OBJECTIVE: This study aims to evaluate the effect of Angelica sinensis polysaccharide (ASP) on the osteogenic differentiation of the bone marrow mesenchymal stem cells (BMSCs) of rats with high glucose levels. METHODS: Rat BMSCs were isolated and identified by osteogenic and adipogenic differentiation. Then, the BMSCs were divided into three groups as follows: normal control group (5.5 mmol·L⁻¹ glucose), high glucose group (25.5 mmol·L⁻¹ glucose), and ASP+high glucose group (25.5 mmol·L⁻¹ glucose +40 mg·L⁻¹ ASP). The proliferation activities of the BMSCs were detected by CCK8. Alizarin red staining, and alkaline phosphatase activity were used in the examination of osteogenic activity. Quantitative real time-polymerase chain reaction was used to detect the expression levels of the osteogenic genes (Runx2, Osx, OCN, Col-Ⅰ) and the key factors of Wnt/β-catenin signal pathway (CyclinD1, β-catenin). In vivo, a type 2 diabetes rat model was established. The rats were divided into three groups, namely, the normal control group (normal rats), diabetes group (diabetic rats), diabetes+ASP group (diabetic rats, ASP feeding). Then, the tibia bone defect was established. The repair of bone defects in each group was observed through histological examination. RESULTS: The proliferation of BMSCs was higher in the high glucose group and ASP+high glucose group than in the normal control group (P<0.05). No significant difference was observed between the high glucose group and ASP+high glucose group (P>0.05). The number of calcium nodules of BMSCs; alkaline phosphatase activity; and the mRNA expression of Runx2, OCN, Osx, Col-Ⅰ, CyclinD1, β-catenin in the high glucose group were lower than those in the normal control and ASP+high glucose groups (P<0.05). No significant difference was observed between the normal control and ASP+high glucose groups (P>0.05). The bone mass was significantly lower in the bone defect of the diabetes group than in the bone defect of the normal control or diabetes+ASP group (P<0.05). No statistical difference was found between the normal control and diabetes+ASP groups (P>0.05). CONCLUSIONS ASP can promote the osteogenic differentiation of rat BMSCs under high glucose culture and induce bone regeneration in rats with type 2 diabetes. These features may be related to the activation of the Wnt/β-catenin signaling pathway.
Angelica sinensis ; chemistry ; Animals ; Bone Marrow Cells ; Cell Differentiation ; drug effects ; Cells, Cultured ; Diabetes Mellitus, Experimental ; Diabetes Mellitus, Type 2 ; Glucose ; Mesenchymal Stem Cells ; Osteogenesis ; drug effects ; Plant Extracts ; pharmacology ; Polysaccharides ; pharmacology ; Rats

BACKGROUNDDendritic cells are professional antigen-presenting cells found in an immature state in epithelia and interstitial space, where they capture antigens such as pathogens or damaged tissue. Matrix metallopeptidase 13 (MMP-13), a member of the collagenase subfamily, is involved in many different cellular processes and is expressed in murine bone marrow-derived dendritic cells (DCs). The function of MMP-13 in DCs is not well understood. Here, we investigated the effect of MMP-13 on DC maturation, apoptosis, and phagocytosis.

METHODSBone marrow-derived dendritic cells were obtained from C57BL/6 mice. One short-interfering RNA specific for MMP-13 was used to transfect DCs. MMP-13-silenced DCs and control DCs were prepared, and apoptosis was measured using real-time polymerase chain reaction and Western blotting. MMP-13-silenced DCs and control DCs were analyzed for surface expression of CD80 and CD86 and phagocytosis capability using flow cytometry.

