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ABSTRACT: The aim of this study was to investigate the effects of interferon (IFN)-γ on biological characteristics and immunomodulatory property of human umbilical cord-derived mesenchymal stem cells (hUC-MSC). hUC-MSC were treated with IFN-γ 10 ng/ml (IFN-γ group) or without IFN-γ (control group). The phenotype of hUC-MSC was detected by flow cytometry. The proliferation status was detected by CCK-8 method, and its differentiation ability was assessed by oil red O and von Kossa staining. The production of PGE-2 was measured by ELISA, and the mRNA expression levels of COX-2, IDO-1 and IDO-2 in hUC-MSC were detected by real-time quantitative PCR. Furthermore, the proliferation of human peripheral blood mononuclear cells (hPBMNC) was evaluated after co-culture with hUC-MSC, IFN-γ pretreatment or not. The results showed that after IFN-γ stimulation, the expression of SSEA-4 on hUC-MSC decreased significantly [(8.15 ± 2.94) vs (16.42 ± 8.5), P < 0.05], and the expression of CD54 increased [(96.64 ± 3.29) vs (84.12 ± 10.73), P = 0.051]. The immunomodulatory property of hUC-MSC on the proliferation of hPBMNC was enhanced (P < 0.05). All the above mentioned effects were IFN-γ concentration-dependent. When hUC-MSC were stimulated by IFN-γ for 24 h, the production of PGE-2 secreted by hUC-MSC decreased significantly (P < 0.01). The mRNA expression level of COX-2 also decreased though the difference did not reach to statistically significant level. Compared with control group, IDO-1 expression level in IFN-γ group increased significantly (P < 0.01), and the mRNA expression level of IDO-2 remained unchanged. It is concluded that IFN-γ can influence the phenotype of hUC-MSC and enhance the immunomodulatory property of hUC-MSC.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 03/2012; 20(2):421-6.
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ABSTRACT: To explore the role of CD72 in the pathogenesis of immune thrombocytopenia (ITP), we detected CD72, Sema4D, IL-2, IL-4, and IFN-γ mRNA expressions and the levels of plasma Sema4D, IL-2, IL-4, IL-6, and IFN-γ in ITP patients (n = 39) and controls (n = 23). The levels of plasma IL-2, IL-4, and IL-6 were assayed by radioimmunoassay, and the levels of plasma IFN-γ and Sema4D were analyzed by enzyme-linked immunosorbent assay. Sema4D, CD72, IL-2, IFN-γ, and IL-4mRNA expressions were analyzed by real-time quantitative reverse-transcription polymerase chain reaction. The expression of CD72 mRNA in ITP patients (n = 23) with active disease was significantly lower than that in patients in remission (p = 0.029) (n = 16) and controls (p = 0.0296) (n = 23). The IFN-γ/IL-4 mRNA (Th1/Th2) expression in ITP patients with active disease and in remission was significantly higher than that in controls (p = 0.0023, p = 0.0125, respectively). The expression of IL-2 mRNA in ITP patients with active disease was significantly lower than that in patients in remission (p = 0.0418) and controls (p = 0.004). The level of plasma IL-2 in ITP patients with active disease was significantly lower than that in patients in remission (p = 0.0029) and controls (p = 0.0101). The levels of plasma IL-4 in ITP patients with active disease and in remission were significantly higher than that of controls (p = 0.0093, p = 0.0053, respectively). CD72 mRNA expression level might correlate with Sema4D mRNA expression in peripheral blood mononuclear cells and level of plasma IL-2 in active ITP patients (p = 0.024 and p = 0.036). Our findings suggest that CD72 might be involved in the pathophysiological process of the ITP disease by increasing B-cell receptor signals.
