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SUMMARY: Senile osteoporosis is mainly caused by reduced osteoblast differentiation and has become the leading cause of fractures in the elderly worldwide. Natural organics are emerging as a potential option for the prevention and treatment of osteoporosis. This study was designed to study the effect of resveratrol on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteoporosis mice. A mouse model of osteoporosis was established by subcutaneous injection of dexamethasone and treated with resveratrol administered by gavage. In vivo and in vitro, we used western blot to detect protein expression, and evaluated osteogenic differentiation of BMSCs by detecting the expression of osteogenic differentiation related proteins, calcium deposition, ALP activity and osteocalcin content. Resveratrol treatment significantly increased the body weight of mice, the level of serum Ca2+, 25(OH)D and osteocalcin, ration of bone weight, bone volume/total volume, trabecular thickness, trabecular number, trabecular spacing and cortical thickness in osteoporosis mice. In BMSCs of osteoporosis mice, resveratrol treatment significantly increased the expression of Runx2, osterix (OSX) and osteocalcin (OCN) protein, the level of calcium deposition, ALP activity and osteocalcin content. In addition, resveratrol treatment also significantly increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT in BMSCs of osteoporosis mice. In vitro, resveratrol increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT, Runx2, OSX and OCN protein, the level of calcium deposition, ALP activity and osteocalcin content in BMSCs in a concentration-dependent manner, while SIRT1 knockdown significantly reversed the effect of resveratrol. Resveratrol can attenuate osteoporosis by promoting osteogenic differentiation of bone marrow mesenchymal stem cells, and the mechanism may be related to the regulation of SIRT1/PI3K/AKT pathway.
La osteoporosis senil es causada principalmente por una diferenciación reducida de osteoblastos y se ha convertido en la principal causa de fracturas en las personas mayores en todo el mundo. Los productos orgánicos naturales están surgiendo como una opción potencial para la prevención y el tratamiento de la osteoporosis. Este estudio fue diseñado para estudiar el efecto del resveratrol en la diferenciación osteogénica de las células madre mesenquimales de la médula ósea (BMSC) en ratones con osteoporosis. Se estableció un modelo de osteoporosis en ratones mediante inyección subcutánea de dexametasona y se trató con resveratrol administrado por sonda. In vivo e in vitro, utilizamos Western blot para detectar la expresión de proteínas y evaluamos la diferenciación osteogénica de BMSC detectando la expresión de proteínas relacionadas con la diferenciación osteogénica, la deposición de calcio, la actividad de ALP y el contenido de osteocalcina. El tratamiento con resveratrol aumentó significativamente el peso corporal de los ratones, el nivel sérico de Ca2+, 25(OH)D y osteocalcina, la proporción de peso óseo, el volumen óseo/ volumen total, el espesor trabecular, el número trabecular, el espaciado trabecular y el espesor cortical en ratones con osteoporosis. En BMSC de ratones con osteoporosis, el tratamiento con resveratrol aumentó significativamente la expresión de las proteínas Runx2, osterix (OSX) y osteocalcina (OCN), el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina. Además, el tratamiento con resveratrol también aumentó significativamente la expresión de SIRT1, p-PI3K/PI3K y p-AKT/AKT en BMSC de ratones con osteoporosis. In vitro, el resveratrol aumentó la expresión de las proteínas SIRT1, p-PI3K/PI3K y p- AKT/AKT, Runx2, OSX y OCN, el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina en BMSC de manera dependiente de la concentración, mientras que La caída de SIRT1 revirtió significativamente el efecto del resveratrol. El resveratrol puede atenuar la osteoporosis al promover la diferenciación osteogénica de las células madre mesenquimales de la médula ósea, y el mecanismo puede estar relacionado con la regulación de la vía SIRT1/PI3K/AKT.
Subject(s)
Animals , Male , Mice , Osteoporosis/drug therapy , Resveratrol/administration & dosage , Osteogenesis/drug effects , Cell Differentiation/drug effects , Blotting, Western , Disease Models, Animal , Sirtuin 1 , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Resveratrol/pharmacology , Mice, Inbred C57BLABSTRACT
OBJECTIVE@#To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ).@*RESULTS@#Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area.@*CONCLUSION@#After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.
