Chronic Injury of Mice Bone Marrow Multipotent Hematopoietic Progenitor Cells Induced by Ionizing Radiation / 中国实验血液学杂志

OBJECTIVE@#To explore the chronic injury and its possible mechanism of ionizing radiation on multipotent hematopoietic progenitor cells (MPPs) by determining the related indicators of MPPs in bone marrow of mice post-radiation.@*METHODS@#Sixteen C57BL/6 adult mice were randomly divided into normal control and irradiation groups, 8 mice in each group. The mice in irradiation group were exposed to 6 Gy X-ray. The proportion of bone marrow MPPs, their apoptosis and proliferation 2 months after irradiation were detected by flow cytometry. Mitochondrial activity and levels of reactive oxygen species (ROS) in each MPPs population were detected by Mitotracker Red and DCFDA probes, and the senescent state of MPPs in the bone marrow was analyzed.@*RESULTS@#Ionizing radiation could reduce the proportion of MPPs in mouse bone marrow. The proportions and numbers of MPP1, MPP3 and MPP4 in the bone marrow were significantly decreased after whole-body irradiation with 6 Gy X-ray (P<0.05). In addition, radiation significantly reduced the colony-forming capacity of MPPs in bone marrow (P<0.05), the proportions of apoptotic cells in the MPP1 and MPP4 cell populations increased significantly in the bone marrow (P<0.05). The activity of mitochondria was significantly reduced in the bone marrow MPP2, MPP3 and MPP4 cell populations compared with that of the control group (P<0.05). It was also found that the radiation could significantly increase the ROS levels of MPPs in bone marrow, and the content of ROS in the MPP2, MPP3 and MPP4 cell population of the bone marrow was significantly increased(P<0.05). The senescent cells ratios of MPP1, MPP3 and MPP4 cells in the bone marrow after irradiation were significantly higher than those in the control group (P<0.05).@*CONCLUSION@#Ionizing radiation can cause chronic MPPs damage in mice, which is closely associated with persistent oxidative stress, cells apoptosis, and cellular senescence.

miR-877-3p causes osteoporosis in mice by inhibiting MCP-1 secretion from mouse bone marrow mesenchymal stem cells and the migration and apoptosis of T lymphocytes / 细胞与分子免疫学杂志

Objective To investigate the effects of miR-877-3p on migration and apoptotic T lymphocytes of bone mesenchymal stem cells (BMSCs). Methods The model of osteoporosis induced by bilateral ovariectomy (OVX) and sham operation was established. At 8 weeks after operation, the bone parameters of the two groups were detected by micro-CT. The levels of monocyte chemotactic protein 1(MCP-1) in BMSCs were detected by ELISA. BMSC in OVX group and sham group were co-cultured with T lymphocytes, respectively. The migration ability of T lymphocytes in the two groups was observed by TranswellTM assay with PKH26 staining and apoptosis of T lymphocytes were detected by flow cytometry. Reverse transcription PCR was used to detect the expression of miR-877-3p in BMSCs. miR-877-3p was overexpressed or down-regulated by cell transfection. The level of MCP-1 secreted by BMSCs in each group was detected by ELISA. The migration and apoptosis of T lymphocytes were detected by the above methods. Results The number of trabecular bone and bone mineral density in OVX group were lower than those in sham group. The levels of MCP-1 secretion, chemotactic and apoptotic T lymphocyte ability of BMSCs in OVX group were also lower than those in sham group. The expression level of miR-877-3p in BMSC in OVX group was higher than that in sham group. After overexpression of BMSC miR-877-3p, the levels of MCP-1 secreted from BMSCs, and apoptotic T lymphocytes decreased, while the results were opposite after down-regulation of miR-877-3p. Conclusion miR-877-3p may be one of the causes of osteoporosis by inhibiting MCP-1 secretion of BMSCs and the migration and apoptosis of T lymphocytes.

