Effect of mesenchymal stem cells on human Th1 cells by flow cytometry / 中国实验血液学杂志

This study was aimed to investigate the effect of fetal bone marrow-derived mesenchymal stem cells (FBM-MSC) on the development of human Th1 cells. FBM-MSC were isolated, cultured and expanded in vitro. The cells were identified by their phenotype profiles and differential capacity. Human CD4(+) T cells from healthy donors were cultured alone or co-cultured with FBM-MSC (FBM-MSC/CD4). In these two cultures, the quantities of Th1 cells (interferon-γ(+)) were analyzed by flow cytometry. The results indicated that the immunophenotype and multilineage differentiation of FBM-MSC satisfied the generally accepted criteria. FBM-MSC played an inhibitory role in the development of Th1 cells. Flow cytometry analysis showed that the percentage of Th1 cells in FBM-MSC/CD4 was significantly lower than that in CD4(+) T cells cultured alone. The protein level of IFN-γ in FBM-MSC/CD4 detected by ELISA was also lower than that in CD4(+) T cells cultured alone. It was also demonstrated that the expression level of IL-6 in FBM-MSC/CD4 was much higher than that in CD4(+) T cells cultured alone or FBM-MSC. The neutralizing antibody of IL-6 could increase the quantities of Th1 cells and the expression levels of IFN-γ. It is concluded that FBM-MSC may play an inhibitory role in the development of human Th1 cells, and the IL-6 pathway may be one of mechanisms involved in the inhibitory role.

Immunoregulatory function of interleukin-6 on human Th17 cells / 中国实验血液学杂志

The aim of this study was to explore the regulatory function of interleukin-6(IL-6) on human Th17 cells. Human peripheral blood CD4(+) T cells were purified from healthy donors by anti-CD4 monoclonal antibody (mAb) conjugated microbeads. The experiment was divided into 2 groups. Test group in which CD4(+) T cells (1 × 10(6)/ml) were stimulated by human recombined IL-6 (20 ng/ml) for 4 days; control group in which CD4(+) T cells did not stimulated by IL-6. The concentrations of IL-17 protein in the supernatants were assayed by enzyme-linked immunosorbent assay (ELISA), and quantity of Th17 cells were detected by flow cytometry. The results showed that as compared to control group, IL-17 protein level in the supernatants of CD4(+) T cells significantly increased in IL-6 stimulated group: (337.05 ± 189.09 pg/ml; vs 15.07 ± 12.70 pg/ml) (p < 0.05). Furthermore, the percentage of Th17 cells in cultures of CD4(+) T cells stimulated by IL-6 was significantly higher than that in control group (4.05% ± 0.30% vs. 2.81% ± 0.44%)(p < 0.01). It is concluded that IL-6 promotes the expansion of Th17 cells in vitro.

Analysis on promoter CpG methylation and expression of HOXB4 gene in cord blood CD34(+) cells and peripheral blood mononuclear cells / 中国实验血液学杂志

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.

Regulatory role of HOXB4 in self-renewal of hematopoietic stem cells - review / 中国实验血液学杂志

Self-renewal and multilineage differentiation of hematopoietic stem cell (HSC) are their functional characteristics. The regulation of HSC self-renewal is governed by a balance between positive regulatory signals promoting growth and negative regulatory signals resulting in apoptosis. Among the positive regulatory signals, HOXB4 activates distinct pathways that enhance self-renewal divisions of HSC without overriding the regulatory mechanisms that maintain normal steady-state hemopoiesis. The upregulation of HOXB4 gene expression can greatly promote the HSC self-renewal, but does not affect the HSC differentiation, the morphology and function of linage-specific cells and terminally-differentiated blood cells. Furthermore, HOXB4 can enhance the hematopoietic potential of embryonic stem cell (ESC), promoting the differentiation of ESC into hematopoietic cells. As a consequence, upregulation of HOXB4 expression and/or corresponding HOXB4 target genes can have enormous therapeutical potential for human HSC in the stem cell transplantation and gene therapy. In this review the regulatory role of HOXB4 in HSC self-renewal, "zero" effect of HOXB4 on differentiation specificity of HSC lines and terminal differentiation cells, and molecular mechanisms of regulating HSC self-renewal by HOXB4 are summarised.

Neovascularization potential of mobilized peripheral mononuclear cells from diabetes patients / 中国医学科学院学报

<p><b>OBJECTIVE</b>To determine whether mobilized peripheral blood mononuclear cells (M-PBMNCs) obtained from patients with diabetes was impaired in therapeutic neovascularization in limb ischemia, and to explore the pathological mechanisms of the impairment.</p><p><b>METHODS</b>Endothelial progenitor cells (EPC) were cultured in EGM-2MV, and then characterized by uptake of 1, 1-dioctadecyl-3, 3, 3, 3-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (Dil-AcLDL) and binding of ulex europaeus agglutinin (UEA). The number of EPC was compared between M-PBMNCs obtained from diabetic patients and those from normal subjects. M-PBMNCs obtained from diabetic patients, M-PBMNCs obtained from normal controls, or PBS were injected into the ischemic limbs of streptozotocin-induced diabetic nude mice. The limb blood perfusion was detected by laser Doppler blood perfusion imaging between these three groups in the following 1, 3, 7, 14, 21, and 28 days. Ambulatory score and ischemia damage were evaluated in the following 4 weeks. Capillary/fiber ratio was detected by CD31 or BS-1 lectin, and arteriole density was detected by alpha-smooth muscle actin (alpha-SMactin).</p><p><b>RESULTS</b>The number of EPC from diabetic patients were positively correlated with the blood perfusion (R = 0.486, P < 0.05) and capillary density (R = 0.491, P < 0.05), and the EPC number in diabetic patient were negatively correlation with their disease courses (R = - 0.587, P < 0.05). Transplantation of diabetic M-PBMNCs augmented the blood perfusion of ischemia hindlimbs, increased the capillary and arteriole densities, and promoted the collateral vessel formation. However, all the improvements were less significant in the diabetic patients than in the non-diabetic patients (P < 0.05).</p><p><b>CONCLUSION</b>Diabetes decreased the capability of M-PBMNCs to augment neovascularization in ischemia.</p>