Subject(s)
Female , Mice , Animals , Core Binding Factor Alpha 1 Subunit/pharmacology , PPAR gamma/metabolism , Steroid 12-alpha-Hydroxylase/metabolism , Mice, Inbred C57BL , Cell Differentiation , Osteogenesis , Mesenchymal Stem Cells , Bile Acids and Salts/pharmacology , Bone Marrow Cells , Cells, Cultured , Azo CompoundsABSTRACT
Objective@#To investigate the osteogenic properties of a methacrylated gelatin (GelMA) / bone marrow mesenchymal stem cells (BMSCs) composite hydrogel applied to the skull defect area of rats and to provide an experimental basis for the development of bone regeneration biomaterials.@*Methods@#This study was approved by the Animal Ethics Committee of Nanjing University. A novel photocurable composite biohydrogel was developed by constructing photoinitiators [lthium phenyl (2,4,6-trimethylbenzoyl) phosphinate, LAP], GelMA, and BMSCs. The surface morphology and elemental composition of the gel were examined using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). The compressive strength of the gel was evaluated using an electronic universal testing machine. After in vitro culture for 1, 2, and 5 days, the proliferation of the BMSCs in the hydrogels was assessed using a CCK-8 assay, and their survival and morphology were examined through confocal microscopy. A 5 mm critical bone deficiency model was generated in a rat skull. The group receiving composite hydrogel treatment was referred to as the GelMA/BMSCs group, whereas the untreated group served as the control group. At the 4th and 8th weeks, micro-CT scans were taken to measure the bone defect area and new bone index, while at the 8th week, skull samples from the defect area were subjected to H&E staining, van Gieson staining, and Goldner staining to evaluate the quality of bone regeneration and new bone formation.@*Results@#SEM observed that the solidified GelMA showed a 3D spongy gel network with uniform morphology, the porosity of GelMA was 73.41% and the pore size of GelMA was (28.75 ± 7.13) μm. EDX results showed that C and O were evenly distributed in the network macroporous structure of hydrogel. The hydrogel compression strength was 152 kPa. On the 5th day of GelMA/BMSCs culture, the cellular morphology transitioned from oval to spindle shaped under microscopic observation, accompanied by a significant increase in cell proliferation (159.4%, as determined by the CCK-8 assay). At 4 weeks after surgery, a 3D reconstructed micro-CT image revealed a minimal reduction in bone defect size within the control group and abundant new bone formation in the GelMA/BMSCs group. At 8 weeks after surgery, no significant changes were observed in the control group's bone defect area, with only limited evidence of new bone growth; however, substantial healing of skull defects was evident in the GelMA/BMSCs group. Quantitative analysis at both the 4- and 8-week examinations indicated significant improvements in the new bone volume (BV), new bone volume/total bone volume (BV/TV), bone surface (BS), and bone surface/total bone volume (BS/TV) in the GelMA/BMSCs group compared to those in the control group (P<0.05). Histological staining showed continuous and dense formation of bone tissue within the defects in the GelMA/BMSCs group and only sporadic formation of new bone, primarily consisting of fibrous connective tissue, at the defect edge in the control group.@*Conclusion@#Photocuring hydrogel-based stem cell therapy exhibits favorable biosafety profiles and has potential for clinical application by inducing new bone formation and promoting maturation within rat skull defects.
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The senescence of bone marrow mesenchymal stem cells (BM-MSCs) will induce age-related bone tissue degeneration and chronic inflammation, and reduce its application effect for cell therapy. More and more active ingredients of traditional chinese medicine have been proved to intervene BM - MSCs senescence, playing an important role in bone diseases prevention and treatment, and improving the therapeutic effect of BM-MSCs. In this paper, the latest research progress on the molecular mechanism of traditional chinese medicine active ingredients interfering BM-MSCs senescence was summarized, in order to provide new direction and reference basis for senescence intervention research and clinical application improvement of BM-MSCs.
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Aim To investigate the effect of miR-141-5p/ZNF705A in chronic myeloid leukemia(CML)cell-derived exosome(Exo)on the adhesion of bone marrow mesenchymal stem cells(BMSCs). Methods The morphology and size of Exo in peripheral blood from CML patients and K562 cells were examined by electron microscopy and NTA particle size analysis. The expressions of Exo and BMSCs marker molecules and adhesion proteins in K562 cells were detected by qRT-PCR and Western blot before and after transfection. The adhesion ability of BMSCs was detected by cell adhesion assay, and the cellular activity of BMSCs was examined using CCK-8. miR-141-5p binding to ZNF705A was detected by luciferase assay. Results qRT-PCR results showed that miR-141-5p expression was significantly reduced in both CML patients and K562 cell-derived Exo. qRT-PCR, Western blot and other results showed that BMSCs in CML patients had significantly reduced the expression of adhesion proteins CD44 and CXCL12, and were able to phagocytose K562 cell-derived Exo. Further, K562-derived Exo was found to reduce CD44 and CXCL12 expression and adhesion in Exo-promoted BMSCs compared with CD34+ cells. Meanwhile, the results of dual luciferase reporter assay verified that miR-141-5p targeted binding to ZNF705A. Finally, we found ZNF705A could be targeted by up-regulating miR-141-5p expression in Exo of K562 cells, which in turn inhibited the adhesion of BMSCs. Conclusions K562 cells down-regulate miR-141-5p expression in Exo and inhibit the adhesion function of BMSCs by targeting ZNF705A, thus regulating the bone marrow hematopoietic function in CML patients.