Platelets 11/2011; · 1.85 Impact Factor
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ABSTRACT: Hematopoietic stem cells (HSC) shift from fetal liver and spleen to bone marrow at neonatal stages and this movement may be due to inductive signals from different microenviroments. Mesenchymal stem cells (MSC) are the precursors of stromal cells in bone marrow microenviroments such as osteoblasts and endothelial cells. Some researchers speculated that fetal bone marrow before birth might be not perfectly suit HSC growth. However, it is still lack of direct evidence to prove this hypothesis. This study was aimed to compare the hematopoietic supportive capacity between human fetal and adult bone marrow MSC in vitro. Adult bone marrow MSC (ABM-MSC) were isolated from three healthy donors and fetal bone marrow MSC (FBM-MSC) were isolated from three fetuses between gestations of 19 to 20 weeks. After irradiation, MSC were co-cultured with CD34(+) cells isolated from umbilical cord blood in long-term culture-initiating cell (LTC-IC) assay. The colony number of colony forming cells (CFC) was counted and the phenotypic changes of co-cultured CD34(+) cells were analyzed by flow cytometry. Cytokine expressions in both kinds of MSC were detected by reverse transcription polymerase chain reaction (RT-PCR). The results showed that ABM-MSC had a stronger hematopoietic supportive capacity than FBM-MSC. Both of them enhanced the differentiation of CD34(+) cells into myeloid lineages. Cytokines were expressed differently in ABM-MSC and FBM-MSC. It is concluded that ABM-MSC possess more potential application in some treatments than FBM-MSC, especially in hematopoietic reconstitution.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 07/2011; 19(4):1028-32.
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ABSTRACT: To explore the effects of stem cell factor (SCF) on proliferation, transmigration, capillary tube formation of human umbilical vein endothelial cells (HUVEC) and on the chemotaxis of CD133(+) cells.
In the presence of blank control, SCF, vascular endothelial growth factor (VEGF), anti-human SCF (anti-SCF) or human IgG, the difference in proliferation capacity of HUVEC was analyzed by MTT and CCK-8 methods, and wound scratch assay and three-dimensional in vitro Matrigel assay were used for transmigration and capillary tube formation of HUVEC, respectively. In addition, the chemotaxis of CD133(+) cells sorted from human umbilical cord blood by flow cytometry was investigated by Transwell migration assay.
SCF didn't improve the proliferative capacity of HUVEC, but significantly enhanced the transmigration capacity, and increased capillary tube formation in a dose-dependent manner. The number of intact tubules [(30.0 ± 3.4)/10(5) HUVEC] formed by HUVECs in the presence of the optimal concentration of SCF (100 ng/ml) was remarkably higher than that in blank control group [(5.0 ± 2.6)/10(5) HUVEC, P < 0.01]. SCF also significantly induced a chemotactic response of CD133(+) cells, the transmembrane migration cell number into Transwell lower chamber was significantly higher in SCF group [(118.0 ± 6.5)/10(4) CD133(+) cells] than in blank control group [(47.0 ± 4.7)/10(4) CD133(+) cells, P < 0.01 ].
SCF significantly promotes the transmigration and capillary tube formation of HUVEC, and induces a chemotactic response of CD133(+) cells. SCF/c-kit signaling possibly plays a critical role in regulating angiogenesis of vascular endothelial cells and vasculogenesis of endothelial progenitor cells.
Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 05/2011; 32(5):326-30.
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ABSTRACT: This study was aimed to investigate whether mesenchymal stem cells (MSC) can be isolated from bone marrow filters which have always been discarded. The bone marrow (BM) particles from BM filters of 2 healthy donors were cultivated by primary explant culture. After expansion, the number of MSC was counted and their immunophenotype and differentiation potential were detected. The results indicated that many MSC were found in bone marrow particles from filters, and nearly 10(7) MSC were obtained at 3 passages of expansion. They not only possessed the characteristics of morphology and immunophenotype of MSC, but also could differentiate into osteoblasts, chondrocytes and adipocytes. In conclusion, a large amount of MSC can be obtained from BM filters if the BM particles were cultivated by primary explant culture.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 03/2011; 19(2):459-63.