Expression Changes of Hypoxia-Inducible Factor-1α in G-CSF Induced Hematopoietic Stem Cell Mobilization / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression and its relative mechanism of hypoxia-inducible factor-1α(HIF-1α) in bone marrow(BM) of mice during G-CSF mobilization of hematopoietic stem cells (HSC) .@*METHODS@#Flow cytometry was used to detect the proportion of Lin-Sca-1+ c-kit+ (LSK) cells in peripheral blood of C57BL/6J mice before and after G-CSF mobilization. And the expression of HIF-1α and osteocalcin (OCN) mRNA and protein were detected by RQ-PCR and immunohistochemistry. The number of osteoblasts in bone marrow specimens of mice was counted under the microscope.@*RESULTS@#The proportion of LSK cells in peripheral blood began to increase at day 4 of G-CSF mobilization, and reached the peak at day 5, which was significantly higher than that of control group (P<0.05). There was no distinct difference in the expression of HIF-1α mRNA between bone marrow nucleated cells and osteoblasts of steady-state mice (P=0.073), while OCN mRNA was mainly expressed in osteoblasts, which was higher than that in bone marrow nucleated cells (P=0.034). After mobilization, the expression level of HIF-1α increased, but OCN decreased, and the number of endosteum osteoblasts decreased. The change of HIF-1α expression was later than that of OCN and was consistent with the proportion of LSK cells in peripheral blood.@*CONCLUSION@#The expression of HIF-1α in bone marrow was increased during the mobilization of HSC mediated by G-CSF, and one of the mechanisms may be related to the peripheral migration of HSC induced by osteoblasts inhibition.

Effect of PKM2 on Osteogenic and Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Myeloma Bone Disease / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of pyruvate kinase M2 (PKM2) in bone marrow mesenchymal stem cells (BMSCs) in myeloma bone disease (MBD) and its effect on osteogenic and adipogenic differentiation of BMSCs.@*METHODS@#BMSCs were isolated from bone marrow of five patients with multiple myeloma (MM) (MM group) and five with iron deficiency anemia (control group) for culture and identification. The expression of PKM2 protein were compared between the two groups. The differences between osteogenic and adipogenic differentiation of BMSCs were assessed by using alkaline phosphatase (ALP) and oil red O staining, and detecting marker genes of osteogenesis and adipogenesis. The effect of MM cell line (RPMI-8226) and BMSCs co-culture on the expression of PKM2 was explored. Functional analysis was performed to investigate the correlations of PKM2 expression of MM-derived BMSCs with osteogenic and adipogenic differentiation by employing PKM2 activator and inhibitor. The role of orlistat was explored in regulating PKM2 expression, osteogenic and adipogenic differentiation of MM-derived BMSCs.@*RESULTS@#Compared with control, MM-originated BMSCs possessed the ability of increased adipogenic and decreased osteogenic differentiation, and higher level of PKM2 protein. Co-culture of MM cells with BMSCs markedly up-regulated the expression of PKM2 of BMSCs. Up-regulation of PKM2 expression could promote adipogenic differentiation and inhibit osteogenic differentiation of MM-derived BMSCs, while down-regulation of PKM2 showed opposite effect. Orlistat significantly promoted osteogenic differentiation in MM-derived BMSCs via inhibiting the expression of PKM2.@*CONCLUSION@#The overexpression of PKM2 can induce the inhibition of osteogenic differentiation of BMSCs in MBD. Orlistat can promote the osteogenic differentiation of BMSCs via inhibiting the expression of PKM2, indicating a potential novel agent of anti-MBD therapy.

Bone Marrow Adipocytes Promote the Survival of Multiple Myeloma Cells and Up-Regulate Their Chemoresistance / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM.@*METHODS@#Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4, FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor.@*RESULTS@#The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4 and FASN, but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells.@*CONCLUSION@#The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.

Connexin 43-modified bone marrow stromal cells reverse the imatinib resistance of K562 cells via Ca 2+ -dependent gap junction intercellular communication / 中华医学杂志(英文版)

BACKGROUND@#Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown.@*METHODS@#Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca 2+ -related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance.@*RESULTS@#Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca 2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments.@*CONCLUSIONS@#Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.