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OBJECTIVE@#To explore the effect of human bone marrow mesenchymal stem cells (MSCs) with ectopic high OCT4 expression on T-cell proliferation, activation and secretion in vitro.@*METHODS@#Peripheral blood mononuclear cells were isolated from healthy children. Anti-CD3 and anti-CD28 monoclonal antibodies were used to activate T lymphocytes, which were stimulated by interleukin (IL)-2 for one week in vitro. Then MSCs with ectopic high OCT4 expression (MSC-OCT4) were co-cultured with activated T lymphocytes. After one week of co-culture, the supernatant was collected and the levels of Th1/Th2 cytokines [IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α and interferon (IFN)-γ] were determined by flow cytometry. The lymphocytes after one week of co-culture were collected and counted by Countstar software. After the proportions of activated/inactivated T cell subsets were determined by flow cytometry, the absolute lymphocyte counts were calculated and expressed as mean ± standard deviation.@*RESULTS@#Compared with control T cell alone culture group, the proliferation of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells were significantly inhibited in MSC group and MSC-OCT4 group. Compared with MSC, MSC-OCT4 could inhibit CD3+CD8+ T cell proliferation better (P =0.049), and mainly inhibited early T cell activation. Compared with control T cell alone culture group, the levels of IL-2 and INF-γ were significantly down-regulated both in MSC group and MSC-OCT4 group.After co-culture with T cells for one week, the level of IL-6 significantly increased in MSC group and MSC-OCT4 group compared with that before co-culture. Compared with control MSC group, MSC-OCT4 group had higher viable cell numbers after 1 week of co-culture (P =0.019), and could resist the inhibition of proliferation by higher concentration of mitomycin C.@*CONCLUSION@#Both MSC and MSC-OCT4 can inhibit the proliferation and activation of IL-2-stimulated T cells in vitro. After overexpression of OCT4, MSC has better proliferation ability in vitro and can inhibit the proliferation of CD3+CD8+ T cells more effectively, which may have a better and more lasting immunosuppressive ability to regulate the balance of Th1/Th2.
Subject(s)
Child , Humans , Bone Marrow Cells , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation , Cells, Cultured , Interleukin-2 , Interleukin-6/metabolism , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation , Mesenchymal Stem Cells , Tumor Necrosis Factor-alpha/metabolismABSTRACT
OBJECTIVE@#To explore the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and the combination of bFGF and EGF in the neural differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and the role of Wnt/β-catenin signaling pathway in this process.@*METHODS@#The identified 4th-generation hBMSCs were divided into five groups according to different induction conditions, namely control group (group A), EGF induction group (group B), bFGF induction group (group C), EGF and bFGF combined induction group (group D), and EGF, bFGF, and Dickkopf-related protein 1 (DKK-1) combined induction group (group E). After 7 days of continuous induction, the cell morphology was observed by inverted fluorescence phase contrast microscopy, levels of genes that were related to neural cells [Nestin, neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP)] and key components of the Wnt/β-catenin signaling pathway (β-catenin and Cyclin D1) were detected by RT-PCR, and the levels of proteins that were related to neural cells (Nestin and GFAP) as well as genes that were involved in Wnt/β-catenin signaling pathway [β-catenin, phosphorylation β-catenin (P-β-catenin), Cytoplasmic β-catenin, and Nuclear β-catenin] were explored by cellular immunofluorescence staining and Western blot.@*RESULTS@#When compared to groups A and B, the typical neuro-like cell changes were observed in groups C-E, and most obviously in group D. RT-PCR showed that the relative expressions of Nestin, NSE, and MAP-2 genes in groups C-E, the relative expressions of GFAP gene in groups D and E, the relative expression of NSE gene in group B, the relative expressions of β-catenin gene in groups C and D, and the relative expressions of Cyclin D1 gene in groups B-D significantly increased when compared with group A ( P<0.05). Compared with group E, the relative expressions of Nestin, NSE, MAP-2, GFAP, β-catenin, and CyclinD1 genes significantly increased in group D ( P<0.05); compared with group C, the relative expression of Nestin gene in group D significantly decreased ( P<0.05), while NSE, MAP-2, and GFAP genes significantly increased ( P<0.05). The cellular immunofluorescence staining showed that the ratio of NSE- and GFAP-positive cells significantly increased in groups C-E than in group A, in group D than in groups C and E ( P<0.05). Western blot assay showed that the relative expression of NSE protein was significantly higher in groups C and D than in group A and in group D than in groups C and E ( P<0.05). In addition, the relative expression of GFAP protein was significantly higher in groups C-E than in group A and in group D than in group E ( P<0.05). Besides, the relative expressions of β-catenin, Cytoplasmic β-catenin, Nuclear β-catenin, and the ratio of Nuclear β-catenin to Cytoplasmic β-catenin were significantly higher in groups C and D than in group A and in group D than in group E ( P<0.05), whereas the relative expression of P-β-catenin protein was significantly lower in groups C and D than in group A and in group D than in group E ( P<0.05).@*CONCLUSION@#Different from EGF, bFGF can induce neural differentiation of hBMSCs. In addition, EGF can enhance the hBMSCs neural differentiation of bFGF, while the Wnt/β-catenin signaling pathway may play a positive regulatory role in these processes.
