁~DMEM+10mg/L OxLDL;C₂~DMEM+10mg/L OxLDL+10 IFUs/L AR-39. 72 h later, morphological changes of the cells were observed and of intracellular cholesterol of content was detected. RESULTS: 1、Morphological studies: there were no lipid particles in A₁, A₂ and B₁ groups, but the lipid particles could be found in B₂、C₁ and C₂ group, Among six groups, the most lipid particles were seen in C₂ groups. 2、Biochemical detection:The ratio of cholesterol ester to total cholesterol was much higher than 50% in B₂、C₁ and C₂ groups, but was less than 50% in A₁、A₂ and B₁ groups. CONCLUSION: Chlamydia pneumoniae may have played a role in promoting C57 BL/6J mouse peritoneal macrophages into foam cells.     

The inhibitory effect of Chinese medicine Jinan injectionon Lewis lung cancer in mice

AIM:To investigate the inhibitory effect of Chinese medicine Jinan injection(JA) on Lewis lung cancer (LLC) in mice.METHODS:The C₅₇BL／6J mice with Lewis lung cancer(LLC) were divided into normal saline(NS), Jinan high dose (JAH), Jinan middle dose (JAM), Jinan low dose (JAL) and cyclophosphamide(CTX) groups. The body weight changes and inhibitory rate of LLC in each group were observed. In addition, flow cytometry and TUNEL were used to detect the anticancer mechanism of Jinan.RESULTS:The body weights were increased significantly in JA-treated groups vs CTX and the resistant rate was 45.79%, 40.90%, 32.48% and 98.96%, respectively. The apoptotic rate was 24.19%, 14.95% and 13.93% in JAH, JAM and JAL, respectively, and the Jinan induced apoptosis of LIC in a dose-dependent manner.CONCLUSION:Jinan injection inhibites the growth of LLC, and the apoptosis induction may be one of mechanisms that Jinan treates LLC in mice.     

Allogeneic antigen-pulsed murine immature CD8α+ dendritic cells prevent T-cells proliferation in vitro

AIM: To explore the optimal condition of predominant immature CD8α⁺ dendritic cells(predo-iDCs) preventing T-cell proliferation pulsed by allogeneic antigen. METHODS: Predo-iDCs, induced with GM-CSF +IL-4 +SCF +Flt3L from SPF healthy C57BL/6 murine bone marrow cells, were pulsed by different doses (0, 2.5, 5, 10 and 20 mg/L)of allogeneic murine splenocyte antigen. Syngeneic T-cells were co-incubated with Ag-pulsed DCs (DCs/T=1∶ 1, 2∶ 1, 4∶ 1) and the T-cell proliferation was measured by MTT. The secretion of cytokines (IFN-γ and IL-10) in the co-incubated supernatants was detected by ELISA. The effect of prevention of T-cell proliferation generated by murine predo-iDCs pulsed by allogeneic antigen was detected. The control derived from mature dendritic cells(mDCs)induced by GM-CSF +IL-4 +TNF-α. RESULTS: The effect of Ag-pulsed predo-iDCs for stimulating T-cell proliferation was the slightest in predo-iDCs/T 1∶ 1 group, compared with that in predo-iDCs/T 2∶ 1 and 4∶ 1 group (P<0.05). The secretion of IFN-γ in mixed lymphocyte reaction(MLR) was significantly lower than the one in mDCs control group, while the secretion of IL-10 was higher than that in control group when low dose of antigen (<2.5 mg/L) was added into MLR. CONCLUSION: Predominant iDCs pulsed by low dose of allogeneic antigen (2.5 mg/L) mixed 1∶ 1 with T-cells is the optimal condition for the prevention of T-cells proliferation.     

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.     

