OBJECTIVE: To investigate the effects of sodium valproate (VPA) in inhibiting Erastin-induced ferroptosis in bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanisms. METHODS: BMSCs were isolated from bone marrow of 8-week-old Spragur Dawley rats and identified [cell surface antigens CD90, CD44, and CD45 were analyzed by flow cytometry, and osteogenic and adipogenic differentiation abilities were assessed by alizarin red S (ARS) and oil red O staining, respectively]. Cells of passage 3 were used for the Erastin-induced ferroptosis model, with different concentrations of VPA for intervention. The optimal drug concentration was determined using the cell counting kit 8 assay. The experiment was divided into 4 groups: group A, cells were cultured in osteogenic induction medium for 24 hours; group B, cells were cultured in osteogenic induction medium containing optimal concentration Erastin for 24 hours; group C, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA for 24 hours; group D, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA, and 8 μmol/L EX527 for 24 hours. The mitochondrial state of the cells was evaluated, including the levels of malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS). Osteogenic capacity was assessed by alkaline phosphatase (ALP) activity and ARS staining. Western blot analysis was performed to detect the expressions of osteogenic-related proteins [Runt-related transcription factor 2 (RUNX2) and osteopontin (OPN)], ferroptosis-related proteins [glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), and solute carrier family 7 member 11 (SLC7A11)], and pathway-related proteins [adenosine monophosphate-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1)]. RESULTS: The cultured cells were identified as BMSCs. VPA inhibited Erastin-induced ferroptosis and the decline of osteogenic ability in BMSCs, acting through the activation of the AMPK/SIRT1 pathway. VPA significantly reduced the levels of ROS and MDA in Erastin-treated BMSCs and significantly increased GSH levels. Additionally, the expression levels of ferroptosis-related proteins (GPX4, FTH1, and SLC7A11) significantly decreased. VPA also upregulated the expressions of osteogenic-related proteins (RUNX2 and OPN), enhanced mineralization and osteogenic differentiation, and increased the expressions of pathway-related proteins (AMPK and SIRT1). These effects could be reversed by the SIRT1 inhibitor EX527. CONCLUSION VPA inhibits ferroptosis in BMSCs through the AMPK/SIRT1 axis and promotes osteogenesis.
Mesenchymal Stem Cells/metabolism* ; Ferroptosis/drug effects* ; Animals ; Valproic Acid/pharmacology* ; Rats ; Rats, Sprague-Dawley ; Sirtuin 1/metabolism* ; Cell Differentiation/drug effects* ; Cells, Cultured ; AMP-Activated Protein Kinases/metabolism* ; Osteogenesis/drug effects* ; Piperazines/pharmacology* ; Bone Marrow Cells/cytology* ; Reactive Oxygen Species/metabolism* ; Signal Transduction/drug effects*

