Comparison of the Biological Functions between Human Bone Marrow Derived CD106 Mesenchymal Stem Cells and CD106 Subgroup / 中国医学科学院学报

Objective To analyze the differences in biological functions between bone marrow(BM)-derived CD106 mesenchymal stem cells(MSCs)and the CD106 subgroup. Methods The MSCs from normal BM were isolated and expanded.The subgroups of CD106 and CD106 MSCs were sorted.The cell proliferation and adhesion functions,chemotactic activities,adipogenic and osteogenic potentials,senescence,and senescence protein 21(p21)were detected.The capacity of translocation into nucleus of nuclear factor-kappa B(NF-κB)when stimulated by tumor necrosis factor(TNF-α)was measured. Results The proliferative ability was higher in CD106 MSCs than that in CD106 MSCs.In 48 hours,the value of optical density(OD)was significantly higher in CD106 MSCs than that in CD106 subgroup(1.004±0.028 0.659±0.023,=3.946,=0.0225).In 72 hours,this phenomenon was even more pronounced(2.574±0.089 1.590±0.074,=11.240,=0.0000).The adhesive capacity of CD106 MSCs was significantly stronger than that of CD106 subgroup(0.648±0.018 0.418±0.023,=7.869,=0.0002).Besides,the metastasis ability of CD106 MSCs were significantly stronger than that of CD106 subgroup(114.500±4.481 71.000±4.435,=6.900,=0.0005).The CD106 MSCs had signifcnatly lower proportions of senescent cells.The expression of aging protein p21 in CD106 MSCs was significantly lower than that in CD106 MSCs [(17.560±1.421)% (45.800±2.569)%,=9.618,=0.0000].Furthermore,there were no visible pigmenting cells after β-galactosidase staining in CD106 MSCs subgroup.However,in CD106 MSCs,some colored green cells were detected.The rate of NF-κB translocation into nucleus after stimulated by TNF-α was significantly higher in CD106 MSCs than CD106 MSCs [(37.780±3.268)% (7.30±1.25)%,=8.713,=0.0001]. Conclusion Bone marrow-derived CD106 MSCs possess more powerful biological functions than CD106 MSCs.

Effect of CD106 Mesenchymal Stem Cell on Bone Marrow Vascular Failure in Patients with Aplastic Anemia / 中国医学科学院学报

Objective To investigate the vascularization ability of mesenchymal stem cells(MSCs)and explore its influencing factors in aplastic anemia(AA) patients. Methods MSCs were isolated from the bone marrow of AA patients(AA MSCs) and normal controls(N MSCs) were cultured and then evaluated by flow cytometry and immunofluorescene staining technique.The expression level of vascular cell adhesion molecule-1(CD106) was detected by gene sequencing,and the content and fluorescene intensity of CD106MSCs was determined by fluorescence-activated cell sorting.The content of CD105CD106MSCs in fresh AA bone marrow was measured,followed by the determination of the capability of endothelial differentiation from AA MSCs and N MSCs with immunofluorescene analysis;finally,the capability of CD31cell differentiation from CD106-blocking N MSCs and its tubular structures formation in matrigel were tested.Results The expression of CD106 in AA patients was defective(decreased by 12.13 times when compared with N MSCs) and the concentration and fluorescene degree of CD106MSCs was also decreased in AA patients [(28.03±17.71)% vs.(59.61±12.26)%,P=0.000].The content of CD105CD106MSCs decreased significantly in the fresh bone marrow [(0.33±0.10)% vs.(2.98±0.46)%,P=0.0005].Besides, the capability of CD31cell differentiation from AA MSCs was significantly delayed [(13.67±1.50)% vs.(43.24±0.96)%,P=0.0004].Also,the capability of CD31cell differentiation and tubular structures formation of CD106-blocking N MSCs was also obviously decreased [(26.00±2.65)% vs.(91.78±2.44)%,P=0.000;(13.81±1.98)mm vs.(68.12±6.78)mm,P=0.0015].Conclusion The deficient or decreased expression of CD106MSCs accelerate the bone marrow vascularization failure in AA patients.

