Role of Gab2 in mouse myeloid abnormal proliferation induced by SHP-2D61G/+ mutation

AIM: To investigate whether Gab2, the key adapter protein in the SHP-2 signaling pathway, is involved in mouse myeloid abnormal proliferation induced by SHP-2D61G/+ mutation.METHODS: Four kinds of mouse model genotyped as SHP-2+/+, Gab2-/-, SHP-2D61G/+ and SHP-2D61G/+/Gab2-/- were generated from crossbreeding of Gab2-/- mice and SHP-2D61G/+ mice. The mouse spleen size was analyzed. The number of peripheral blood leukocytes was counted by cell counting and the percentage of Mac-1 or Gr-1 positive myeloid cells in the bone marrow was detected by flow cytometry. The proliferation ability of bone marrow hematopoietic stem/progenitor cells in response to cytokines was assayed by colony formation. The expression of p-ERK and p-Akt and the binding capacity of SHP-2 with Gab2 in the bone marrow-derived mast cells stimulated with IL-3 were detected by Western blotting and immunoprecipitation.RESULTS: The phenotype of myeloproliferative disorder, such as enlarged spleen size, increased leukocyte number and high percentage of myeloid cells, in SHP-2D61G/+ mutant mice was found, and was dramatically improved in SHP-2D61G/+/Gab2-/- double mutation mice. Furthermore, compared with SHP-2D61G/+ mutation mice, significantly decreased colony formation ability of the bone marrow cells with IL-3 stimulation was observed in SHP-2D61G/+/Gab2-/- double mutation mice. A reduced phosphorylation level of ERK/Akt, and SHP-2 without binding of Gab2 were found in SHP-2D61G/+/Gab2-/- bone marrow-derived mast cells with IL-3 stimulation.CONCLUSION: Gab2 knockout significantly reduces mouse myeloid abnormal proliferation induced by SHP-2D61G/+ mutation. The molecular mechanism may be associated with reduced binding of SHP-2D61G/+ under Gab2 knockout, and further weakened the activation of downstream signaling pathways of ERK and Akt.     

Panaxadiol saponins induce activation of MAPK/ERK signaling pathway in bone marrow cells of aplastic anemia mice

AIM: To observe the effects of panaxadiol saponins (PDS) on up-regulation of MAPK/ERK signal pathway in bone marrow cells and increase in regulatory T (Treg) cells in spleen tissue of aplastic anemia (AA) mice, and to explore the mechanisms. METHODS: For preparation of immune-mediated AA model, BALB/c mice were exposed to sublethal dose (5.0 Gy) of[⁶⁰Co]-γ radiation, followed by transplantation of lymphocytes from DBA/2 donor mice. BALB/c mice (n=60) were randomly divided into 6 groups, including normal mouse group, AA model group, PDS treatment groups at low, medium and high doses, and cyclosporine group as positive control. PDS and cyclosporine were given by gavage for 14 d. The peripheral blood cell counts and bone marrow pathological examination were tested. The protein levels of MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 in the bone marrow cells were analyzed by Western blot and immunohistochemistry experiment. Flow cytometry was used to detect the proportion of Treg cells in spleen tissue of each group. RESULTS: The peripheral blood cell counts were significantly decreased in AA mouse group as compared with normal mouse group (P<0.05). The bone marrow sections showed markedly inhibition status of hematopoiesis and the decrease in cellularity. In response to PDS treatment, the peripheral blood cell counts and Treg cells in the spleen tissues of AA mouse treated with PDS were significantly increased in a dose-dependent manner (P<0.05). Treatment with PDS at medium and high doses up-regulated the protein levels of MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 in the bone marrow of AA mice (P<0.05). CONCLUSION: PDS is effective to enhance recovery of hematopoietic function in AA mice. This effect may be related to up-regulating multiple protein kinases of MAPK/ERK signal pathway in the bone marrow cells of AA mice. In addition, PDS has an impact on immune function of AA mice.     