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Wen Xing,
Peng-Xia Liu,
Meng Liu,
Shao-Guang Yang,
Qin-Jun Zhao,
Jian-Ping Li, Shi-Hong Lu,
Hong-Ying Ren,
Ying Huang,
Hao Wu,
Bin Liu,
Lei Zhang,
Zhong-Chao Han
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ABSTRACT: Bone marrow (BM) is the major source of mesenchymal stem cells (MSC). In most experiments, MSC were classically cultured from mononuclear cells (MNC) isolated by density gradient centrifugation method. However, several studies have demonstrated that this method was less efficient for MSC recovery. This study was aimed to investigate whether BM particles were the cause resulting in less efficiency of this method and how to isolate them. A total of 20 patients were enrolled in this study. MNC were cultured by standard adherence and BM particles were cultivated by primary explant culture. For BM from patients 1-10, MNC were first isolated and BM particles were then filtered out. The morphology and the fibroblastic colony number were compared between cultures of MNC and BM particles. For BM from patients 11-20, MNC isolation and BM particle filtration were processed in opposite order, then the immunophenotype and function between adherent cells expanded from MNC and BM particles were compared. In addition, for patients 11-20, the left BM aspirates were cultured too after BM particles and MNC were isolated separately. The results showed that adherent cells from BM particles were MSC. After BM particles were filtered out and cultured separately, MSC could be recovered completely from MNC isolated by density gradient centrifugation and no MSC were left in the residual BM aspirates. BM particles, which have been mostly discarded by the method of density gradient centrifugation, are another important source of MSC and they can be cultivated reliably by primary explant culture. It is concluded that more MSC are recovered from a single BM sample by culturing BM particles and MNC separately.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 11/2010; 18(6):1552-9.
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ABSTRACT: This study was aimed to construct an adenovirus hybrid system with high transduction efficiency and site-specific integration. By a series of DNA manipulation, a hybrid system of two adenovirus vectors was constructed. One vector contains loxP-flanked transgene expression cassette, in which there are hFIX and DsRed coding sequences and attB for phiC31 recolonization. The other vector carries Cre and phiC31 gene. Vectors only expressing Cre or phiC31 were used as controls. 293A cells were constructed and transfected with the adenoviral vectors by Lipofectamine 2000, and the expression of target genes was identified by fluorescence microscopy and RT-PCR. The results showed that after being identified by PCR, restriction analysis and sequencing, an adeno-integrase hybrid system was successfully constructed. The system expressed RFP, GFP, hFIX, Cre and phiC31 in 293A cells in vitro. It is concluded that the adeno-integrase hybrid system is successfully constructed, which lays a good foundation for further investigation of its therapeutic application.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 10/2010; 18(5):1229-34.
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Hao Wu,
Feng-Xia Ma,
Jian-Ping Li,
Shao-Guang Yang,
Hai-Rong Jia, Shi-Hong Lu,
Qian Ren,
Qin-Jun Zhao,
Wen Xing,
Lei Zhang,
Zhong-Chao Han
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ABSTRACT: This study was aimed to investigate the enhancement of all-trans retinoic acid-induced HL-60 leukemia cell differentiation by human umbilical cord mesenchymal stem cells (hucMSC). The HL-60 cells were divided into 4 groups: control group (HL-60 cells treated without ATRA), hucMSC group (HL-60 cells co-cultured with hucMSCs), ATRA group (HL-60 cells treated with ATRA) and ATRA + hucMSC group (HL-60 cells treated with ATRA and co-cultured with hucMSCs). The proliferations of control group and hucMSC group were compared by Cell Counting Kit-8 (CCK8). The morphology of HL-60 cells and NBT positive rate in 4 groups were observed and compared by means of microscopy, the c-myc expression of HL-60 cells in different groups was evaluated by real-time PCR, and the CD11b expression on HL-60 cells in different groups were detected by flow cytometry. The results showed that in the co-culturing system, hucMSCs could inhibit the proliferation of HL-60 (hucMSC:HL-60 is 1:1, 48 hours p < 0.05, 72 hours p < 0.01; hucMSC:HL-60 is 1:5, 72 hours p < 0.05). In condition of stimulation with 2 micromol/L ATRA, the neutrophil like HL-60 cells and NBT positive rate in ATRA + hucMSC group were higher than those in ATRA group (p < 0.05). The c-myc expression of HL-60 cells in ATRA + hucMSC group was lower than that in ATRA group (p < 0.05). Furthermore, HL-60 cells in ATRA + hucMSC group had stronger CD11b expression than ATRA group (48 hours p < 0.05, 72 hours p < 0.01). It is concluded that hucMSC not only can inhibit the proliferation of HL-60 cells, but also can enhance the differentiation effect of HL-60 cells induced by ATRA.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 08/2010; 18(4):877-81.