Effect of wheat-grain moxibustion on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition / 中国针灸

OBJECTIVE@#To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition, and to explore the possible mechanism of wheat-grain moxibustion in treating bone marrow inhibition.@*METHODS@#Forty-five SPF male CD1(ICR) mice were randomly divided into a blank group, a model group and a wheat-grain moxibustion group, 15 mice in each group. The bone marrow inhibition model was established by intraperitoneal injection of 80 mg/kg of cyclophosphamide (CTX). The mice in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), 3 moxa cones per acupoint, 30 s per moxa cone, once a day, for 7 consecutive days. The white blood cell count (WBC) was measured before modeling, before intervention and 3, 5 d and 7 d into intervention. After intervention, the general situation of mice was observed; the number of nucleated cells in bone marrow was detected; the serum levels of interleukin-3 (IL-3), interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF) were measured by ELISA; the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc in bone marrow cells was measured by Western blot and real-time PCR method.@*RESULTS@#Compared with the blank group, the mice in the model group showed sluggish reaction, unstable gait, decreased body weight, and the WBC, number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were decreased (P<0.01), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was decreased (P<0.01). Compared with the model group, the mice in the wheat-grain moxibustion group showed better general condition, and WBC, the number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were increased (P<0.01, P<0.05), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was increased (P<0.05).@*CONCLUSION@#Wheat-grain moxibustion shows therapeutic effect on bone marrow inhibition, and its mechanism may be related to activating Wnt/β-catenin signaling pathway in bone marrow cells, improving bone medullary hematopoiesis microenvironment and promoting bone marrow cell proliferation.

Research progress in biological characteristics and influencing factors of jaw bone marrow mesenchymal stem cell / 中华口腔医学杂志

Jaw bone marrow mesenchymal stem cell (JBMMSC), which exists in the maxilla and mandible, is adult stem cells with strong proliferation ability and multiple differentiation potential. Pathological, physicochemical and biological factors can affect the biological characteristics of JBMMSC. Compared with bone marrow mesenchymal stem cells derived from long bone, the biological characteristics of JBMMSC are site-specific because of the different sources of tissue and osteogenesis of bone. The same influencing factors have different effects on these two kinds of cells. Besides, JBMMSC also has the advantages of easier access, less trauma and lower immunogenicity. It has broad application prospects in craniomaxillofacial defect repair, periodontal tissue regeneration, and improving the success rate after implantation and so on. It has attracted wide attention in the basic and clinical studies. However, the regulation mechanism of its proliferation and differentiation is not clear, which affects its application as seed cell. Therefore, this paper reviews the biological characteristics influencing factors of JBMMSC and application progress in clinical and basic research, aiming to provide reference for further research and clinical application.

Morphological Characteristics of Bone Marrow Cells in Patients with EB Virus Infection / 中国实验血液学杂志

OBJECTIVE@#Review and analyze the characteristics of bone marrow cell morphology in patients with Epstein-Barr virus (EBV) infection, and explore the diagnostic value of bone marrow cell morphology for the early identification of EBV infection.@*METHODS@#A total of 33 patients with EBV-DNA positive detection in the First Affiliated Hospital of Guangxi Medical University from January 2018 to May 2021 were collected as the research objects. Bone marrow cell morphology and peripheral blood cell analysis were performed, and the significance in disease diagnosis was analyzed by statistical methods.@*RESULTS@#The sampling satisfaction of 33 patients with EBV infection was 100%. In the clinical diagnosis of all cases, 7 cases were IM, 17 cases were EBV-HLH, 3 cases were lymphoma, 2 cases were EBV-associated lymphoid hyperplasia, and 4 cases were not diagnosed. Among them, 31 patients had active bone marrow hyperplasia or above, 26 patients had active granulocytic hyperplasia or above, 21 patients had active erythroid hyperplasia or above, and 17 cases of megakaryocyte production platelet function decreased. The abnormal components of bone marrow mainly indude atypical lymphocyte cells (33 cases), hemophagocytic cells (22 cases), abnormal histiocyte (10 cases).@*CONCLUSION@#According to the proliferation of granulocytes, erythrocytes and megakaryocytes in the bone marrow, and the emergence of abnormal components such as atypical lymphocytes, hemophagocyte, abnormal histiocyte. Bone marrow cell morphological examination can indicate the possibility of EBV infection, which is certain diagnostic value for early identification of EBV infection.