Remaining residual bone marrow cells through bone marrow transplantation from female to male in mice

AIM:To investigate the remaining residual bone marrow cells after bone marrow transplantation (BMT) from female to male in mice by detecting the male Y chromosome from the blood cells. METHODS:Bone marrow cells from either male or female C57BL/6 mice were injected via tail vein to the corresponding male or female mice at 1×107 per animal 6 h after irradiation exposure to different doses of ［137Cs］. The survival rate of BMT was calculated after 14 d. The numbers of leukocytes in peripheral blood were measured and Y chromosome levels were also assayed in the male recipent mice. RESULTS:With radiation doses of 1 000 rad and 950 rad, the hematopoietic function of the female recipient mice quickly restored, but the male recipient mice had only 48% survival rate. With the radiation dose of 900 rad, the male recipient mice all survived and their hematopoietic function quickly restored. The peripheral leukocyte counts returned to normal 13 d after BMT. The Y chromosome genes in the peripheral blood cells were detected in 5 weeks after BMT in the male recipient mice, suggesting that the bone marrow cells in the male mice were completely destroyed by radiation, and the bone marrow cells from female mice completely replaced those in the male mice. CONCLUSION: After irradiation at a dose of 900 rad, the male mice can be used as BMT recipients without endogenous bone marrow cells. This study warrants male recipient mouse model in BMT for further investigation on the function of bone marrow cell-specific genes after global gene manipulations of the animals.     

Establishment of a human chronic myeloid leukemia-like mouse model

AIM：To establish a new model of chronic myeloid leukemia (CML) for further studying the pathogenesis mechanisms and discovering new therapeutic targets of this disease. METHODS：The retroviral packaging technique was improved by increasing retroviral titer with higher-quality plasmid, better cell state and appropriate cell seeding density for further studying. Donor BALB/c mice were pretreated with 5-fluorouracil (5-FU), and their bone marrow cells were infected with p210 BCR/ABL retroviral supernatant (BCR/ABL group) or empty retroviral vector (MigR1) supernatant (MigR1 group). Infected bone marrow cells were transplanted into lethally irradiated recipient BALB/c mice via tail vein. The activities of the mice were observed after bone marrow transplantation (BMT). The morphology of peripheral blood cells and bone marrow cells, and the pathological changes of the liver and spleen in dying mice were also determined. RESULTS：Retroviral packaging efficiency was improved by optimizing the experimental conditions with higher-quality plasmid, better cell state and appropriate cell seeding density. All mice in BCR/ABL group died at 19 to 25 d after BMT. Their myelocytes, metamyelocytes and mature granulocytes in peripheral blood and bone marrow were abundant. Hepatosplenomegaly and granulocyte infiltration in the liver and spleen were also observed. All mice in MigR1 group were normal. CONCLUSION: We have successfully set up a mouse model of CML by increasing retroviral titer with improved retroviral packaging technique, transfecting BCR/ABL into mouse bone marrow cells in vitro and transplanting the cells to the recipients of the same lineage.     

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.     

SHP-2 regulates PC12 cell survival and apoptosis in response to NGF via activation of ERK and JNK

AIM: To investigate the central role of the protein tyrosine phosphatase SHP-2 in the survival of PC12 cells upon NGF treatment and apoptosis after NGF withdrawal. METHODS: PC12 cells were treated with SHP-2 inhibitor (NSC87877). Cell survival rate was detected by MTT method and apoptosis was determined by flow cytometry. Moreover, pIRES-GFP (vector alone), pIRES-GFP-SHP-2 (wild type) and pIRES-GFP-SHP-2C459S (dominant negative mutant form) were transfected into PC12 cells. ERK, p-ERK, JNK and p-JNK were immunoblotted at 1 h in the presence of NGF and 5 h after NGF withdrawal. RESULTS: SHP-2 potentially enhanced survival and attenuated apoptosis of PC12 cells. The activation of ERK was significantly enhanced with NGF treatment either in the group without SHP-2 inhibitor or the SHP-2 wild type group. On the other hand, phosphorylation level of JNK was significantly increased after NGF withdrawal when PC12 cells were treated with SHP-2 inhibitor or transfected with SHP-2 mutant. CONCLUSION: SHP-2 may play a central role in mediating the survival and apoptosis of PC12 cells upon NGF exposure and withdrawal. The underlying mechanisms may be through the activation of ERK and JNK.     

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.     

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.     