OBJECTIVE: To investigate the antioxidant and osteogenic induction capabilities of calcium phosphate nanoflowers (hereinafter referred to as nanoflowers) in vitro at different concentrations. METHODS: Nanoflowers were prepared using gelatin, tripolyphosphate, and calcium chloride. Their morphology, microstructure, elemental composition and distribution, diameter, and molecular constitution were characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive spectroscopy. Femurs and tibias were harvested from twelve 4-week-old Sprague Dawley rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured using the whole bone marrow adherent method, followed by passaging. The third passage cells were identified as stem cells by flow cytometry and then co-cultured with nanoflowers at concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, and 3.6 mg/mL. Cell counting kit 8 (CCK-8) assay was performed to screen for the optimal concentration that demonstrated the best cell viability, which was subsequently used as the experimental concentration for further studies. After co-culturing BMSCs with the screened concentration of nanoflowers, the biocompatibility of the nanoflowers was verified through live/dead cell staining, scratch assay, and cytoskeleton staining. The antioxidant capacity was assessed by using reactive oxygen species (ROS) fluorescence staining. The in vitro osteoinductive ability was evaluated via alkaline phosphatase (ALP) staining, alizarin red staining, and immunofluorescence staining of osteocalcin (OCN) and Runt-related transcription factor 2 (RUNX2). All the above indicators were compared with the control group of normally cultured BMSCs without the addition of nanoflowers. RESULTS: Scanning electron microscopy revealed that the prepared nanoflowers exhibited a flower-like structure; transmission electron microscopy scans discovered that the nanoflowers possessed a multi-layered structure, and high-magnification images displayed continuous atomic arrangements, with the nanoflower diameter measuring (2.00±0.25) μm; energy-dispersive spectroscopy indicated that the nanoflowers contained elements such as C, N, O, P, and Ca, which were uniformly distributed across the flower region; Fourier transform infrared spectroscopy analyzed the absorption peaks of each component, demonstrating the successful preparation of the nanoflowers. Through CCK-8 screening, the concentrations of 0.8, 1.2, and 1.6 mg/mL were selected for subsequent experiments. The live/dead cell staining showed that nanoflowers at different concentrations exhibited good cell compatibility, with the 1.2 mg/mL concentration being the best (P<0.05). The scratch assay results indicated that the cell migration ability in the 1.2 mg/mL group was superior to the other groups (P<0.05). The cytoskeleton staining revealed that the cell morphology was well-extended in all concentration groups, with no significant difference compared to the control group. The ROS fluorescence staining demonstrated that the ROS fluorescence in all concentration groups decreased compared to the control group after lipopolysaccharide induction (P<0.05), with the 1.2 mg/mL group showing the weakest fluorescence. The ALP staining showed blue-purple nodular deposits around the cells in all groups, with the 1.2 mg/mL group being significantly more prominent. The alizarin red staining displayed orange-red mineralized nodules around the cells in all groups, with the 1.2 mg/mL group having more and denser nodules. The immunofluorescence staining revealed that the expressions of RUNX2 and OCN proteins in all concentration groups increased compared to the control group, with the 1.2 mg/mL group showing the strongest protein expression (P<0.05). CONCLUSION The study successfully prepares nanoflowers, among which the 1.2 mg/mL nanoflowers exhibits excellent cell compatibility, antioxidant properties, and osteogenic induction capability, demonstrating their potential as an artificial bone substitute material.
Animals ; Osteogenesis/drug effects* ; Mesenchymal Stem Cells/drug effects* ; Calcium Phosphates/pharmacology* ; Rats, Sprague-Dawley ; Rats ; Antioxidants/chemistry* ; Cells, Cultured ; Cell Differentiation/drug effects* ; Nanostructures/chemistry* ; Tissue Engineering/methods* ; Bone Marrow Cells/cytology* ; Coculture Techniques ; Tissue Scaffolds/chemistry* ; Male ; Biocompatible Materials/chemistry* ; Cell Survival ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Cell Proliferation

The healing of the tendon-bone interface is a complex dynamic process involving the interaction of multiple cellular and molecular signaling pathways. Bone mesenchymal stem cells (BMSCs) have the potential to differentiate into various types of cells, including osteoblasts, chondrocytes and adipocytes, etc., and have the potential to regenerate damaged tissues. They are potential seed cells for promoting tendon-bone healing. How to precisely regulate the proliferation and differentiation of BMSCs to accelerate the process of tendon-bone healing is a current research hotspot. Monomers of traditional Chinese medicine can promote tendon-bone healing by regulating signaling pathways such as Wnt/β-catenin and BMP/Smad to induce osteogenic and chondrogenic differentiation of BMSCs. This article reviews from several aspects such as the regulatory role of related signaling pathways on tendine-bone healing, traditional Chinese medicine monomers and their mechanism of regulating BMSCs to promote tendine-bone healing in order to providing new ideas for promoting tendine-bone healing.
Mesenchymal Stem Cells/cytology* ; Humans ; Animals ; Bone Marrow Cells/cytology* ; Bone and Bones/drug effects* ; Wound Healing/drug effects* ; Medicine, Chinese Traditional ; Tendons/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Signal Transduction/drug effects* ; Cell Differentiation/drug effects*

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 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

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

BACKGROUNDSteady-state bone marrow (SS-BM) and granulocyte colony-stimulating growth factor-primed BM/peripheral blood stem-cell (G-BM/G-PBSC) are the main stem-cell sources used in allogeneic hematopoietic stem-cell transplantation. Here, we evaluated the treatment effects of SS-BM and G-BM/G-PBSC in human leucocyte antigen (HLA)-identical sibling transplantation.