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ABSTRACT: OBJECTIVE: To investigate whether the plasmid bearing attB and human coagulation factor IX (hFIX) coding sequence could insert into hemophilia B mice genome and persistently express hFIX with co-injected integrase. METHODS: The plasmid attB-hFIX-pIRES2-EGFP was constructed, which bore attB site and hFIX coding sequence and was proved in vitro to express hFIX. The plasmid and CMV-int expressing integrase was co-infused rapidly in a large-volume solution through tail vein of hemophilia B mice. Mice infused with the plasmid alone served as controls. ELISA was performed to determine serum hFIX level. Correction of coagulation defect in vivo by plasmid infusion was assessed by bleeding time. Genomic integration of the plasmid was determined by nested PCR. RESULTS: The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed. The hemophilia B mice produced (1533 ± 239) ng/ml hFIX at 24 hour after infusion of the hFIX encoding plasmid and the bleeding diathesis of the hemophilia B mice was significantly corrected as measured by clotting assays. However, whether or not co-injected with CMV-int, the serum hFIX level decreased to background level in 10 days after infusion. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in the mouse liver chromosomes. CONCLUSION: Integrase phiC31 can catalyze recombination of 34 bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed after infusion, but rapidly silenced in vivo.
Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 05/2010; 31(5):294-299.
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Jian-Ping Li,
Shao-Guang Yang,
Chun-Lan Dong,
Hao Wu,
Hai-Rong Jia,
Yan-Jin Zhao,
Tong Wang, Shi-Hong Lu,
Qian Ren,
Qin-Jun Zhao,
Wen Xing,
Lei Zhang,
Zhong-Chao Han
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ABSTRACT: The present study was aimed to investigate the pathways, by which IL-27 regulates the expression of adherent molecule Mac-1, chemotactic factor receptor fMLP-R and pro-inflammatory cytokine IL-1beta in human neutrophils. Highly purified human neutrophils were isolated from peripheral blood using Ficoll-Hypaque gradients centrifugation and erythrocyte lysis. The mRNA expression of IL-27 receptor components (WSX-1/TCCR and gp130) in human neutrophils was detected by reverse transcription polymerase chain reaction (RT-PCR). After incubation with IL-27 and specific inhibitors (p38 MAPK inhibitor SB203580, PI3K inhibitor LY294002 and ERK inhibitor U0126), the mRNA levels of fMLP-R and IL-1beta were determined by real time RT-PCR, and the adherent molecule Mac-1 expression in human neutrophils was determined by flow cytometry. The IL-1beta level in culture supernatant of human neutrophils was assayed by radioimmunoassay. The results showed that IL-27 receptor components (WSX-1/TCCR and gp130) were constitutively expressed in human neutrophils. IL-27 down-regulated Mac-1 expression in human neutrophils (p<0.05). After incubation with specific inhibitors, SB203580, not LY294002 and U0126, inhibited the down-regulation of Mac-1 expression by IL-27. However, IL-27 up-regulated the mRNA expression of fMLP-R and IL-1beta, and increased the release of IL-1beta (p<0.05). Interestingly, LY294002, not SB203580 and U0126, inhibited the up-regulation of fMLP-R and IL-1beta by IL-27. It is concluded that the IL-27 may regulate the expression of Mac-1, fMLP-R and IL-1beta in human neutrophils through p38 MAPK and PI3K signal pathways.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 04/2010; 18(2):391-5.