Distribution and identification of immunocytes in humanized SCID mouse model

AIM: Humanized-NOD/SCID(hu-NOD/SCID) mouse model was established and the level of immune reconstitution was assessed in this model. METHODS: Mononuclear cells (MNC) and CD34⁺ cells were isolated or sorted from cord blood(CB). Human CD45, CD19, CD3 markers on cells from NOD/SCID murine peripheral blood(PB), bone marrow(BM), thymus were detected by FCM from 4 to 10 weeks after hematopoietic stem cell transplantation. After 10 weeks, the gene expressions of the human β₂M and RAG2 were detected by RT-PCR in PB or bone marrow of mice model. RESULTS: Human CD45, CD19, CD3 cells populations in PB and BM were found by flow cytometry in mice model transplanted with CD34+ cells or CB MNC from 4 to 10 weeks. The highest positivity of human lymphocytes was at 8 week after transplantation. The levels of human cell engraftment in mice transplanted with CD34⁺ cells were higher than those in mice transplanted with CB MNC. The mRNA of human β₂M and RAG2 were found by RT-PCR in BM.CONCLUSION: The higher level of human lymphocyte engraftment is established in NOD/SCID mouse model transplanted with CD34⁺ compared with CB MNC. The maturation of T lymphocytes could be happened in bone marrow of mice model.     

Effect of hydrogen sulfide on airway inflammation induced by ozone in mice

AIM: To investigate the effect of hydrogen sulfide (H₂S) on airway inflammation induced by ozone (O₃) exposure and its mechanisms.METHODS: C57BL/6 mice (n=32) were randomly divided into control group, O₃ group, NaHS+O₃ group and NaHS group. The mice in O₃ group and O₃+NaHS group were exposed to 2.14 mg/m³ O₃ for 3 h on days 1, 3 and 5, while the mice in control group and NaHS group were exposed to filtered air. NaHS (14 μmol/kg) was administered intraperitoneally to the mice in NaHS group and O₃+NaHS group 30 min before each exposure. After the last exposure for 24 h, the airway responsiveness was determined, and bronchoalveolar lavage fluid (BALF) was collected for counting inflammatory cells and measuring total protein concentration. The lung tissues were collected for observing the morphological changes with HE staining. The levels of interleukin-6 (IL-6), interleukin-8 (IL-8), malondialdehyde (MDA) and NF-κB p65 protein in the lungs were determined.RESULTS: Compared with control group, the airway responsiveness, inflammatory cells, protein concentration, inflammation score, levels of IL-6, IL-8, MDA and NF-κB p65 in O₃ group increased significantly, but these in NaHS+O₃ group decreased compared with O₃  group.CONCLUSION: The present findings suggest that H₂S attenuates O₃ induced airway inflammation by inhibiting NF-κB expression and preventing lipid peroxidation.     

An activated mutant of SHP-2 tyrosine phosphatase causes murine myeloid abnormal proliferation

AIM: To investigate whether an activated mutant of SHP-2 tyrosine phosphatase is involved in abnormal proliferation of murine myeloid.METHODS: Wild-type (WT) and SHP-2^D61G/+mutant mice aged 8 weeks and 16 weeks were used. The number of peripheral blood leukocytes and the spleen sizes were measured by cell counting and weighing methods,respectively. The surface markers (Mac-1 and Gr-1 for myeloid, Ter119 for erythroid, CD3 for T-lymphocyte and B220 for B-lymphocyte) of hematopoitic cells in peripheral blood and bone marrow were detected by flow cytometry. The rate of Mac-1 or Gr-1 positive cells in the peripheral blood and the rate of Mac-1, Gr-1, Ter119, CD3 or B220 positive cells in bone marrow were analyzed. The ability of colony formation unit (CFU) of the bone marrow was also observed by CFU assay. Finally, the expression of p-Akt and p-ERK in the peripheral blood leukocytes induced by interleukin-3 (IL-3, 5 μg/L) was detected by Western blotting.RESULTS: The number of leukocytes in peripheral blood of 16-week-old mice was more (P<0.01) and the spleens were bigger in mutant SHP-2^D61G/+ mice than those in WT mice. The rate of Mac-1 and Gr-1 positive cells in peripheral blood leukocytes of 16-week-old SHP-2^D61G/+ mice were dramatically increased (P<0.05). Mac-1 and Gr-1 positive cell rates in bone marrow of SHP-2^{D61G / +} mice were much higher (P<0.05) than those in WT mice and no statistic significance was found in the erythroid or lymphocyte cells. The number of CFU-GM (represents myeloid) was increased in mutant mice. The expression of p-Akt and p-ERK in peripheral blood leukocytes of mutant mice was significantly enhanced after stimulated with IL-3.CONCLUSION: These results suggest that activated mutant SHP-2 results in the disorder of mouse myeloid proliferation via MAPK and PI3K activation.     