Subject(s)
Humans , beta Catenin/metabolism , Bone Marrow Cells , Cell Differentiation , Cells, Cultured , Epidermal Growth Factor/metabolism , Mesenchymal Stem Cells , Wnt Signaling Pathway , Neurons , Fibroblast Growth Factor 2/metabolismABSTRACT
Bone marrow mesenchymal stem cells （BMSCs） are derived from stem cells isolated from bone marrow and have the potential for multidirectional differentiation and self-renewal. Under certain conditions， BMSCs can be induced to differentiate into osteoblast （OB）， chondrocyte， adipocyte， fibroblast， etc. BMSCs play an important role in maintaining the stability of bone structure and balancing bone metabolism. Promoting the proliferation of BMSCs and inducing their differentiation into OB of great significance for the clinical prevention and treatment of osteoporosis， bone defects， fracture healing， and other diseases. Because the proliferation and osteogenic differentiation of BMSCs are complex processes controlled by multiple genes and regulated by multiple signal transduction pathways， traditional Chinese medicine （TCM） happens to have the advantages of multi-bioactive component， multi-target， and multi-pathway synergism， which can affect the proliferation and differentiation of BMSCs through multiple channels and induce the proliferation of BMSCs. The transcription and expression of genes related to osteogenesis can be enhanced to promote the differentiation of BMSCs into OB， so as to achieve the purpose of preventing and treating osteoporosis， bone defects， and other bone diseases. Based on the literature on the intervention of TCM monomers and compounds in the proliferation and osteogenic differentiation of BMSCs， this study reviewed TCM monomers and compounds in promoting the proliferation and osteogenic differentiation of BMSCs by regulating secreted glycoprotein （Wnt）， neurogenic locus notch homolog protein （Notch）， mitogen-activated protein kinase （MAPK）， phosphatidylinositol-3 kinase （PI3K） /protein kinase B （Akt）， bone morphogenetic protein （BMP）/Smad， Janus kinase （JAK）/signal transducer and activator of transcription protein （STAT）， osteoprotegerin （OPG）/receptor activator of nuclear factor-kappa B （RANK）/RANK ligand （RANKL）， and other signaling pathways to provide new ideas for the research and clinical application of Chinese medicine in the prevention and treatment of orthopedic diseases.
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Objective:To investigate the effect of bone marrow derived mesenchymal stem cells (BMSC) transplantation on bone metabolism and its mechanism in ovariectomized osteoporosis rats.Methods:Forty clean SD female rats aged 7 weeks were divided into 4 groups according to the random number table method: sham operation group, model group, the transplantation group, positive control group, in addition to control the rest of the group were performed bilateral oophorectomy build osteoporosis rats model, after 2 months of model establishment, rats in transplantation group were injected with 80 μl/kg PBS solution containing bone marrow mesenchymal stem cells through tail vein, rats in sham operation group and model group were injected with the same amount of PBS solution through tail vein, and rats in positive control group were given Xianlinggubao (0.5 g/100 g) by gavage every day. Serum and femur were collected 14 days after treatment. Hematoxylin and eosin staining (HE) was used to observe the histopathological changes of femur. Micro-CT was used to measure bone mineral density and bone parameters. The expression levels of osteocalcin, osteoprotegerin, alkaline phosphatase and insulin-like growth factor 1 were detected by enzyme-linked immunosorbent assay (ELISA) kit. The serum levels of calcium, phosphorus and magnesium were measured by spectrophotometer. The protein expressions of RANKL, OPG, TRAF6 and NF-KB1 in femur of each group were detected by Western blot.Results:Compared with the sham operation group, the bone mineral density (BMD) of the model group was decreased by (0.28±0.01) g/cm 3, bone volume fraction (BMD) was decreased by (0.28±0.01) g/cm 3. BV/TV) decreased by (19.73±2.02) %, trabecular thickness (Tb.Th) decreased by (0.082±0.008) mm, trabecular number (Tb.N) decreased by (1.60±0.17) mm -1 and trabecular separation/spacing (Tb.Sp) increased (0.273±0.024) mm, osteoprotegerin (489.49±55.29) ng/L, alkaline phosphatase (229.13±15.05) U/L, insulin-like growth factor-1 (236.64±14.32) μg/L, and osteocalcin were decreased (1.866±0.109) μg/L, calcium (11.98±1.09) mg/dl, phosphorus (6.85±0.68) mg/dl, and magnesium decreased (0.62±0.04) mg/dl) , the relative expression level of RANKL increased (1.05±0.09) , the relative expression level of OPG decreased (0.58±0.08) , the relative expression level of RANKL increased (0.74±0.10) , and the relative