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ABSTRACT: To investigate the biological function of hepatocyte-like cells derived from mesenchymal stem cells that isolated from human umbilical cord UC-MSCs in vitro, and to detect the changes in the immunogenicity of the differentiated hepatocyte-like cells (DHC).
Transdifferentiation of UC-MSCs into hepatic lineage in vitro was induced in modified two-step induction medium. The expressions of hepatic specific markers were detected by RT-PCR analysis and immunofluorescence staining at different time points after induction. The levels of albumin and urea in the supernatants of cultures were measured by enzyme-linked immunosorbent assay. Furthermore, the immunosuppressive property of DHC was detected by one-way mixed lymphocyte culture.
The mRNA and proteins of alpha fetoprotein (AFP), albumin (ALB),and cytokeratin-19 (CK-19) were expressed in naive UC-MSCs at low levels. DHC highly expressed hepatic markers AFP, ALB, CK-19, and tryptophan 2, 3-dioxygenase 14 and 28 days after hepatic differentiation and were accompanied by an increased production of ALB and urea in supernatant in a time-dependent manner. DHC did not express human leukocyte antigen DR antigen and significantly decreased the lymphocyte proliferation.
UC-MSCs are able to differentiate into functional hepatocyte-like cells in vitro, while the immunogenicity of DHC remains low.
Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae 04/2010; 32(2):190-4.
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ABSTRACT: Batroxobin, a thrombin-like enzyme from Bothrops atrox moojeni venom, is associated with the reduction of fibrinogen levels in plasma and the enhancement of anticoagulation and fibrinolysis. In this study, 15 patients with deep vein thrombosis (DVT) achieved successful limb salvage after the administration of batroxobin. We found that the levels of CD34+, CD31+, CD34+/CD31+, and vascular endothelial cadherin (VE-cadherin+) cells had increased in the peripheral blood of patients at 7 days and 14 days after treatment. At 0 day, 7 days, and 14 days, the percentages of CD34+ cells, which are assumed to be hematopoietic stem cells, are 0.39% ± 0.43%, 0.71% ± 0.50%, and 1.11% ± 0.66%, respectively. The levels of CD34+ cells at 14 days are significantly higher than the levels on the first day (P = .004). The levels of CD31+ cells and VE-cadherin+ cells, which represent mature endothelial cells, at 7 days (34.15% ± 11.32%, P = .013; 1.25% ± 1.39%, P = .014) and 14 days (35.21% ± 7.66%, P = .071; 1.85% ± 2.60%, P = .117) were slightly elevated compared with those at 0 day (27.55% ± 8.65%; 0.25 ± 0.39%). The double positive of CD34 and CD31 cells are assumed to be endothelial progenitor cells (EPCs). The levels of CD34+/CD31+ cells at 7 days (0.69% ± 0.50%, P = .001) and 14 days (1.07% ± 0.66%, P = .006) are significantly higher than that on the initial day (0.28% ± 0.30%). The number of CD34+/CD31+ cells significantly increased, indicating that in addition to its role in anticoagulation and fibrinolysis, treatment with batroxobin might simultaneously activate circulating EPCs that might promote the recanalization of the damaged vessel wall.