Human Umbilical Cord-derived Mesenchymal Stem Cells Secrete Interleukin-6 to Influence Differentiation of Leukemic Cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effect of human umbilical cord-derived mesenchymal stem cells (UC-MSC) on the differentiation of leukemic cells.</p><p><b>METHODS</b>The co-culture system of UC-MSC with acute promyelocytic leukemic cell line NB4 cells was constructed in vitro,and the differentiation status of the leukemic cells was assessed by cell morphology,nitroblue tetrazolium reduction test,and cell surface differentiation marker CD11b.</p><p><b>RESULTS</b>UC-MSC induced the granulocytic differentiation of NB4 cells. When UC-MSC and a small dose of all-trans retinoic acid were applied together,the differentiation-inducing effect was enhanced in an additive manner. Interleukin (IL)-6Ra neutralization attenuated differentiation and exogenous IL-6-induced differentiation of leukemic cells.</p><p><b>CONCLUSION</b>UC-MSC can promotd granulocytic differentiation of acute promyelocytic leukemia cells by way of IL-6 and presented additive effect when combined with a small dose of all-trans retinoic acid.</p>

Influence of MicroRNA-382 on Biological Properties of Human Umbilical Cord-Derived Mesenchymal Stem Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of microRNA-382 (miR-382) on the biological properties of human umbilical cord-derived mesenchymal stem cells (hUC-MSC).</p><p><b>METHODS</b>The mimics and inhibitor of miR-382 were transfected into hUC-MSC with lipo2000. Inverted microscopy was used to observe the morphology change of hUC-MSC. The proliferation of hUC-MSC was detected by CCK-8. Oil red O and alizarin red staining were applied to assess the adipogenic and osteogenic differentiation of hUC-MSC. Cetylpyridinium chloride was used to the quantitative analysis of osteogenic differentiation. The expression of Runx2 and some cytokines were detected by RT-PCR.</p><p><b>RESULTS</b>miR-382 did not influence the morphology, proliferation and adipogenic differentiation of hUC-MSC miR-382 inhibited the expression of Runx2, thus could inhibit the osteogenesis of hUC-MSC, being confirmed by alizarin red stain; miR-382 could influence the expression of key cytokines secreted from hUC-MSC, such as IL-6, IDO1, G-CSF, M-CSF, GM-CSF.</p><p><b>CONCLUSION</b>miR-382 decreases the expression of Runx2 and inhibites the osteogenesis of hUC-MSC. In addition, it also affects the expression of some key cytokines secreted from hUC-MSC.</p>

Biological characteristics of exosomes secreted by human bone marrow mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to explore the immunoregulatory function and capability supporting the angiogenesis of exosomes secreted by bone marrow mesenchymal stem cells (BMMSC) from healthy persons. Supernatant of BMMSC (P4-P6) was collected for exosome purification. Transmission electron microscopy (TEM) and Western blot were used to identify the quality of isolated exosomes. The amount of exosomes was quantified through bicinchoninic acid (BCA) protein assay. Human peripheral blood mononuclear cells (PBMNC) were isolated from healthy donor and added with isolating exosomes. After co-cultured for 72 h, IFN-γ from the co-culture system was detected by ELISA. The expression of miRNA-associated with immunity were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). The interactions between exosomes and human umbilical vein endothelial cells (HUVEC) were observed with confocal microscopy. Subconfluent HUVEC were harvested and treated with the indicated concentration of exosomes. Nude mice were injected subcutaneously with exosomes or PBS as control to verify the ability of angiogenesis. The results showed that diameter range of exosomes was range from 40 to 160 nm. The isolated exosomes expressed the CD9. There was approximately linear relation between the secretion of exosomes and cell density. The exosomes suppressed the production of IFN-γ from PBMNC, and contained miRNA associated with immune regulation such as miR301, miR22 and miR-let-7a. Exosomes induced vascular tube formation in vitro and vascularization of Matrigel plugs in vivo. It is concluded that the BMMSC-derived exosomes can regulate immunity and support vascularization.

Effects of rapamycin on biological characteristics of bone marrow mesenchymal stem cells from patients with aplastic anemia / 中国实验血液学杂志