expression level of NF-kB1 increased (1.01±0.11) ( P<0.05) ; bone mineral density, bone mineral density, bone mineral density BMD (0.38±0.04 g/cm 3, BV/TV (26.73±2.74) %, Tb.Th (0.094±0.006) mm, Tb.N (2.67±0.09) mm-1 and Tb.Sp were decreased (0.241±0.026) mm) , osteoprotegerin (720.09±67.41) ng/L, alkaline phosphatase (269.48±14.15) U/L, insulin-like growth factor 1 (IGF-1) decreased (335.95±24.13) μg/L, and osteocalcin increased (1.392±0.153) μg/L, calcium (7.12±0.53) mg/dl, phosphorus (4.54±0.32) mg/dl, magnesium (0.87±0.08) mg/dl. RANKL relative expression level increased (0.59±0.05) , OPG relative expression level decreased (0.97±0.10) , RANKL relative expression level increased (0.45±0.06) , NF-kB1 relative expression level increased (0.72±0.06) ( P<0.05) ;bone mineral density, bone mineral density, bone mineral density BMD (0.36±0.05) g/cm 3, BV/TV (28.72±3.20) %, Tb.Th (0.096±0.011) mm, Tb.N (2.85±0.24) mm -1 Tb.Sp was basically unchanged (0.241±0.027) mm, osteoprotegerin was decreased (716.78±36.90) ng/L, alkaline phosphatase was basically unchanged (270.65±18.59) U/L, and insulin-like growth factor 1 was decreased (336.94±17.50) μg/L, osteocalcin (1.377±0.101) μg/L, calcium (7.13±0.80) mg/dl, phosphorus (4.58±0.71) mg/dl, and magnesium (0.89±0.04) remained unchanged mg/dl, the relative expression level of RANKL increased (0.55±0.08) , the relative expression level of OPG decreased (0.98±0.13) , the relative expression level of RANKL was basically unchanged (0.40±0.05) , and the relative expression level of NF-kB1 increased (0.65±0.09) ( P<0.05) . Conclusion:Bone marrow mesenchymal stem cell transplantation can improve osteoporosis in ovariectomized rats by regulating bone metabolism and serum levels of calcium, phosphorus and magnesium, which may be related to RANKL/OPG/TRAF6 pathway.
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ObjectiveTo explore the mechanism of Buyang Huanwutang combined with bone marrow mesenchymal stem cell （BMSC） transplantation in the treatment of spinal cord injury （SCI）. MethodDifferent concentrations （12.5， 25， 50 g·kg-1） of Buyang Huanwutang were administrated to rats by gavage. The spinal cord function of rats was measured by modified Tarlov score， and the most suitable concentration of Buyang Huanwutang was screened out. SD rats were then divided into 6 groups， namely， the sham operation group （gavage of equal amount of normal saline）， the model group （gavage of equal amount of normal saline）， the Buyang Huanwutang group （gavage of 25 g·kg-1 Buyang Huanwutang）， the BMSC transplantation group （tail vein injection of BMSCs 1 mL）， the Buyang Huanwutang+BMSC group （gavage of 25 g·kg-1 Buyang Huanwutang and tail vein injection of BMSCs 1 mL）， the Buyang Huanwutang+BMSC+LY294002 group （gavage of 25 g·kg-1 Buyang Huanwutang and tail vein injection of BMSCs 1 mL and 40 mg·kg-1 LY294002）， with 10 rats in each group. The spinal cord function was measured by the modified Tarlov score， inclined plate test， and latency of cortical somatosensory evoked potential. Immunohistochemistry was used to detect the number of 5-bromo-2-deoxyuracil nucleoside （Brdu）-labeled positive cells in the spinal cord tissue. The protein expression levels of phosphorylated protein kinase B （p-Akt）， glycoprotein 130 （gp130）， and interleukin-6 （IL-6） in spinal cord were detected by Western blot. ResultAs compared with the sham operation group， the Tarlov score and the critical angle of tilt plane in the model group were significantly decreased （P<0.05）， and the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 were significantly increased （P<0.05）. As compared with the model group， the Tarlov score and the critical angle of tilt plane in the sham operation group and each treatment group were significantly increased （P<0.05）， and the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 were significantly decreased （P<0.05）. As compared with the BMSC group， the Tarlov score and the critical angle of inclined plane in the Buyang Huanwutang+BMSC group increased （P<0.05）， the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 decreased （P<0.05）， and the number of Brdu-labeled positive cells increased 5 weeks after transplantation （P<0.05）. As compared with the Buyang Huanwutang+BMSC group， the Tarlov score and the critical angle of the inclined plane in the Buyang Huanwutang+BMSC+LY294002 group increased （P<0.05）， and the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 decreased significantly （P<0.05）. Five weeks after transplantation， the number of Brdu-labeled positive cells increased significantly in the Buyang Huanwutang+BMSC+LY294002 group （P<0.05）. ConclusionBuyang Huanwutang can promote BMSCs migration and restore spinal cord function by inhibiting phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signal.