ROS promotes adhesion of neutrophils to bone marrow stromal cells and its mechanism

AIM:To investigate the effect of reactive oxygen species (ROS) on the adhesion of neutrophils to bone marrow stromal cells (BMSCs) and its mechanism. METHODS:Murine bone marrow neutrophils were isolated from mouse tibia and femur by density gradient centrifugation. HL60 cells were induced into human mature neutrophils (dHL60 cells) by DMSO treatment. Murine bone marrow neutrophils and dHL60 cells were labeled with CFDA-SE. The adhesion of the CFDA-SE-labeled cells to BMSC monolayer was tested by microplate reader after H₂O₂ treatment. The level of glutaredoxin 1 (Grx1) in dHL60 cells infected by lentivirus carrying Grx1 expression vector was examined by fluorescence microscopy and Western blot. The genotype of Grx1^-/- mice was identified by PCR. RESULTS:Diff-Quick staining result displayed that the purity of murine bone marrow neutrophil was higher than 90%. The adhesion of H₂O₂-pretreated neutrophils to BMSCs was higher than that of the control cells (P<0.01). The expression of Grx1 in Grx1 stably transfected dHL60 cells was significantly higher than that in the control cells. After H₂O₂ treatment, the results of in vitro adhesion assay showed that the adhesion of dHL60 cells with Grx1 over-expression to BMSCs was lower than that of the control cells (P<0.01). The results of PCR showed no Grx1 was detected at the whole gene level in Grx1^-/- mice. Compared with the neutrophils from wild-type mice, the neutrophils from Grx1^-/- mice displayed increased adhesion to BMSCs after H₂O₂ treatment. Vascular cell adhesion molecule-1 (VCAM-1) antibody pretreatment induced the adhesion rate back to non-H₂O₂-treated condition. CONCLUSION:ROS promotes the adhesion of neutrophils to BMSCs in bone marrow, which might be regulated by VCAM-1 adhesion signaling-related S-glutathionylation.     

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.     

Expression of Akt and ERK1/2 in human osteoarthritic chondrocytes

AIM: To investigate the expression of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) in normal and osteoarthritic chondrocytes. METHODS: The samples of knee cartilage were obtained from the normal donors (n=5) and the patients (n=18) undergoing total knee arthroplasty with the diagnosis of osteoarthritis (OA). The expression of p-Akt and p- ERK1/2 in the normal and osteoarthritic cartilage tissues was detected by the method of immunohistochemistry. The chondrocytes were isolated and identified by toluidine blue staining and immunohistochemical method. The expression levels of Akt, p-Akt, ERK1/2,p-ERK1/2,phosphorylated 70-kD ribosomal protein S6 kinase(p-p70S6K) and proliferating cell nuclear antigen(PCNA) were tested in normal and osteoarthritic chondrocytes by Western blotting. Real-time fluorescence quantitative PCR was used to measured the expression levels of aggrecan and type II collagen gene in normal and osteoarthritic chondrocytes. RESULTS: The expression of p-Akt in normal cartilage was higher than that in OA cartilage. The expression of p- ERK1/2 in OA cartilage was higher than that in normal cartilage. Compared with the normal chondrocytes, the expression of p-Akt and p-p70S6K, and the mRNA levels of aggrecan and type II collagen were increased (P<0.05), and the expression of p-ERK1/2 and PCNA was decreased in OA chondrocytes (P<0.05). CONCLUSION: Akt might regulate aggrecan and type II collagen synthesis via p-p70S6K, and ERK1/2 might regulate OA chondrocyte proliferation through PCNA. Both Akt and ERK1/2 play important roles in the pathogenesis of OA.     