Effect and Mechanism of Cxcr4 Gene-Modified BMSC-Derived Exosomes on Aplastic Anemia / 中国实验血液学杂志

OBJECTIVE@#To explore the improvement effect of CXC chemokine receptor 4 (Cxcr4) gene-modified bone marrow mesenchymal stem cell (BMSC)-derived exosomes on aplastic anemia (AA), and make a preliminary exploration of the mechanism.@*METHODS@#Mouse BMSCs were isolated and cultured, then infected by recombinant lentivirus carrying Cxcr4 gene. The expression of green fluorescence was observed through fluorescence microscope, the expression of Cxcr4 mRNA was detected by real-time fluorescence quantitative PCR, and the BMSC-derived exosomes modified with Cxcr4 gene were extracted. Mouse models of AA were constructed, and control group, model group (AA), AA+BMSC group, AA+NC-BMSC group, AA+Cxcr4-BMSC group were set up. Except control group and model group, the other three groups of mice were injected 400 μl exosomes from different sources via the tail vein, after 2 weeks, the routine blood indices and the number of bone marrow nucleated cells were detected, the pathological changes of bone marrow were observed by HE staining, and the expression level of Treg cells was detected by flow cytometry.@*RESULTS@#Mouse BMSCs were successfully isolated, and BMSCs with high expression of Cxcr4 and their exosomes were obtained. Compared with the control group, the number of red blood cell (RBC), white blood cell (WBC), and platelet (PLT), the hemoglobin (Hb) content and proportion of Treg cells in the peripheral blood of mice in the model group significantly decreased (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01). The proliferation level of nucleated cells was low, and the medullary cavity was filled with a large number of fat cells. Compared with the model group, the number of RBC, WBC, PLT, the Hb content and proportion of Treg cells in the peripheral blood of mice in the AA+BMSC group, AA+NC-BMSC group, and AA+Cxcr4-BMSC group significantly increased (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01), and pathological changes of bone marrow were improved. In addition, the number of RBC, WBC, PLT, the Hb content and proportion of Treg cells in the peripheral blood of mice in the AA+Cxcr4-BMSC group were significantly higher than those in the AA+BMSC group (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01).@*CONCLUSION@#Injection of Cxcr4 gene-modified BMSC-derived exosomes has a certain improvement effect on AA mice, and the mechanism may be related to an increase of the proportion of Treg cells.

A 3D hydrogel loaded with exosomes derived from bone marrow stem cells promotes cartilage repair in rats by modulating immunological microenvironment / 南方医科大学学报

OBJECTIVE@#To assess the efficacy of GelMA hydrogel loaded with bone marrow stem cell-derived exosomes for repairing injured rat knee articular cartilage.@*METHODS@#The supernatant of cultured bone marrow stem cells was subjected to ultracentrifugation separate and extract the exosomes, which were characterized by transmission electron microscopy, particle size analysis and Western blotting of the surface markers. The changes in rheology and electron microscopic features of GelMA hydrogel were examined after loading the exosomes. We assessed exosome release from the hydrogel was detected by BCA protein detection method, and labeled the exosomes with PKH26 red fluorescent dye to observe their phagocytosis by RAW264.7 cells. The effects of the exosomes alone, unloaded hydrogel, and exosome-loaded hydrogel on the polarization of RAW264.7 cells were detected by q-PCR and immunofluorescence assay. We further tested the effect of the exosome-loaded hydrogel on cartilage repair in a Transwell co-culture cell model of RAW264.7 cells and chondrocytes in a rat model of knee cartilage injury using q-PCR and immunofluorescence assay and HE and Masson staining.@*RESULTS@#GelMA hydrogel loaded with exosomes significantly promoted M2-type polarization of RAW264.7 cells (P < 0.05). In the Transwell co-culture model, the exosome-loaded GelMA hydrogel significantly promoted the repair of injured chondrocytes by regulating RAW264.7 cell transformation from M1 to M2 (P < 0.05). HE and Masson staining showed that the exosome-loaded hydrogel obviously promoted cartilage repair in the rat models damage.@*CONCLUSION@#GelMA hydrogel loaded with bone marrow stem cell-derived exosomes can significantly promote the repair of cartilage damage in rats by improving the immune microenvironment.