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Aim To elucidate the biological effects of insulin-like growth factor-1 ( IGF-1 ) and basic fibro-blast growth factor (bFGF) alone or in combination on the differentiation of bone mesenchymal stem cells (BMSCs) into cardiomyocytes (CMs), and to explore the mechanism in the differentiation process of BMSCs into CMs induced by IGF-1 or bFGF. Methods After four weeks of BMSCs induced by induction differentiation medium ( with or without LY294002) containing IGF-1 and/or bFGF, the expression levels of proteins associated with the cardiomyogenic phenotype in BMSCs were detected via immunocytochemistry, immuno-fluorescence staining, and Western blot. Meanwhile, the expression levels of pathway related proteins were detected by Western blot. The cell ultrastructure was observed by transmission electron microscopy (TEM) . The expression levels of myocardial specific genes were measured via RT-qPCR. Results Compared with the control group, the expression levels of myocardial specific proteins and genes significantly increased in the IGF-1, bFGF and combination groups and were the highest in the coinduction group. The TEM of the conduction group showed parallel myofilaments, mitochondria, endoplasmic reticulum and so on. The additon of LY294002 decreased the expression levels of myocardial specific proteins, genes and phosphorylation Akt. Conclusions The effect of IGF-1 combined with bFGF on the differentiation of BMSCs into CMs is markedly better than that induced by IGF-1 or bFGF a-lone, and the differentiation process may depend on the PI3K/Akt signaling pathway.
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【Objective】 To study the effects of miR-30e-5p from bone marrow mesenchymal stem cell-derived exosomes(BMSC-exos) on high glucose (HG)-induced HK-2 cell pyroptosis and explore an alternative strategy to manage diabetic kidney disease (DKD). 【Methods】 BMSC-exos were isolated and internalized into HK-2 cells treated with HG to measure viability and cytotoxicity. The secretion of IL-1β and IL-18 was measured by ELISA. Pyroptosis was assessed by flow cytometry. The levels of miR-30e-5p, IL-1β, and IL-18 were measured. The expression of pyroptosis-associated cytokine proteins was determined. 【Results】 BMSC-exos decreased LDH, IL-1β, and IL-18 secretion and inhibited the expression of the pyroptosis-related factors (IL-1β, caspase-1, GSDMD-N, and NLRP3) in HG-induced HK-2 cells. Moreover, miR-30e-5p depletion in BMSC-exos promoted HK-2 cell pyroptosis. 【Conclusion】 BMSC-derived exosomal miR-30e-5p inhibits caspase-1-mediated pyroptosis in HG-induced HK-2 cells, which might provide a new strategy for treating DKD.
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Objective To study the mechanical effects of cyclic strain on neural differentiation of rat bone marrow mesenchymal stem cells (rBMSCs). Methods The rBMSCs were subjected to cyclic strain for 24 hours andthen cultured for 5 days. The expression of neural markers and the phosphorylation of relative signaling pathway proteins were evaluated. The stress distribution on cell surface was analyzed by finite element method. The differentially expressed genes induced by strain were identified by RNA sequencing analysis. Results The 0. 5 Hz strain with 5% magnitude could significantly induce higher expression of neural markers and elevated phosphorylation level of extracellular-signal-regulated kinase (ERK), protein kinase B (AKT) and mammalian target of rapamycin ( mTOR). KEGG pathway analysis showed that the focal adhesion and ECM-receptor interaction were significantly enriched under cyclic strain. Conclusions Cyclic strain could change the interaction of cells with the extracellular matrix ( ECM) and enhance the AKT/ mTOR and ERK pathway, finally promote rBMSC neural differentiation. Knowledge about the impact of mechanical stimulation on BMSC neural differentiation is expected to improve the efficiency of stem cell differentiation, shed light on device design for tissue engineering, and promote clinical application of mesenchymal stem cells in neural issue repair and regeneration.