Anti-aging roles of Human Umbilical Cord Mesenchymal Stem Cell in hypoxic conditioned-medium on the mouse hematopoietic system

AIM：To explore Anti-aging roles of human umbilical cord mesenchymal stem cells (hUC-MSC) in hypoxic conditioned-medium on the natural senescence of hematopoietic system in aging mice. METHODS：The 6 month old Balb / c mice were randomly divided into experimental and control groups. Experimental group were injected with primary culture medium of hUC-MSC, 1 per 4 days, continuous infusion of 6 months; while control group were injected with medium. After treated with hUC-MSC primary culture medium, observe the mice’s general situation had been taken for 6 months. On the third month and sixth month, compared the two groups on peripheral blood (WBC, Hb), bone marrow nucleated cell count (BMNC), hematopoietic progenitor cell colony culture (CFU-GM, CFU-E, CFU-MK), fibroblast colony culture (CEU-F), and exogenous spleen colony-forming units count (CFU-S). Bone marrow biopsy, immunohistochemistry detection of P16 expression, and the capability of hematopoietic reconstruction of bone marrow cell were compared only on the sixth month. RESULTS ：The general physical status of experimental group was better than that of control group. Comparing with the control group on BMNC, CFU-GM and CFU-MK, the data were much higher in the experimental group on the third month, with no significant difference on the peripheral blood, CFU-F, CFU-E and CFU-S; on the sixth month, all the indexes mentioned above were higher than control group with exception of peripheral blood. All the indexes had a similar descending-tendency during six months, but the tendency of experimental group was much slower than that of control group with significant difference (p<0.05). Experimental group had more hematopoietic tissue but less adipose tissue than that of control group from bone marrow biopsy, had less expression of P16 but positive hUC-MSC implantation. The capability of hematopoietic reconstruction of bone marrow cell from experimental group was better than that of control group. CONCLUSION：The cytokines from human umbilical cord mesenchymal stem cells (hUC-MSC) could slow down the natural recession of hematopoietic system in aging mice, which may be considered to be a potential anti-aging research strategy in the future.     

Role of P-selectin in intestinal tumorigenesis in ApcMin/+ mice

AIM: To investigate the role of P-selectin in intestinal tumorigenesis in Apc^Min/+ mice (C57BL/6J-Apc^Min/J). METHODS: Female P-selectin knockout mice (B6.129S7-Selp^tm1Bay /J, that was P-selectin ^-/-) were mated with male Apc^Min/+ mice. The offspring was genotyped for Min ^+/- and P-selectin null mutantions, which were Apc^Min/+ P-selectin ^-/- mice. The tumor number and gross tumor volume in the small and large intestines of the Apc^Min/+ P-selectin ^-/- mice and Apc^Min/+ mice were determined. RESULTS: P-selectin deficiency in Apc^Min/+ mice resulted in significant decreases in the tumor number and gross tumor volume in the small intestine and total intestine. CONCLUSION: Deletion of P-selectin significantly inhibits the tumorigenesis in mouse intestines. In addition, the results also suggest that P-selectin may be a marker for colon cancer.     

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.     

Molecular mechanism of BMSC intracerebral transplantation in improving learning and memory abilities of AD mice

AIM: To investigate the effect of bone marrow mesenchymal stem cell (BMSC) transplantation on learning and memory abilities and pathological changes of Alzheimer disease (AD) mice and the molecular mechanisms. METHODS: C57/BL6 wild-type (WT) and transgenic (Tg) mice were randomly divided into 4 groups:WT/PBS group, WT/BMSCs group, Tg/PBS group and Tg/BMSCs group. The mice were administered with PBS or BMSCs via intracerebroventricular injection. Spatial learning and memory abilities of the mice were evaluated by Morris water maze test on the 3rd day after surgery. Real-time PCR was applied to detect the mRNA expression of CX3C chemokine ligand 1 (CX3CL1), CX3C chemokine receptor 1 (CX3CR1), IL-1β, TNF-α, Nurr1, YM1, insulin-degrading enzyme (IDE) and matrix metalloproteinase 9 (MMP9). The protein levels of CX3CL1 and Aβ42 were measured by ELISA. Western blot was used to detect the protein expression of postsynaptic density protein 95 (PSD95) and synaptophysin (SYP). RESULTS: The transplanted BMSCs were observed near the hippocampus of APP/PS1 mice on the 10th postoperative day. The escape latency of the mice in Tg/PBS group was significantly longer than that in the WT/PBS mice (P<0.05). Compared with Tg/PBS group, the escape latency of Tg/BMSCs group was significantly shorter (P<0.05), and the mRNA and protein levels of CX3CL1 in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.01). The results of immunohistofluorescence staining showed that BMSC transplantation promoted the activation of microglia in the brain of WT and Tg mice. The mRNA expression of YM1 was up-regulated in WT/BMSCs group and Tg/BMSCs group (P<0.05). Compared with WT/PBS mice, the mRNA expression of TNF-α in the cortex and hippocampus of Tg/PBS group was significantly increased (P<0.05), and the mRNA expression of Nurr1 in the cortex was significantly decreased (P<0.01). Meanwhile, the mRNA expression of TNF-α in the cortex of Tg/BMSCs mice was decreased (P<0.01) and the mRNA expression of CX3CR1 and Nurr1 was up-regulated compared with Tg/PBS group (P<0.05). The results of Western blot showed that the protein levels of PSD95, p85, p110 and p-Akt in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.05). Finally, BMSC transplantation reduced the protein level of Aβ42 in APP/PS1 mice (P<0.05), and increased the mRNA expression of IDE and MMP9 in the hippocampus (P<0.05). CONCLUSION: BMSC transplantation modulates neuroinflammatory responses and promotes neuroprotective factor and synaptic protein expression, thus improving the learning and memory abilities in the APP/PS1 mice, which may be achieved by up-regulating the expression of CX3CL1.     