METHODSA total of 226 patients (acute myelogenous leukemia-complete remission 1, chronic myelogenous leukemia-chronic phase 1) received SS-BM, G-BM, or G-PBSC from an HLA-identical sibling. Clinical outcomes (graft-versus-host disease [GVHD], overall survival, transplant-related mortality [TRM], and leukemia-free survival [LFS]) were analyzed.

RESULTSWhen compared to SS-BM, G-BM gave faster recovery time to neutrophil or platelet (P < 0.05). Incidence of grade III-IV acute GVHD and extensive chronic GVHD (cGVHD) was lower than seen with SS-BM (P < 0.05) and similar to G-PBSC. Although the incidence of cGVHD in the G-BM group was similar to SS-BM, both were lower than G-PBSC (P < 0.05). G-BM and G-PBSC exhibited similar survival, LFS, and TRM, but were significantly different from SS-BM (P < 0.05). There were no significant differences in leukemia relapse rates among the groups (P > 0.05).

CONCLUSIONSG-CSF-primed bone marrow shared the advantages of G-PBSC and SS-BM. We conclude that G-BM is an excellent stem-cell source that may be preferable to G-PBSC or SS-BM in patients receiving HLA-identical sibling hematopoietic stem-cell transplantation.

Adolescent ; Adult ; Aged ; Bone Marrow ; drug effects ; Bone Marrow Transplantation ; methods ; Child ; Female ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; therapy ; Leukemia, Myeloid, Acute ; therapy ; Male ; Middle Aged ; Retrospective Studies ; Stem Cells ; cytology ; Young Adult

The efficiency of dendritic cell-activated and cytokine-induced killer cell (DC-CIK) therapy on children with acute myeloid leukemia (AML) after chemotherapy was investigated. Mononuclear cells were collected from children achieving complete remission after chemotherapy, cultured in vitro and transfused back into the same patient. Interleukin-2 (IL-2) was injected subcutaneously every other day 10 times at the dose of 1 × 10(6) units. Peripheral blood lymphocyte subsets and minimal residual disease (MRD) were detected by flow cytometry. Function of bone marrow was monitored by methods of morphology, immunology, cytogenetics and molecular biology. The side effects were also observed during the treatment. The average follow-up period for all the 22 patients was 71 months and relapse occurred in two AML patients (9.1%). The percentage of CD3(+)/CD8(+) cells in peripheral blood of 15 patients at the 3rd month after DC-CIK treatment (36.73% ± 12.51%) was dramatically higher than that before treatment (29.20% ± 8.34%, P < 0.05). The MRD rate was >0.1% in 5 patients before the treatment, and became lower than 0.1% 3 months after the treatment. During the transfusion of DC-CIK, side effects including fever, chills and hives appeared in 7 out of 22 (31.82%) cases but disappeared quickly after symptomatic treatments. There were no changes in electrocardiography and liver-renal functions after the treatment. MRD in children with AML can be eliminated by DC-CIK therapy which is safe and has fewer side effects.
Adolescent ; Antineoplastic Agents ; therapeutic use ; Bone Marrow ; drug effects ; immunology ; pathology ; Child ; Child, Preschool ; Cytokine-Induced Killer Cells ; cytology ; immunology ; transplantation ; Dendritic Cells ; cytology ; immunology ; transplantation ; Female ; Humans ; Immunotherapy, Adoptive ; methods ; Injections, Subcutaneous ; Interleukin-2 ; therapeutic use ; Leukemia, Myeloid, Acute ; immunology ; pathology ; therapy ; Male ; Neoplasm, Residual ; Primary Cell Culture ; Recurrence ; Remission Induction ; Treatment Outcome