Calreticulin expression is necessary for protective immune effect of T-cell vaccine in EAE mice

AIM:To study the role of cell membrane ectopic calreticulin (CALR) expression on the protective immunie effect of T-cell vaccine (TCV) on experimental autoimmune encephalomyelitis (EAE). METHODS:EAE model was established by myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) immunization in C57BL/6 mice, and the mice were immunized with MOG_35-55-specific CALR⁺ and CALR^- T-lymphocytes. Symptomatic scores were compared at the maximum of the disease. On the 15th day after immunization, the proportion of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Treg) in the spleen, and the expression of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10 and IL-17A in the serum were measured. RESULTS:Increased expression of CALR in activated T cells after γ-irradiation was observed. Blockade of CALR on the vaccinating T-cell surface reduced the protective effect of TCV. Furthermore, blockade of CALR reduced the number of Treg in the spleen and up-regulated pro-inflammatory cytokines. CONCLUSION:CALR expression in the T cells is necessary for the protective immunity induced by TCV in EAE mice.     

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.     

Neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells on mice with experimental autoimmune encephalomyelitis

AIM: To explore the neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells (BM-NSCs) on the mice with experimental autoimmune encephalomyelitis (EAE). METHODS: Female C57BL/6 mice (8~10 weeks old, n=32) were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) to establish chronic EAE model. The mice were randomly divided into control (ddH₂O) group, fasudil group, BM-NSCs group, and fasudil+BM-NSCs group. The clinical score and body weight were recorded every other day. The expression of neurotrophic factors was determined by immunofluorescence staining. RESULTS: In comparison with ddH₂O group, fasudil combined with BM-NSCs delayed onset and ameliorated severity of EAE. The numbers of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and ciliary neurotrophic factor positive cells in fasudil group, BM-NSCs group and fasudil+BM-NSCs group were all increased in various extents. In particularly, the expression of these neurotrophic factors in fasudil+BM-NSCs group was significantly higher than that in the mice treated with fasudil or BM-NSCs alone (P<0.01). CONCLUSION: Fasudil combined with BM-NSCs promotes the expression of neurotrophic factors and improves microenvironment of central nervous system, thus playing a positive role in neural restoration and regeneration through a synergistic and superimposed effect.     

Changes of interleukin-6 in pulmonary hypertension mice induced by chronic hypoxic-hypercapnia

AIM: To investigated the changes of interleukin-6 (IL-6) in the pulmonary hypertension mice induced by chronic hypoxic hypercapnia. METHODS: Sixteen male C57BL/6 mice were randomly divided into 2 groups (8 mice in each group): normal control group and chronic hypoxic hypercapnia group. The mice in chronic hypoxic hypercapnia group were placed in a sealed chamber where O₂ concentration was kept at 9%~11%, and the CO₂ concentration at 5% ~6%, 8 h a day, 6 days a week for 4 weeks. The right ventricular (RV) weight, the weight of left ventricle plus ventricular septum (LV+S) were measured and right ventricular hypertrophy index was calculated. The structural changes of the pulmonary arteries were assessed by the method of histology with HE staining. The vessel wall diameter/total diameter (WT%) and the vessel wall area/total area (WA%) were analyzed by Image-Pro Plus 6.0 software. The protein expression of IL-6 in the lungs of the mice was determined by immunohistochemistry and ELISA, and the mRNA expression of IL-6 in the lungs was determined by RT-PCR. RESULTS: Compared with control group, RV/(LV+S), MT%, MA% and the expression of IL-6 at mRNA and protein levels were significantly increased in chronic hypoxic hypercapnia group. CONCLUSION: In the environment of chronic hypoxia and hypercapnia, the expression of interleukin-6 was elevated in mouse lungs, which may closely related to the development of pulmonary hypertension.     