This study was aimed to investigate the effects of rapamycin on biological function and autophagy of bone marrow mesenchymal stem cells (BM-MSC) from patients with aplastic anemia so as to provide experimental basis for the clinical treatment of aplastic anemia (AA) with rapamycin. BM-MSC were treated with different concentrations of rapamycin (0, 10, 50, 100 nmol/L) for 48 h, the expression of LC3B protein was detected by Western blot to observe the effect of rapamycin on cell autophagy; cell apoptosis and cell cycles were detected by flow cytometry; the proliferation of BM-MSC of AA patients was measured by cell counting kit-8; the adipogenic differentiation of BM-MSC were tested by oil red O staining after adipogenic induction for 2 weeks; the adipogenic related genes (LPL, CFD, PPARγ) were detected by real-time PCR. The results showed that the proliferation and adipogenesis of BM-MSC of AA patients were inhibited by rapamycin. Moreover, the autophagy and apoptosis of BM-MSC were increased by rapamycin in a dose-dependent way.Rapamycin arrested the BM-MSC in G0/G1 phase and prevented them into S phase (P < 0.05). It is concluded that rapamycin plays an critical role in inhibiting cell proliferation, cell cycles, and adipogenesis, these effects may be related with the autophagy activation and mTOR inhibition resulting from rapamycin.

Human umbilical cord mesenchymal stem cells reduce the sensitivity of HL-60 cells to cytarabine / 中国实验血液学杂志

This study was purposed to investigate the impact of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) on the sensitivity of HL-60 cells to therapeutic drugs so as to provide more information for exploring the regulatory effect of hUC-MSC on leukemia cells. Transwell and direct co-culture systems of HL-60 and hUC-MSC were established. The apoptosis and cell cycle of HL-60 cells were detected by flow cytometry. RT-PCR and Western blot were used to detect the mRNA and protein levels of Caspase 3, respectively. The results showed that the apoptosis of HL-60 induced by cytarabine (Ara-C) decreased significantly after direct co-cultured with hUC-MSC cycle mRNA (P < 0.05). The similar phenomenon was observed in transwell co-culture system. Cell cycle of HL-60 cells were arrested at G0/G1 phase and did not enter into S phase (P < 0.05) and the expression of Caspase-3 mRNA and protein in HL-60 cells were reduced (P < 0.05). It is concluded that hUC-MSC protected HL-60 from Arc-C induced apoptosis through regulating the cell cycle and down-regulating expression of Caspase 3 in HL-60 cells. In addition, this effect is caused by the soluble factors from hUC-MSC.

Effect of 15-deoxy-Δ(12), 14-prostaglandin J2 on the cytokines in the culture supernatant of bone marrow mesenchymal stem cells / 中国实验血液学杂志

15-Deoxy-Δ(12), 14-prostaglandin J2 (15d-PGJ2), a well known peroxisome proliferator activated receptor (PPAR) γ ligand, has been shown to inhibit cellular proliferation and induce apoptosis and differentiation. However, whether 15d-PGJ2 influences the cytokines in the culture supernatant of bone marrow mesenchymal stem cells (BM-MSC) is unknown. This study was purposed to investigate the influence of 15d-PGJ2 on cytokines in the culture supernatant of BM-MSC. The fibroblast-like cells attached to the culture dish from bone marrow of healthy donors were isolated. The immunophenotype and differentiation potential of the obtained cells were detected by flow cytometry and oil red O and von kassa staining respectively to confirm that these cells were BM-MSC. Thereafter, the BM-MSC were cultured with complete medium supplemented with 10, 20, 40 and 60 µmol/L 15d-PGJ2 for 24 hours respectively. The real-time PCR was used to assay the PPARγ mRNA level, the confocal immuno fluorescence technique was used to detect the expression level of PPARγ. The results showed that the BM-MSC underwent apoptosis and got detached from the culture dish when the concentration of 15d-PGJ2 was no less than 20 µmol/L. The PPARγ mRNA level of BM-MSCs cultured with medium containing 10 µmol/L 15d-PGJ2 was higher than that cultured without 15d-PGJ2, and the difference was statistically significant (P < 0.05). The enhancement of PPARγ expression was observed after stimulated by 15d-PGJ2. The protein chip detecting the culture supernatants of BM-MSC cultured with 10 µmol/L 15d-PGJ2 or without 15d-PGJ2 for 24 hours demonstrated that expression levels of some of the cytokines varied. It is concluded that the down-regulation of TIMP-2 exists after treatment of 15d-PGJ2, which is statistical significant.