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Objective:To explore the effects of conditioned medium of human bone marrow mesenchymal stem cells (BMSCs) on the proliferation, adhesion and differentiation of immortalized human Müller cell line (MIO-M1).Methods:The differentiation was induced in the third-passage BMSCs with osteogenic, chondrogenic and adipogenic medium and identified by alizarin red, alcian blue and oil red O staining, respectively.The expression levels of mesenchymal stem cell markers CD73, CD90 and CD105 and hematopoietic cell markers CD34, CD45 and human leukocyte antigen-DR (HLA-DR) were assayed by flow cytometry.The expressions levels of Müller cell markers SOX9, glutamine synthetase (GS), vimentin and cellular retinaldehyde-binding protein (CRALBP), retinal stem cell markers SOX2, nestin and CHX10, and cell proliferation marker cyclin D3 (CCND3) in MIO-M1 cells were detected by immunofluorescence staining.The MIO-M1 cells were divided into standard medium group, 293T conditioned medium group, and BMSC conditioned medium group and were incubated in the medium according to grouping.The cellular area, circularity, elongation factor and perimeter were analyzed quantitatively.The cell cycle was detected by flow cytometry, and the cell proliferation was determined by neurospora experiment and 5-ethynyl-2'-deoxyuridine (EdU) staining.The expression of vascular cell adhesion molecule 1 (VCAM-1) at protein and mRNA levels in the culture supernatant was detected by enzyme linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR), respectively.The expression of retinal neuron markers protein kinase C (PKCα), Rhodopsin, microtubule-associated protein 2 (MAP2) and β-tubulin (Tuj1) was detected by immunofluorescence staining and qRT-PCR.Results:CD73, CD90, CD105 showed an enhanced expression, and CD34, CD45 and HLA-DR showed weakened expression in the BMSCs.The BMSCs differentiated into osteoblasts, chondrocytes and adipocytes.Expression of SOX9, GS, vimentin and CRALBP, SOX2, CHX10, nestin and CCND3 was found in the MIO-M1 cells.Compared with standard medium group and 293T conditioned medium group, MIO-M1 cells cultured in BMSC conditioned medium group changed into an elongated spindle-shaped or multipolar morphology with reduced cell area, increased elongation index and decreased circularity, showing statistically significant differences among them ( F=6.973, 12.370, 6.311; all at P<0.01). There were increased neurospheres formed by MIO-M1 cells in BMSC conditioned medium group compared with standard medium group and 293T conditioned medium group at different time points ( Fgroup=134.300, P<0.001; Ftime=82.910, P<0.001). Compared with the standard medium group and 293T conditioned medium group, the EdU-positive rate and proliferation index of MIO-M1 cells in BMSC conditioned medium group were significantly increased, with statistically significant differences ( F=6.973, 74.110; all at P<0.05); the VCAM-1 protein expression in cell supernatant and the relative expression level of VCAM-1 mRNA in BMSC conditioed medium group were significantly increased ( F=13.720, 7.896; all at P<0.05); the mRNA expression levels of PKCα, Rhodopsin, Tuj1 and MAP2 were higher in MIO-M1 cells of BMSC conditioned medium group under the condition of differentiation ( F=14.490, 5.424, 14.330, 7.405; all at P<0.05). Conclusions:BMSCs conditioned medium can change the morphology of MIO-M1 cells and promote their proliferation, adhesion and differentiation into retinal neurons.
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Objective@#To investigate the effect of brain and muscle arant-like-1 (Bmal1) and miRNA-155-5p on the proliferation ability and aging of bone marrow mesenchymal stem cells (BMMSCs) to provide an experimental basis for elucidating the mechanism of bone senescence.@*Methods @# BMMSCs were extracted from the femur medullary cavity of 1-month-old mice, purified and cultured via the whole bone marrow mesenchymal adherent method and passed to P3. The characteristics of BMMSCs were detected by flow cytometry. BMMSCs were transfected with lentivirus to construct stable miR-155-5p and Bmal1 overexpression/interference BMMSCs. shRNA-transfected BMMSCs were identified by qRT-PCR. The proliferation activities of miR-155-5p and Bmal1 overexpression/interference BMMSCs were detected via CCK-8 assay. The apoptosis rates were measured by flow cytometry. The aging status of BMMSCs was identified with the senescence-associated β-galactosidase (SA-β-Gal) test. The expression of senescence-related genes P16 and P53 was detected by qRT-PCR.@*Results@#The shRNA-transfected BMMSCs were successfully generated. The proliferation ability decreased, and the apoptosis rates, the activity of SA-β-Gal and the relative expression levels of P53 and P16 increased when miRNA-155-5p was overexpressed. The proliferation ability increased, and the apoptosis rates, the activity of SA-β-Gal and the relative expression levels of P53 and P16 decreased when miRNA-155-5p was inhibited. The effect of Bmal1 is opposite to that of miRNA-155-5p.@*Conclusions @# The expression of Bmal1 promotes the proliferation and antiaging ability of BMMSCs, while miRNA-155-5p inhibits the proliferation and accelerates the aging of BMMSCs.
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In recent years, studies have found that mitochondrial transfer between leukemic cells and different types of cells in their bone marrow microenvironment, especially mesenchymal stem cells, plays a key role in the occurrence, development and drug resistance of hematological malignant tumors. This paper mainly introduces the role and latest research progress of mitochondrial transfer in acute and chronic myeloid leukemia, acute lymphoblastic leukemia and multiple myeloma, and briefly describes the mechanism of drug resistance caused by mitochondrial transfer in leukemic cells during chemotherapy. The aim is to provide a new idea and theoretical basis for using intercellular mitochondrial transfer as a potential therapeutic target.