Effect of Acute Myeloid Leukemia Cells on the Proliferation and Apoptosis of Bone Marrow-Derived Mesenchymal Stromal Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of acute myeloid leukemia cells in leukemia-microenvironment on proliferation and apoptosis of bone marrow-derived mesenchymal stromal cells (BM-MSC).@*METHODS@#Acute myeloid leukemia (AML) murine models overexpressing MLL-AF9 were established. The number of BM-MSC of wild type (WT) and AML-derived mice were analyzed by flow cytometry. Morphology and growth differences between WT and AML-derived BM-MSC were analyzed by inverted fluorescence microscope. Proliferation and apoptosis of BM-MSC between these two groups were detected by Brdu and Annexin V/PI.@*RESULTS@#Compared with WT-derived BM-MSC, the number and proliferation rate of AML-derived BM-MSC significantly increased (P<0.01, P<0.001), while apoptosis rate decreased (P<0.05). When cultured in vitro, BM-MSC grew faster under conditional medium.@*CONCLUSION@#AML cells can promote proliferation and inhibit apoptosis of BM-MSC.

Research Advances on the Role of Bone Marrow Stromal Cell in Acute Lymphoblastic Leukemia --Review / 中国实验血液学杂志

Acute lymphoblastic leukemia (ALL) is a kind of the most common hematopoietic malignancy, its recurrence and drug resistance are closely related to the bone marrow microenvironment. Bone marrow stromal cell (BMSC) is an important part of the bone marrow microenvironment and their interaction with leukemia cells cannot be ignored. BMSC participates in and regulate signaling pathways related to proliferation or apoptosis of ALL cells by secretes cytokines or extracellular matrix proteins, thus affecting the survival of ALL cells. In this review, the research advance of several signaling pathways of the interaction between BMSC and ALL cells was summarized briefly.

Establishment of a platelet production model by bone marrow cavity transplantation of mouse primary megakaryocytes / 中华血液学杂志

Objective: To establish an intramedullary transplantation model of primary megakaryocytes to evaluate the platelet-producing capacity of megakaryocytes and explore the underlying regulatory mechanisms. Methods: Donor megakaryocytes from GFP-transgenic mice bone marrow were enriched by magnetic beads. The platelet-producing model was established by intramedullary injection to recipient mice that underwent half-lethal dose irradiation 1 week in advance. Donor-derived megakaryocytes and platelets were detected by immunofluorescence staining and flow cytometry. Results: The proportion of megakaryocytes in the enriched sample for transplantation was 40 to 50 times higher than that in conventional bone marrow. After intramedullary transplantation, donor-derived megakaryocytes successfully implanted in the medullary cavity of the recipient and produce platelets, which showed similar expression of surface markers and morphology to recipient-derived platelets. Conclusion: We successfully established an in vivo platelet-producing model of primary megakaryocytes using magnetic-bead enrichment and intramedullary injection, which objectively reflects the platelet-producing capacity of megakaryocytes in the bone marrow.

Knockdown of long non-coding RNA MIR4697 host gene inhibits adipogenic differentiation in bone marrow mesenchymal stem cells / 北京大学学报(医学版)

OBJECTIVE@#To preliminarily investigate the role of long non-coding RNA (lncRNA) MIR4697 host gene (MIR4697HG) in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#For adipogenic differentiation, BMSCs were induced in adipogenic media for 10 days. The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhanced binding protein α (CEBP/α) and adiponectin (ADIPQ) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) at different time points (0, 1, 2, 3, 5, 7, 10 days). The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses. To avoid off-target effect, two target sequences (shMIR4697HG-1, shMIR4697HG-2) were designed. And then cells were induced to differentiate in adipogenic medium. Oil red O staining, Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs.@*RESULTS@#The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation (P < 0.01), and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ. Observed by fluorescence microscopy, more than 90% transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection for 72 h. And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%. Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P < 0.01), while the expression levels of PPARγ and CEBP/α proteins were decreased remarkably as well (P < 0.01). Consistently, MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs, which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs.@*CONCLUSION@#lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs, and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs. These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.