Clinical and Applied Thrombosis/Hemostasis 10/2009; 17(1):75-9. · 1.33 Impact Factor
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ABSTRACT: The present study was aimed to isolate and identify human mesenchymal stem cells from adult bone marrow (BM-MSC) and umbilical cord (UC-MSC), and to compare their ability to support in vitro long-term hematopoiesis. MSC from bone marrow and umbilical cord were isolated by using density gradient centrifugation or enzyme digestion. MSC were further purified by adherent culture. Immunophenotype, adipogenic and osteogenic differentiation potential of BM-MSC and UC-MSC were detected. The hematopoietic supporting capacity of BM-MSC and UC-MSC was assessed by LTC-IC assay. Nonadherent cells in each group were collected for phenotypic analysis at 3, 5 and 7th week of culture. The results showed that BM-MSC and UC-MSC in culture shared a similar spindle-shaped morphology and adhered to the tissue culture substrate. They were both positive for CD90, CD105, CD73, CD29, CD54, CD166, HLA-ABC, and negative for HLA-DR, CD34 and CD45. BM-MSC and UC-MSC could differentiate into adipocytes or osteoblasts confirmed by oil red O staining and von Kossa staining, separately. LTC-IC assay showed that at 5th week of culture, the difference of the CFC yields between UC-MSC group and BM-MSC group was not statistically significant (p>0.05). At 6, 7, 9th week of culture, the CFC yields in the UC-MSC group were lower than those of BM-MSC (p<0.05). The phenotypic analysis of nonadherent cells at 3, 5, 7th week of culture indicated that along with prolongation of time, the percentages of CD34+ cells and CD117+ cells in each group decreased markedly, and the percentages of CD33+ cells, CD13+ cells and CD11b+ cells increased gradually. It is concluded that MSC from human adult bone marrow and umbilical cord can be successfully isolated and identified. UC-MSC are able to support long-term hematopoiesis in vitro, but its hematopoietic supportive capacity is weaker than those of BM-MSC.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 10/2009; 17(5):1294-300.
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ABSTRACT: The purpose of this study was to explore the effect of intra-bone marrow infusion (iBMI) of cord blood (CB)-derived hematopoietic stem/progenitor cells (HS/PCs) on human hematopoietic reconstitution in xenotransplanted NOD-SCID mouse model. Aliquots containing 5 x 10(5) CB CD34(+) cells were transplanted into sublethally irradiated NOD-SCID mouse via intravenous infusion (iVI) or iBMI routes. 64 female NOD-SCID mice were divided randomly into 3 groups: iBMI group, iVI group and negative control group. The engraftment levels of human hematopoietic cells at 3, 5 and 8 weeks after xenotransplantation were detected by fluorescence-activated cell sorter (FACS), polymerase chain reaction (PCR), immunohistochemistry and HPC colony formation assay, and long-term hematopoietic reconstitution capacity of HSC was tested by secondary transplantation. The results showed that the percentages of human CD45(+) cells in bone marrow, peripheral blood and spleen of recipient in iBMI group at 8 weeks after xenotransplantation were significantly higher than those in iVI group (p < 0.05). HS/PCs given through both iVI and iBMI methods had the ability of multilineage differentiation, the percentages of CD45(+)CD19(+) cells, CD45(+)CD33(+) cells, CD45(+)CD56(+) cells and CD45(+)CD34(+) cells of recipient bone marrow in iBMI group at 8 weeks after xenotransplantation were significantly higher than those in iVI group (p < 0.05), while other lineages in iBMI group were also higher than that in iVI group (p > 0.05). alpha-satellite-specific fragment of human chromosome 17 could be detected by PCR in liver, spleen, lung, peripheral blood and bone marrow cells of long-term survival recipients in both iVI and iBMI groups. Human CD45 antigen could be detected by immunohistochemical method in spleen, liver and lung of recipients in iBMI group at 8 weeks after xenotransplantation. Total colony count in iBMI group at 8 weeks after xenotransplantation was significantly higher than that in iVI group (p < 0.05). alpha-satellite-specific fragment of human chromosome 17 could be detected in all above organs of both group recipients at 6 weeks after secondary xenotransplantation. It is concluded that iBMI of CB CD34(+) cells improves hematopoietic reconstitution in xenotransplanted NOD-SCID mouse model.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 08/2009; 17(4):1010-5.