Effect of mesenchymal stem cells infusion on hematopoietic recostitution after peripheral blood stem cell transplantation in mice

AIM: To study the effect of mesenchymal stem cells (MSCs) infusion on hematopoietic recovery after peripheral blood stem cell transplantation in mice. METHODS: BALB/c mice conditioned by high dose chemotherapy/radiotherapy were infused with10⁶ peripheral blood mononuclear cells (PBMC) mobilized by granulocyte colony-stimulating factor (PBSCT group),10⁴ MSCs culture-expanded in vitro and10⁶ PBMC(experimental group1),10⁶ MSCs and10⁶ PBMC(experimental gruop 2). Survival rate within 4 weeks, white blood cell count, bone marrow nucleated cells (BMNC), granulocyte-macrophage colony forming unit(GM-CFU) and fibroblast colony forming unit (F-CFU) were examined. RESULTS: Survival rate, BMNC, GM-CFU, F-CFU were significantly higher in experimental group 2 than that in PBSCT group (P<0.05), WBC recovery was faster (P<0.01) and F-CFU level was higher in experimental group1than that in PBSCT group (P<0.05). CONCLUSION: Mesenchymal stem cells infusion enhanced hematopoietic reconstitution after peripheral blood stem cell transplantation.     

Effects of iNKT cells from OVA-induced asthmatic mice combined with OVA on phenotype and function of bone marrow-derived dendritic cells in vitro

AIM:To investigate the effects of invariant natural killer T-cells (iNKT cells) from ovalbumin (OVA)-induced asthmatic mice combined with OVA on the phenotypic and functional characteristics of bone marrow-derived dendritic cells (BMDCs) in vitro. METHODS:The BMDCs from wild-type (WT) BLAB/c mice were co-cultured with purified iNKT cells from WT mice immunized and challenged with OVA in the presence of 100 mg/L OVA (iNKT cells plus OVA group) or PBS (iNKT cells plus PBS group) for 20 h, and were also cultured with 50 mg/L LPS (LPS group), 100 mg/L OVA (OVA group) or PBS (PBS group) for 20 h. The expression of MHC-Ⅱ, CD40, CD86, and CD80 on the BMDCs was measured by flow cytometric analysis, and the levels of interleukin-12 (IL-12) p70, IL-6, tumor necrosis factor-α (TNF-α) and IL-10 in the culture supernatant were measured by ELISA. Splenic CD4⁺ T cells from DO11.10 transgenic mice were co-cultured for 48 h with the above mentioned BMDCs, and then the concentrations of IL-4 and interferon-γ (IFN-γ) in culture supernatants were measured by ELISA. RESULTS:The expression of MHC-Ⅱ, CD80, CD86 and CD40, and releases of proinflammatory cytokines by BMDCs in iNKT cells plus OVA group were comparable to those in the LPS group (P>0.05), but significantly higher than those in iNKT cells plus PBS group, OVA group, and PBS group (P<0.05 or P<0.01). The concentration of IL-4 in culture supernatants from BMDCs in iNKT cells plus OVA group co-cultured with DO11.10 CD4⁺ T cells was similar to that in LPS group (P>0.05), but markedly higher than that in iNKT cells plus PBS group, OVA group, and PBS group (P<0.01). CONCLUSION:Bone marrow-derived dendritic cells undergo immunogenic maturation upon interaction with iNKT cells in the presence of OVA.