Analysis of Proliferation Characters of Bone Marrow Mesenchymal Stem Cells Derived from Patients with Myelodysplastic Syndrome / 中国实验血液学杂志

OBJECTIVE@#To analyze the proliferation potential of bone marrow-derived mesenchymal stem cells (MSC) in patients with myelodysplastic syndrome (MDS).@*METHODS@#The MSC derived from the 24 patients with newly diagnosed MDS (MDS-MSC group) and MSC derived from 15 patients with nutritional anemia (control group) in the Affiliated Hospital of Hebei University were used as the research objects. The proliferation potential of MSC was analyzed by colony-forming unit assay, doubling time, cumulative passaging, cell number after 10 days of culture with equal amount of MSC and MTT experiment. The mechanism of abnormal proliferation was analyzed by cell cycle experiment, apoptosis experiment and p21 gene expression assay.@*RESULTS@#In the colony forming unit assay, the number of MDS-MSC colonies was 4.44±2.51, which was significantly lower than that of the control group (12.44±2.55)(P<0.01); the doubling time of MDS-MSC group was significantly longer than that of the control group (7.80±3.26 vs 3.63±0.85) (P<0.01); the number of MDS-MSC in 5×10@*CONCLUSION@#The proliferative capability of MDS-MSC is significantly reduced, which relates with the arrest of cell cycle in G

Construction and identification of recombinant adenovirus vector and observation of its transfecting rabbit bone marrow mesenchymal stem cells / 中国骨伤

OBJECTIVE@#To construct and identify adenovirus vector co-expressing hBMP2 and hVEGF165 fusion protein which labeled with green fluorescence protein, and laying the foundtion of the effect of hBMP2 and hVEGF165 gene inducing BMMSCs differentiation to osteoblast and bone defect repaired in the body.@*METHODS@#BMP2 and VEGF165 gene was amplified from cDNA library by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. Ad-BMP2- VEGF165 was recombinated and propagated in HEK293 cells by co-transfecting with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ E1, 3Cre. The recombinant adenovirus was purified and virustiter was determined, and then to research GFP expression and to calculate the adenovirus transfection rate in rabbit BMMSCs.@*RESULTS@#The recombinant adenovirus vector Ad-BMP2-VEGF165 was successfully constructed by the methods of gene analyzing, colony PCR, Western blotting and observing GFP expression, and the titer of the adenovirus was 1×10@*CONCLUSION@#Recombinant adenovirus vector containing hBMP2 and hVEGF165 gene was successfully constructed and its high titer was obtained.

Role of Bone Marrow Cell Morphology Combined with Immunohistochemistry Examinations in the Diagnosis of Patients with Primary Bone Marrow Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To explore the diagnostic value of bone marrow cell morphology combined with immunohistochemistry in patients with primary bone marrow lymphoma.@*METHODS@#The clinical data of 23 patients with primary bone marrow lymphoma diagnosed in the First Affiliated Hospital of Xi'an Jiaotong University from January 2010 to December 2019 were collected. The characteristics of bone marrow aspiration, bone marrow biopsy and immunohistochemistry results were analyzed retrospectively, and the diagnostic value of bone marrow cell morphology combined with immunohistochemistry in primary bone marrow lymphoma were clarified.@*RESULTS@#Most of primary bone marrow lymphoma was B-cell lymphoma, among which diffuse large B-cell lymphoma was the most common pathological type. Typical lymphoma cells could be found in all the patients. 78.26% of the patients could be diagnosed as lymphoma with pathological type, while 91.30% were diagnosed as lymphoma through combined with the bone marrow immunohistochemistry.@*CONCLUSION@#Bone marrow cell morphology combined with immunohistochemistry shows very important diagnostic value in patients with primary bone marrow lymphoma.