RESULTSCompared to the control DCs, MMP-13-silenced DCs increased expression of anti-apoptosis-related genes, BAG1 (control group vs. MMP-13-silenced group: 4.08 ± 0.60 vs. 6.11 ± 0.87, P = 0.008), BCL-2 (control group vs. MMP-13-silenced group: 7.54 ± 0.76 vs. 9.54 ± 1.29, P = 0.036), and TP73 (control group vs. MMP-13-silenced group: 4.33 ± 0.29 vs. 5.60 ± 0.32, P = 0.001) and decreased apoptosis-related genes, CASP1 (control group vs. MMP-13-silenced group: 3.79 ± 0.67 vs. 2.54 ± 0.39, P = 0.019), LTBR (control group vs. MMP-13-silenced group: 9.23 ± 1.25 vs. 6.24 ± 1.15, P = 0.012), and CASP4 (control group vs. MMP-13-silenced group: 2.07 ± 0.56 vs. 0.35 ± 0.35, P = 0.002). Protein levels confirmed the same expression pattern. MMP-13-silenced groups decreased expression of CD86 on DCs; however, there was no statistical difference in CD80 surface expression. Furthermore, MMP-13-silenced groups exhibited weaker phagocytosis capability.

CONCLUSIONThese results indicate that MMP-13 inhibition dampens DC maturation, apoptosis, and phagocytosis.

Animals ; Apoptosis ; drug effects ; physiology ; Bone Marrow Cells ; cytology ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Female ; Lipopolysaccharides ; pharmacology ; Matrix Metalloproteinase 13 ; metabolism ; physiology ; Mice ; Mice, Inbred C57BL ; RNA, Small Interfering

No abstract available.
Aged ; Antineoplastic Combined Chemotherapy Protocols/adverse effects ; Bone Marrow Cells/cytology/pathology ; Female ; Flow Cytometry ; Fusion Proteins, bcr-abl/*genetics ; Granulocyte Colony-Stimulating Factor/therapeutic use ; Humans ; Immunophenotyping ; Leukemia/*diagnosis/etiology ; Lymphoma, Large B-Cell, Diffuse/*drug therapy ; Phenotype ; Rituximab/administration & dosage

OBJECTIVETo investigate the effects of panaxadiol saponins component (PDS-C) isolated from total saponins of panax ginseng on proliferation, differentiation and corresponding gene expression profile of megakaryocytes.

METHODSBone marrow culture of colony forming assay of megakaryocytic progenitor cells (CFU-MK) was observed for the promoting proliferation mediated by PDS-C, and differentiation of megakaryocytic blasts caused by PDS-C was analyzed with flow cytometry in CHRF-288 and Meg-01 cells, as well as proliferation, differentiation-related genes expression profile and protein expression levels were detected by human gene expression microarray and western blot.

RESULTSIn response to PDS-C 10, 20 and 50 mg/L, CFU-MK from 10 human bone marrow samples was increased by 28.9%±2.7%, 41.0%±3.2% and 40.5%±2.6% over untreated control, respectively (P <0.01, each). Flow cytometry analysis showed that PDS-C treated CHRF-288 cells and Meg-01 cells significantly increased in CD42b, CD41, TSP and CD36 positive ratio, respectively. PDS-C induced 29 genes up-regulated more than two-fold commonly in both cells detected by human expression microarray representing 4000 known genes. The protein expression levels of ZNF91, c-Fos, BTF3a, GATA-1, RGS2, NDRG2 and RUNX1 were increased with western blot in correspond to microarray results.

CONCLUSIONPDS-C as an effective component for hematopoiesis, play the role to enhance proliferation and differentiation of megakaryocytes, also up-regulated expression of proliferation, differentiation-related genes and proteins in vitro.

Blotting, Western ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Colony-Forming Units Assay ; Drugs, Chinese Herbal ; pharmacology ; Flow Cytometry ; Gene Expression Profiling ; Ginsenosides ; pharmacology ; Humans ; Megakaryocytes ; cytology ; drug effects ; metabolism ; Patents as Topic ; Saponins ; pharmacology ; Stem Cells ; cytology ; drug effects ; Transcription Factors ; metabolism ; Up-Regulation ; drug effects ; genetics

OBJECTIVETo observe the effects of Compound Zhebei Granule (, CZBG) combined with chemotherapy on surface markers of leukemia stem cell (LSC) in the bone marrow of patients with acute myeloid leukemia (AML).