High expression of PDX-1 in bone marrow mesenchymal stem cells mediated by superfect

AIM: To construct a eukaryotic expression vector containing pancreatic duodenal homebox-1 (PDX-1) and to elevate the expression efficiency of exogenous gene in rat bone marrow mesenchymal stem cells (MSCs). METHODS: Recombinant vector containing PDX-1 was constructed. Flow cytometry was used to identify the cell cycle of bone marrow mesenchymal stem cells (MSCs) cultured in vitro. Recombinant vector containing PDX-1 was transfected into bone marrow MSCs using superfect in medium. After being selected by G418, RT-PCR and Western blotting were used to investigate the expression of PDX-1 in MSCs. RESULTS: Restricted enzyme analysis and sequencing showed that PDX-1 gene segment was consistent with that in GenBank. Flow cytometry showed that there were about 85.9% cells at the cell cycle of G0/G1. The whole cells transfected emitted green fluorescence under flow cytometry. The efficiency of transfection was above 40%. RT-PCR and Western blotting demonstrated that there was expression of PDX-1 in transfected bone marrow MSCs. CONCLUSION: Recombinant vector containing PDX-1 was constructed successfully. Superfect mediated expression of exogenous gene in bone marrow MSCs in a high efficiency, and bone marrow MSCs containing exogenous gene are an ideal cells for gene therapy.     

SUMO-specific protease 3 aggravates CaPO4-induced mouse abdominal aortic aneurysm formation by promoting macrophage polarization

AIMTo investigate the role of SUMO-specific protease 3 (SENP3) in macrophage polarization and calcium phosphate (CaPO₄)-induced abdominal aortic aneurysm (AAA) formation in mice. METHODS（1） Bone marrow-derived monocytes (BMDMs) in Senp3^flox/flox (wild-type, WT) mice and Senp3^flox/flox; Lyz2-Cre (monocyte-specific SENP3 knockout, i.e. conditioned knockout, cKO) mice were isolated and induced for M1 and M2 polarization. The mRNA and protein expression level of SENP3 were detected by RT-qPCR, Western blot and immunocytofluorescence, and the differential distribution of M1/M2 BMDMs from WT and cKO mice was analyzed. （2） CaPO₄ was administrated to induce AAA model in 8~12-week-old male WT and cKO mice. The AAA incidence, survival rate and maximal aortic diameter were analyzed between the 2 groups. Aortic aneurysm tissues were collected for pathological analysis, and the expression levels of SENP3, interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), IL-6 and matrix metalloproteinases-9 (MMP-9) were measured by RT-qPCR and Western blot. Dihydroethidium staining in situ in frozen sections was used to analyze the production of reactive oxygen species (ROS). （3） To explore the potential mechanisms, Western blot and co-immunoprecipitation were used to verify the de-SUMO modification of mitogen-activated protein kinase kinase 7 (MKK7) induced by SENP3. Besides, BMDMs were transfected with Flag-MKK7 wild type (Flag-MKK7 WT) and SUMO-modified site K18 mutant (Flag-MKK7 K18R mutant), and then M1 polarization of the cells was induced. The protein levels of p-JNK and MMP-9 in the 2 groups were determined by Western blot. RESULTS(1) SENP3 expression was up-regulated in M1 polarized macrophages (P<0.01), but was down-regulated in M2 polarized macrophages (P<0.01). The expression of SENP3 was decreased during the transformation of M1 to M2 in the macrophages (P<0.01), but was significantly up-regulated during the opposite process (P<0.01). Besides, more M1 macrophages and less M2 macrophages after induction were observed in the BMDMs from cKO mice than those from WT mice. (2) SENP3 expression was up-regulated in AAA tissues (P<0.05). The AAA incidence of cKO mice was significantly reduced after CaPO₄ induction (P<0.01), the survival rate was significantly improved (P<0.05), and maximal aortic diameter was significantly reduced in cKO group (P<0.01). The levels of IL-1β, IL-6 and TNFα, and the production of ROS were significantly down-regulated (P<0.01), meanwhile MMP-9 expression was also down-regulated in cKO mice (P<0.05). (3) the SUMO2/3 modification of MKK7 was reduced during M1 polarization, and MKK7 interaction with SENP3 was enhanced. Significantly up-regulated protein level of p-JNK and MMP-9 were verified in the M1 macrophages transfected with Flag-MKK7 K18R mutant (P<0.05). CONCLUSION SENP3 activates the MAPK/JNK pathway via de-SUMOylation of MKK7, regulates the M1/M2 polarization of macrophages and promotes the protein level of MMP-9, thus aggravating AAA formation.     