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Humans , Bone Marrow , Hematologic Neoplasms/metabolism , Mesenchymal Stem Cells , Mitochondria , Multiple Myeloma/metabolism , Tumor MicroenvironmentABSTRACT
OBJECTIVE@#To investigate the effect of RUNX2 gene overexpression vector modified exosomes derived from bone marrow mesenchymal stem cells (BMSCs) combined with calcium carbonate scaffold system in bone defect.@*METHODS@#Rabbit BMSCs were used as the research object, and BMSCs were identified by flow cytometry. Construct RUNX2 gene overexpression vector, transfect BMSCs with lentivirus, and collect exosomes by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscope, the expression of exosome marker CD63 was detected by Western blot, and the calcium carbonate scaffold was constructed by three chamber parallel automatic temperature control reaction system. According to whether the RUNX2 gene overexpression vector was transfected or not, the complex of BMSCs and calcium carbonate scaffold was divided into three groups, namely BMSCs group, RUNX2 overexpression group and exosome group. The osteogenic differentiation of BMSCs was detected by oil red O staining and RT-PCR. There were 9 clean adult healthy male New Zealand white rabbits, aged (12.97±1.21) months, with a body weight of (19.3±3.6) kg, with 3 rabbits in each group. The animal model of skull defect was constructed by surgical method, and the repair of bone defect was evaluated by imaging, he staining and Masson staining.@*RESULTS@#The results of flow cytometry showed that the expression of CD29 protein, CD44 protein, CD11b protein and CD45 protein on the surface of BMSCs were 99.5%, 100%, 0.1% and 0.1%, respectively. Transmission electron microscopy showed that the exosomes were bilayer vesicles with a diameter of 50 to 150 nm. Western blot showed that the molecular marker CD63 of exosomes was positive. Oil red O staining showed that the osteogenic differentiation of BMSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group (P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group (P<0.05). MSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group(P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group(P<0.05).@*CONCLUSION@#Compared with RUNX2 gene overexpression vector transfection, extraction of exosomes directly can promote the differentiation of BMSCs into osteoblasts more efficiently, and the combination with calcium carbonate scaffold can better promote the healing of bone defects. So as to provide new ideas and methods for the clinical treatment of bone defects.
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Animals , Humans , Male , Rabbits , Calcium Carbonate/metabolism , Core Binding Factor Alpha 1 Subunit/metabolism , Exosomes/metabolism , Osteogenesis/genetics , RNA, Messenger/metabolismABSTRACT
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.
Subject(s)
Adipogenesis/genetics , Bone Marrow Cells/metabolism , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells , Osteogenesis , PPAR gamma/pharmacology , RNA, Long Noncoding/genetics , RNA, Messenger/metabolismABSTRACT
Objective:To explore the effect and possible mechanism of normal temperature mechanical perfusion(NMP)plus bone marrow mesenchymal stem cells(BMMSCs)on early endoplasmic reticulum stress(ERs)and cellular apoptosis after donation after cardiac death(DCD)donor liver transplantation.Methods:BMMSCs were isolated and cultured by adherence method.Sixty Sprague-Dawley(SD)rats were randomly divided into five groups of sham operation(sham), static cold storage(SCS), NMP, BMMSC and NMP plus BMMSCs(BP)( n=12 each). Liver tissue and serum sample of each group were harvested at Day 1/7 post-operation.Hematoxylin-eosin(HE)staining was employed for observing pathological changes of liver tissue; TdT-mediated dUTP nick end labeling(TUNNEL)staining for detecting cellular apoptosis; immunohistochemistry for detecting the expression of hepatocyte transcription factor C/EBP homologous protein(CHOP); Western blot for detecting the expressions of GRP-78, p-PERK, ATF4, CHOP and cleaved caspase-3. Results:Compared with SCS group, hepatic injury and inflammation significantly declined in NMP, BMMSC and BP groups and improvement of hepatic injury was the most pronounced in BP group.Cellular apoptosis lessened markedly in BP group at Day 1/7 as compared with SCS group and the difference was statistically significant.The expressions of ERs-related proteins GRP-78, p-PERK and ATF4 spiked in SCS group and the expressions of pro-apoptotic proteins CHOP and cleaved caspase-3 were significantly elevated and declined markedly in BP group.And the difference was statistically significant.Conclusions:BMMSCs plus NMP can significantly improve hepatocyte apoptosis and inflammatory response after DCD donor liver transplantation.And its mechanism may be correlated with suppressing early endoplasmic reticulum stress of hepatocytes.
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ObjectiveTo systematically review the efficacy and safety of exosomes derived from bone marrow mesenchymal stem cells (BMSC) on animal models of spinal cord injury. MethodsAnimal studies about BMSC-derived exosomes for spinal cord injury were retrieved from databases of PubMed, Cochrane Library, Web of Science, CNKI and Wangfang Data, from establishment to October, 2021. Two researchers independently screened and extracted the data, and evaluated the risk of bias. The studies were qualitatively analyzed. ResultsA total of 21 studies were included, involving 1 342 animals. Male or female Sprague-Dawley rats were selected for 18 studies, and the body mass of the rats was (200±50) g in 19 studies. The injury nodes focused on T9-11 spinal cord, with various methods. The types, medication time, frequency, concentration and dose of the exosomes were heterogeneous. ConclusionsThe BMSC-derived exosomes can improve the motor function after spinal cord injury, reduce the damage of spinal cord, resist apoptosis and inflammation, reduce the permeability of blood-spinal cord barrier, and promote the growth of axons and blood vessels. More high-quality studies are needed for further verification.