METHODSSeventy-eight patients with AML received bone marrow aspiration and the percentages of CD34(+) CD123(+) and CD33(+) CD123(+) cells were tested using flow cytometry method. A total of 24 refractory or relapsed AML patients were enrolled and treated with one cycle of standard chemotherapy combined with CZBG. Bone marrow samples were obtained before and after treatment, and the percentages of CD34(+) CD123(+) and CD33(+) CD123(+) cells were examined by flflow cytometry.

RESULTSCompared with refractory or relapsed AML patients, patients achieved remission had a significant lower percentage of CD34(+) CD123(+) cells(P<0.01) and CD33(+) CD123(+) cells (P<0.01), indicating that controlling the LSC percentage may be important for patients with AML to achieve sustainable remission. Compared with those before treatment, the expression levels of CD34(+) CD123(+) were significantly decreased after CZBG combined with chemotherapy treatment (P<0.01). The percentages of CD34(+) CD123(+) cells and CD33(+) CD123(+) in patients achieving complete remission after CZBG combined with chemotherapy treatment were both significantly lower than those in patients with nonremission (P<0.01).

CONCLUSIONCZBG combining chemotherapy could reduce the percentages of CD34(+) CD123(+) and CD33(+) CD123(+) LSC, which might improve the clinical efficacy of refractory or relapsed AML.

Antigens, CD ; metabolism ; Antineoplastic Agents ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Biomarkers, Tumor ; metabolism ; Bone Marrow Cells ; drug effects ; metabolism ; pathology ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Leukemia, Myeloid, Acute ; drug therapy ; pathology ; Male ; Middle Aged ; Neoplastic Stem Cells ; metabolism ; pathology ; Remission Induction

OBJECTIVETo study the effects of graphene quantum dots (GQDs) on hematopoietic system in rats.

METHODSThirty male SD rats were randomly divided into three groups (n = 10): control group, high dose group (10 mg/kg · d), low dose group (5 mg/kg · d), The rats in experimental group were intravenous injected with GQDs for 28 days and those in control group were injected with normal saline at the same volume. Routine blood and the function of liver and kidney were detected by instrument analysis. The cycle and apoptosis of bone marrow mononuclear cells (BMCs) were detected by FCM. The other three only healthy male SD rat bone marrow mononuclear cells (BMCs) were cultured by joining GQDs for 24 h, 48 h,72 h in vitro, the proliferation was assayed by CCK-8, the content of granulocyte macrophage colony stimulating factor (GM-CSF) from cultural supernatants were detected by ELISA.

RESULTSThe amount of red blood cell and concentration of hemoglobin from experimental group were increased significantly compared with those of control groups (P < 0.05), the concentration of triglyceride and high density lipoprotein were decreased. DNA synthesis period was prolonged (P < 0.01), there was no significant difference in apoptosis. BMCs were promoted proliferation clearly after using GQDs for 72 h (P < 0.05). The content of GM-CSF was increased (P < 0.01) .

CONCLUSIONGQDs may promote hematopoietic function in rats.

Animals ; Apoptosis ; Bone Marrow Cells ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Graphite ; pharmacology ; Hematopoiesis ; drug effects ; Male ; Quantum Dots ; chemistry ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate the therapeutic effects of combined administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human thrombopoietin (rhTPO) and recombinant human interleukin-2 (rhIL-2) on radiation-induced severe haemopoietic acute radiation sickness (ARS) in rhesus monkeys, so as to provide experimental evidences for the effective clinical treatment.

METHODSSeventeen rhesus monkeys were exposed to 7.0 Gy (60)Co γ-ray total body irradiation (TBI) to establish severe haemopoietic ARS model, and were randomly divided into supportive care group, rhG-CSF+rhTPO treatment group and rhG-CSF+rhTPO+rhIL-2 treatment group. Survival time, general signs such as bleeding and infections, and peripheral blood cell counts in each group were monitored. Bone marrow cells were cultivated to examine the colony formation ability. The histomorphology changes of bone marrow were observed at 45 d post irradiation.