Effect of ligustrazine on expression of LFA-1 and ICAM-1 in bone marrow of radiation injured mice

AIM: To investigate the effect of ligustrazine on the expression of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) in bone marrow and on the mechanism of hematopoietic reconstitution in radiation injured mice.METHODS: The 24 mice (clean class) were randomly divided into 3 groups: normal group, radiation injured group and ligustrazine group. After irradiation by 6.0Gy ［⁶⁰Co］ γ-ray, the radiation injured animals were given normal saline (0.2 mL, twice a day) through gastric tube, while the ligustrazine group was given ligustrazine through gastric tube (0.2 mL, twice a day). The mice in normal group received no treatment. At the 7th, 14th, 21st day after irradiation, the femur were taken and the bone marrow mononuclear cells (BMNCs) suspension were made to culture bone marrow stromal cells (BMSCs). The mRNA and protein expressions of ICAM in BMSCs were assayed by RT-PCR and Western blotting. The expression levels of LFA-1 in BMNCs were evaluated by flow cytometry analysis.RESULTS: In ligustrazine group the expression levels of LFA-1 at the 7th, 14th and 21st days after irradiation were higher than those in radiation injured group (P<0.01 or P<0.05). However, the expression level of ICAM-1 was lower than that in the compared group (P<0.01 or P<0.05).CONCLUSION: Ligustrazine can increase the LFA-1 expression level of BMNCs, decrease the ICAM-1 expression level in BMSCs, indicating that ligustrizine promotes the recovery of hematopoietic cells in bone marrow, then improves the bone marrow microenvironment and enhances hematopoietic reconstitution.     

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 bradykinin on proliferation of porcine pulmonary artery smooth muscle cells induced by TGF-β1

AIM: To investigate the effects of bradykinin (BK) on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by transforming growth factor beta 1 (TGF-β1) and its possible mechanisms. METHODS: Primary porcine PASMCs were isolated, cultured and identified, and the cells at passages 2~6 were used in this study. The viability of PASMCs was determined by Cell Counting Kit-8 assay. The protein expression of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was detected by Western blotting. RESULTS: TGF-β1 promoted the proliferation of PASMCs in a dose-dependent manner (P<005). BK significantly inhibited the proliferation of PASMCs induced by TGF-β1 (P<005), and attenuated the elevated expression of PI3K, p-Akt and p-ERK1/2 proteins (P<005). HOE-140, a BK type 2 receptor (B2R) inhibitor, reversed the effects of BK (P<005). CONCLUSION: BK inhibits TGF-β1-induced proliferation of PASMCs, which may be associated with inactivation of PI3K/Akt and ERK1/2 signaling pathways.