Culture of pancreatic progenitor cells in hanging drop and on floating filter / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct a method to culture pancreatic progenitor cells in hanging drop and on floating filter,and to examine if pancreatic progenitor cells can differentiate into mature endocrine cells with this method.</p><p><b>METHODS</b>Murine embryos at day 12.5 were isolated and digested into single cells,which were then cultured in hanging drop for 24h and formed spheres.Spheres were cultured on the filter for 6 days,which floated in the dish containing medium.During culture,the expressions of pancreas duodenum homeobox-1(PDX-1)and neurogenin3(Ngn3)were determined.The expressions of endocrine and exocrine markers,insulin,glucagon,and carboxypeptidase(CPA)were determined on day 7 by immunohistochemistry.Insulin secretion of spheres stimulated by glucose was detected by ELISA.The changes of pancreatic marker expressions during culture were monitored by real-time polymerase chain reaction(PCR).</p><p><b>RESULTS</b>One day after the culture,there were still a large amount of PDX-1 positive cells in pancreatic spheres,and these cells proliferated.On day 3,high expression of Ngn3 was detected,and the Ngn3-positive cells did not proliferate.On day 7,The expressions of endocrine and exocrine markers in the differentiated pancreatic progenitor cells were detected,which were consistent with that in vivo.Insulin was secreted by spheres upon the stimulation of glucose.</p><p><b>CONCLUSION</b>In hanging drop and on floating filter,pancreatic progenitor cells can differentiate into mature endocrine cells.</p>

Effects of interferon-γ on biological characteristics and immunomodulatory property of human umbilical cord-derived mesenchymal stem cells / 中国实验血液学杂志

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.

Establishment of a new method to induce the differentiation of embryonic pancreatic cells into mature endocrine cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a new culture method to induce the differentiation of embryonic pancreatic cells into mature endocrine cells.</p><p><b>METHODS</b>Mouse embryos at day 12.5 were used and embryonic pancreata were isolated. The isolated embryonic pancreata were cultured on the filter for 7 days, which floated in the dish containing medium. During culture, the expression of pancreas duodenum homeobox-1 (PDX-1), a pancreatic stem cell marker, was examined at day 1. The expression of neurogenin 3 (Ngn3), a pancreatic progenitor cell marker, was examined at day 3. The expressions of endocrine and exocrine markers, insulin, glucagon, and carboxypeptidase (CPA) were examined at day 7 by immunohistochemistry. The kinetics of pancreatic marker expression during culture was assayed by real-time PCR.</p><p><b>RESULTS</b>Many pancreatic stem cells still existed in embryonic pancreata cultured for 1 day; meanwhile, these pancreatic stem cells proliferated in high rate. A large amount of pancreatic progenitor cells were found in embryonic pancreata cultured for 3 days.Pancreatic stem/progenitor cells differentiated into mature endocrine and exocrine cells in embryonic pancreata after having been cultured for 7 days. Furthermore, the expression pattern of pancreatic marker is consistent with that in vivo.</p><p><b>CONCLUSION</b>We successfully established a new culture method, with which embryonic pancreatic cells can efficiently differentiate into mature endocrine cell.</p>

Comparison of hematopoietic supportive capacity between human fetal and adult bone marrow mesenchymal stem cells in vitro / 中国实验血液学杂志

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 microenvironments. Mesenchymal stem cells (MSC) are the precursors of stromal cells in bone marrow microenvironments 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.

The effects of stem cell factor on proliferation, transmigration, capillary tube formation of endothelial cells and on the chemotaxis of CD133(+) cells / 中华血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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 ].</p><p><b>CONCLUSIONS</b>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.</p>

Isolation of mesenchymal stem cells from bone marrow filters by primary explant culture / 中国实验血液学杂志

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.

Differentiation of human umbilical cord derived mesenchymal stem cells into low immunogenic and functional hepatocyte-like cells in vitro / 中国医学科学院学报

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>UC-MSCs are able to differentiate into functional hepatocyte-like cells in vitro, while the immunogenicity of DHC remains low.</p>

Construction and identification of a novel adeno-integrase hybrid system for hemophilia B / 中国实验血液学杂志

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.

More mesenchymal stem cells enriched from bone marrow aspirates by culturing bone marrow particles and mononuclear cells separately / 中国实验血液学杂志

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.

IL-27 regulates the expression of Mac-1, fMLP-R and IL-1beta in human neutrophils through p38 MAPK and PI3K signal pathways / 中国实验血液学杂志

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.

Enhancement of all-trans retinoic acid induced HL-60 leukemia cell differentiation by human umbilical cord mesenchymal stem cells / 中国实验血液学杂志

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.

Human coagulation factor IX gene therapy in murine hemophilia B by hydrodynamic delivery and site-specific genomic integration / 中华血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>