RESULTSAfter 7.0 Gy (60)Co γ-ray TBI, monkeys of supportive care group underwent tarry stool and emesis, then died in 12~18 d. The overall survival rate in this group was 16.7%. Gastrointestinal reactions of monkeys in two combined-cytokines treatment groups were inapparent. Combined-cytokines treatment induced 100% survival. Complete blood cells declined sharply after irradiation in each group, but two combined-cytokines treatment schemes could elevate the nadir of all blood cells, shorten the duration of pancytopenia and accelerate the recovery of hemogram. Compared with rhG-CSF+ rhTPO treatment, rhG-CSF+ rhTPO+ rhIL-2 treatment could increase the counts of lymphocytes and monocytes. The colony-formation rate of haemopoietic stem/progenitor cells in bone marrow dropped markedly at 2 d after irradiation. Combined-cytokines treatment promoted the ability of colony formation on day 29. Hematopoietic cells mostly disappeared in bone marrow of animals in supportive care group, but hematopoietic functions were recovered after cytokines were administrated.

CONCLUSIONrhG-CSF+ rhTPO and rhG-CSF+ rhTPO+ rhIL-2 treatment can significantly promote hematopoiesis recovery, improve the quantity of life, simplify the supportive therapy, and enhance the survival rate of rhesus monkeys with severe haemopoietic ARS induced by 7.0 Gy (60)Co γ-ray exposure. Especially the application of rhIL-2 can accelerate the recovery of lymphocytes and monocytes and restore the immunological function. Thus, combination of rhG-CSF, rhTPO and rhIL-2 on the basis of supportive care is an efficient strategy to treat severe haemopoietic ARS.

Animals ; Bone Marrow ; pathology ; Bone Marrow Cells ; pathology ; Gamma Rays ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoiesis ; drug effects ; Hematopoietic Stem Cells ; cytology ; Humans ; Interleukin-2 ; pharmacology ; Macaca mulatta ; Radiation Injuries ; drug therapy ; Random Allocation ; Recombinant Proteins ; therapeutic use ; Thrombopoietin ; pharmacology ; Whole-Body Irradiation

To isolate bone marrow mesenchymal stem cells (BM-MSCs) and establish the model of chronic kidney disease (CKD) of Wuzhishan (WZS) mini-pig, and to study the repairment effect of BM-MSCs on CKD-induced renal fibrosis in vitro.
 Methods: Density gradient method was used to isolate and culture BM-MSCs. The cells were verified by morphology, phenotype, differentiation and so on. The left partial ureteral obstruction (LPUUO) was used to establish the CKD model, which was evaluated by B-ultrasound, single-photon emission computed tomography (SPECT), HE and Masson staining. The cells were divided into 3 groups, the tissue plus BM-MSCs group, the tissue group, and the BM-MSCs group, respectively. Seven days later, the supernatants were collected to observe the changes of hepatocyte growth factor (HGF) cumulative release. HE and Masson staining was used to observe the changes of renal tissue.
 Results: The isolated BM-MSCs possessed the features as follow: fibroblast-like adherent growth; positive in CD29 and CD90 expression while negative in CD45 expression; osteogenic induction and alizarin red staining were positive; alcian blue staining were positive after chondrogenic induction. Twelve weeks after the operation of LPUUO, B-ultrasound showed the thin renal cortical with pelvis effusion; SPETCT showed the left kidney delayed filling and renal impairment. The accumulation of HGF in the tissue plus BM-MSCs group was significantly higher than that in the tissue alone group at the 1st, 5th, 6th, 7th day, respectively (P<0.05). HE staining showed the different degree of renal lesions between the tissue plus BM-MSCs+CKD group and the tissue alone group, which was aggravated with the time going. Masson staining showed that the cumulative optical density of blue-stained collagen fibers in tissue plus BM-MSCs group was significantly lower than that in the tissue group at the 5th to 7th day (P<0.05).
 Conclusion: BM-MSCs from WZS mini-pig can inhibit or delay the progress of CKD-induced renal fibrosis through autocrine HGF in vitro.
Animals ; Autocrine Communication ; physiology ; Bone Marrow Cells ; Cells, Cultured ; Fibrosis ; physiopathology ; prevention & control ; Hepatocyte Growth Factor ; metabolism ; Kidney ; drug effects ; pathology ; physiopathology ; Mesenchymal Stem Cells ; drug effects ; Renal Insufficiency, Chronic ; complications ; physiopathology ; Swine ; Swine, Miniature ; Ureteral Obstruction ; complications