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ABSTRACT: This study was aimed to examine the expression and promoter CpG island methylation of homeobox B4 (HOXB4) gene in CD34(+) cells from cord blood and peripheral blood mononuclear cells (PBMNCs) from health adult, and to investigate the expression level of HOXB4 in these two cells and its relationship with the promoter methylation. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression of HOXB4 in CD34(+) cells and PBMNCs, and bisulfite sequencing technique was used to detect the methylation status of the promoter CpG sites of HOXB4 gene in CD34(+) cells and PBMNCs. The results indicated that highly expressed HOXB4 and unmethylation of HOXB4 promoter CpG island occurred in CD34(+) cells. However, loss of HOXB4 expression and the methylated CpG island of HOXB4 were observed in PBMNCs, and the methylated C residue was positioned at -129 bp in the upstream of ATG. It is concluded that the methylation status of HOXB4 gene promoter may be one negative regulatory mechanism for HOXB4 gene expression. The unmethylation of CpG island in the promoter region of HOXB4 gene may be correlated with the high expression of HOXB4 gene in CD34(+) cells, while the promoter methylation of HOXB4 gene may be associated with HOXB4 gene silencing in PBMNCs. The preliminary identification of HOXB4 promoter methylation site would provide a basis for further study and a novel approach to expand hematopoietic progenitor cells.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 07/2009; 17(3):674-8.
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ABSTRACT: This study was aimed to design and screen short hairpin RNA (shRNA) molecules targeting multidrug resistance gene (mdr1), as well as to investigate the effects of shRNA expression vector on K562/A02 cells. Mdr1-shRNA expression vector was transfected into K562/A02 cells by lipofectamine 2000, and G418 was added to screen and establish the stable expression cell strain. The expressions of mdr1 mRNA and protein were detected by real-time RT-PCR and Western blot respectively. The sensitivity of cells to chemodrugs after interference were tested by CCK8 assay. The function of p-glycoprotein was determined by Rhodamine 123 efflux experiment. The results showed that all of 4 mdr1-shRNA expression vectors could significantly knockdown the expression of p-glycoprotein as compared with control vector, moreover, the vector targeting 508 - 526 sites of mdr1 gene was the best one. It is concluded that the mdr1-shRNA expression vector gained by screening can significantly knockdown the expression of mdr1 gene and reverse leukemia drug resistance, paving the way for the application of RNAi in the following animal experiments.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 07/2009; 17(3):563-7.
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ABSTRACT: The present study was purposed to evaluate the safety of mesenchymal stem cell (MSC)-based therapy impacting on atherosclerosis. Allogeneic MSCs were obtained from rabbit bone marrow aspirates and expanded in vitro. New Zealand white rabbits were divided into three groups: 24 rabbits with hypercholesterolemia receiving intravenous injection of either 5 x 10(7) MSCs (n = 12) or saline (n = 12) after 5 weeks on a high lipid diet and additional rabbits (n = 6) fed with standard rabbit diet were served as controls. Body weight and blood lipids were measured at weeks 0, 5, 9 and 13 during the study. All rabbits were sacrificed at week 13. Atherosclerotic lesion size and vasa vasorum were evaluated by using pathological analysis and immunocytochemical technique. The results showed that the aortic sinus lesion size significantly increased in rabbits infused with MSCs as compared with controls receiving saline (23.35 +/- 3.51% and 11.39 +/- 3.08% respectively). The lesion size in whole aortas of MSC-treated rabbits was 76.64 +/- 12.70% versus 57.61 +/- 9.00% in saline-treated animals (p < 0.05). Moreover, vasa vasorum networks in MSC-treated aortas were more numerous and had increased capillary density. It is concluded that the allogeneic MSC transfusion may result in an increase in atherosclerotic lesion size. In cell therapy with MSCs or cell populations containing MSCs a strategy to attenuate the high potential of MSCs involved in atherogenesis of atherosclerosis should be taken in account.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 06/2009; 17(3):700-5.