Effects of HMGB1 on Proliferation and Secretion of Human Bone Marrow Mesenchymal Stem Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of high mobility group protein 1(HMGB1) on the proliferation and cytokine expression of human bone marrow mesenchymal stem cells (MSC).@*METHODS@#Different concentrations of recombinant human HMGB1 protein (100, 200, 400, 800 and 1000 ng/ml) were incubated with MSC for 24, 48, 72 h and the proliferation of MSC were detected respectively by using the CCK-8 method and flow cytometry. The best concentrations of HMGB1 incubated with MSC was determined (200 ng/ml, 1000 ng/ml), and the flow cytomerty was used to determine the effect of HMGB1 on the proliferation of MSC. The mRNA expression levels of IL-10, TGF- β1, TSG-6 and IFN-γ in MSC incubated with HMGB1 protein were detected by real-time quantitative PCR and ELISA.@*RESULTS@#The result of MSC identification and flow cytometry showed that the CD105, CD73 and CD90 were expressed, but did not expression CD45, CD34, CD11b, CD19 and HLA-DR; CCK-8 showed that HMGB1 at the concentrations of 100 ng/ml, 200 ng/ml and 400 ng/ml could promote the proliferation of MSC incubated for 24, 48 and 72 h as compared with the control group (P<0.05), and the most effective concentration was 200 ng/ml; flow cytometry showed that the compared with the control group, HMGB1 200 ng/ml could induce MSC from G1 phase to S phase to promote the proliferation of MSC; QPCR showed that the mRNA expression of MSC cytokines IL-10, TGF-β1, TSG-6 increased while IFN-γ decreased at the concentration of 200 ng/ml HMGB1 as compared with the control group. ELISA experiments showed that the HMGB1 200 ng/ml acting on MSC for 48 h could significantly promoted the secretion of IL-10, TGF-β 1 and TSG-6(P<0．05), while IFN-γ showed no significant difference as compared with control group.@*CONCLUSION@#Recombinant human HMGB1 can promote the proliferation and secretion of MSC in healthy people.

Proliferation kinetics of immune cells during early phase of bone marrow transplantation in mouse model based on chemotherapy conditioning / 中南大学学报(医学版)

OBJECTIVES@#To establish mouse bone marrow transplantation by pretreatment with chemotherapy, and to explore the dynamic changes of immune cells in the early stage of allogeneic transplantation in the spleen of mice.@*METHODS@#Mice were divided into 4 groups (80 mg/kg group, 100 mg/kg group, 120 mg/kg group, and 150 mg/kg group) according to the difference in dose of busulfan. The mice were treated with busulfan and cyclophosphamide combined chemotherapy, and the appropriate dosage was determined by evaluating the myeloablative effect and drug toxicity. According to the type of the genetic transplantation, the mice were also divided into 4 groups: An allogeneic transplantation group, a homogenic transplantation group, a chemotherapy alone group, and a normal control group. The mice were pretreated with busulfan and cyclophosphamide before bone marrow transplantation. In the allogeneic transplantation group, the suspension of splenocytes was prepared at the first day, the 3rd day, the 5th day, and the 8th day after transplantation for flow cytometry detection, and the dynamic changes of splenic immune cells were analyzed. The homogeneic transplantation group served as the concurrent control, the normal control group served as the control of basic value of spleen immune cells, and the chemotherapy alone group was used to evaluate the myeloablative effect.@*RESULTS@#1) The optimal dose of busulfan was 100 mg/kg. The combination of busulfan and cyclophosphamide can restore the hematopoiesis of transplanted mice, and the toxicity associated with pretreatment is small. 2) In the allogeneic transplantation group: The hematopoietic reconstitution and high donor chimerism rate were achieved after transplantation. In the early phase of bone marrow transplantation, the T lymphocytes were the main cell group, while the recovery of B lymphocytes was relatively delayed. The dendritic cells and natural killer cells from donors were the earliest cells to recover and achieve high chimerism rate compared with T cells and B cells. Most T cells were in the initial T cell state within 5 days after allogeneic transplantation. However, in the 5th day after transplantation, these cells were mainly in the effective memory phenotype. The reconstruction of donor-derived naive T cells was slow, but the reconstruction of donor-derived effective memory T cells and regulatory T cells was relatively fast. 3) In the homogeneic transplantation group: The mice could recover hematopoiesis and the recovery of B lymphocytes was delayed. 4) In the chemotherapy alone group: All mice died in 12-15 days after chemotherapy, and the peripheral blood routine showed pancytopenia before death.@*CONCLUSIONS@#Pretreatment with chemotherapy can successfully establish the mouse model of bone marrow transplantation. There are difference in the proportion of T cells, B cells, natural killer cells, dendritic cells, effector memory T cells, initial T cells, and regulatory T cells after transplantation, and the relationship between donor and recipient is also changed.