Severe graft-versus-host disease (GVHD) is an often lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). The safety of clinical-grade mesenchymal stem cells (MSCs) has been validated, but mixed results have been obtained due to heterogeneity of the MSCs. In this phase I study, the safety of bone marrow-derived homogeneous clonal MSCs (cMSCs) isolated by a new subfractionation culturing method was evaluated. cMSCs were produced in a GMP facility and intravenously administered to patients who had refractory GVHD to standard treatment resulting after allogeneic HSCT for hematologic malignancies. After administration of a single dose (1x10(6) cells/kg), 11 patients were evaluated for cMSC treatment safety and efficacy. During the trial, nine patients had 85 total adverse events and the rate of serious adverse events was 27.3% (3/11 patients). The only one adverse drug reaction related to cMSC administration was grade 2 myalgia in one patient. Treatment response was observed in four patients: one with acute GVHD (partial response) and three with chronic GVHD. The other chronic patients maintained stable disease during the observation period. This study demonstrates single cMSC infusion to have an acceptable safety profile and promising efficacy, suggesting that we can proceed with the next stage of the clinical trial.
Bone Marrow ; Drug-Related Side Effects and Adverse Reactions ; Graft vs Host Disease* ; Hematologic Neoplasms ; Hematopoietic Stem Cell Transplantation ; Humans ; Mesenchymal Stromal Cells* ; Myalgia ; Population Characteristics

OBJECTIVETo explore the role of the basic fibroblast growth factor (bFGF) in the directional differentiation of bone marrow mesenchymal stem cells (BMSCs) into Leydig cells.

METHODSAfter purification and identification, we inoculated the third-generation BMSCs of SD rats onto a six-orifice board and then randomly divided them into groups A (normal saline control), B (human chorionic gonadotropin [hCG] + platelet-derived growth factor [PDGF] induction), C (hCG + PDGF + 5.0 ng/ml bFGF induction), D (hCG + PDGF + 10.0 ng/ml bFGF induction), and E (hCG + PDGF + 20.0 ng/ml bFGF induction). On the 7th, 14th and 21st day of induction, we observed the morphological changes of the cells and measured the level of testosterone (T) and expression of 3 beta hydroxy steroid dehydrogenase (3β-HSD) in the supernatant by immunofluorescence staining.

RESULTSAfter induction, the BMSCs of groups B, C, D, and E exhibited microscopic features of enlarged size, inter-connection, long-shuttle or irregular shape, adherent growth, and large round nuclei, all characteristic of Leydig cells. With the prolonging of time and enhanced concentration of bFGF, gradual increases were observed in the T level and the count of 3β-HSD-positive BMSCs in the four induction groups, with statistically significant differences between group B and groups C, D, and E (P < 0.05), as well as between group C and groups D and E (P < 0.05), but not between D and E (P > 0.05).

CONCLUSIONThe bFGF has an obvious promoting effect in the in vitro induced differentiation of rat BMSCs into Leydig cells.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Differentiation ; Cells, Cultured ; Chorionic Gonadotropin ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Leydig Cells ; cytology ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Testosterone ; metabolism