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ABSTRACT: We investigated the role of stem cells from human umbilical cord tissue in cardiomyocyte regeneration. The umbilical cord stem cells were initially characterized and differentiated in a myocardial differentiation medium containing 5-azacytidine for 24 h. Differentiation into cardiomyocytes was determined by expression of cardiac specific markers, like cardiac alpha-actin, connexin43, myosin, Troponin T, and ultrastructural analysis. In vivo, the transplanted umbilical cord stem cells were sprouting from local injection and differentiated into cardiomyocyte-like cells in a rat myocardial infarction model. Echocardiography revealed increasing left ventricular function after umbilical cord stem cell transplantation. These results demonstrate that umbilical cord stem cells can differentiate into cardiomyocyte-like cells both in vitro and in vivo. Therefore, human umbilical cord might represent a source of stem cells useful for cellular therapy and myocardial tissue engineering. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon.
Journal of Cellular Biochemistry 06/2009; 107(5):926-32. · 2.87 Impact Factor
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ABSTRACT: Interleukin-27 (IL-27) is an IL-12-related cytokine that can promote both anti- and pro-inflammatory immune responses. In this study, we used the promonocytic cell line THP-1, an established model for monocytes to investigate if the immunoregulatory role of IL-27 is in part due to effects on major histocompatibility complex (MHC) Ag presentation. We find that IL-27 induces mRNA and surface expression of class II MHC in THP-1 cells. IL-27 also increases class I MHC heavy chain, beta2m, and TAP-1 transcripts, leading to an increased surface expression of class I MHC. In addition, IL-27 enhances expression of costimulatory molecules CD80 and CD86 and adhesion molecule CD54. Expression of the class II transactivator (CIITA) isoforms III and IV, but not I, transcripts increases in response to IL-27. Our data suggest that the pro-inflammatory role of IL-27 is mediated in part through increased expression of key molecules involved in the class II and class I MHC pathways.
Biochemical and Biophysical Research Communications 04/2008; 367(3):553-9. · 2.48 Impact Factor
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ABSTRACT: This study was aimed to investigate the expression of neuropilin-1 (NRP-1) and NRP-2 mRNA in myeloid leukemia cells and the effect of NRP-1 on cell proliferation and migration. The expressions of NRP-1 and NRP-2 mRNA in bone marrow mononuclear cells of 24 patients with acute myeloid leukemia and in 7 myeloid leukemic cell lines (HL-60, KGIa, NB4, U937, HEL MEG01 and K562) were detected by RT-PCR. The effects of NRP-1 interfered with siRNA on proliferation and migration in leukemic cell line HEL were examined by MTT and migration test. The results showed that the expression of NRP-1 mRNA was found in bone marrow mononuclear cells (BMMNCS) of 24 AML patients, the positive rate was 100% and significantly higher than that in control group (positive rate 67%). The expressions of NRP-2 mRNA were seen in 79% AML patients and in 67% health control, there was no significant difference between them. The increased NRP-1 expression was directly correlated with the blast percentage in both peripheral blood and bone marrow of AML patients (r=05, r=0.4, p<0.05). The expressions of NRP-1 and NRP-2 mRNA were observed in 6/7 and 3/7 myeloid leukemic cell lines respectively. After HEL cells were transfected with siRNA for 24 hours, the expression levels of NRP-1 mRNA and protein decreased obviously. Under VEGF action, the cell number in control group significantly increased, while the cell proliferation in interfered group had been not changed. After being transfected for 24 hours, the migration in interfered group decreased significantly. It is concluded that the higher level of NRP-1 mRNA is expressed in bone marrow mononuclear cells of leukemia patients and plays a pivotal role in proliferation and migration of myeloid leukemic cells. Inhibition of NRP-1 functions may provide a new therapeutic strategy for AML.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 12/2007; 15(6):1150-5.
