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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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Stroke is one of the major diseases threatening human health， with ischemic stroke accounting for about 70%. Ischemic stroke is characterized by complex pathological mechanism and high incidence， mobility and mortality. At present， the effective clinical treatment measures for ischemic stroke are limited， and it is urgent to develop new and effective treatment measures to improve the prognosis of patients. In recent years， bone marrow mesenchymal stem cells （BMSCs） transplantation has shown great therapeutic potential for a variety of diseases， including ischemic stroke， and has become a new research hotspot. However， due to the low homing and survival rate of BMSCs in human body after transplantation， their clinical effect on ischemic stroke needs to be further improved. With the characteristics of multi-components， multi-channels and multi-targets， Chinese medicine displays desirable curative effect on ischemic stroke， which has been widely concerned. Both Chinese medicine and BMSCs transplantation have good overall brain protection， and their combined effect on ischemic stroke is significantly better than that of single application. The mechanisms include improving the transplantation efficiency of BMSCs， promoting angiogenesis， enhancing neuroplasticity， ameliorating neuroinflammation， enhancing neuroprotection， and regulating the blood-brain barrier and exosomes. The combination of Chinese medicine and modern cutting-edge cell therapy reflects the advantages of integrative medicine， providing a new model and idea for preventing and treating ischemic stroke and improving the efficacy of BMSCs transplantation.
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Macrophages are typically identified as classically activated (M1) macrophages and alternatively activated (M2) macrophages, which respectively exhibit pro- and anti-inflammatory phenotypes, and the balance between these two subtypes plays a critical role in the regulation of tissue inflammation, injury, and repair processes. Recent studies indicate that tissue cells and macrophages interact
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Objective: Head and neck cancer is the sixth leading cancer by incidence worldwide and eighth by death. Recent reports revealed that, not only radiotherapy but also chemotherapy may induce xerostomia. The aim of this study was to compare the possible regenerative effect of BMSCs through systemic and local injections. Material and Methods: 52 male Albino rats were randomly divided into 4 groups: Group 1: 10 rats received 0.5 ml of PBS by injection. Group 2: 14 rats received an intraperitoneal injection of 5-FU drug. Group 3: 14 rats were injected the same dose of 5-FU then received an intraglandular transplantation of BMSCs suspended in 0.5 ml of PBS at day 1 after 5-FU administration. Group 4: 14 rats were injected the same dose of 5-FU then received an intravenous injection of BMSCs suspended in 0.5 ml of PBS via the tail vein at day 1 after 5-FU administration. Results: Histological examination showed that group 2 showed features of severe degenerative changes which increased over time. Group 3 showed increasing amelioration in the ductal structure overtime. Group 4 also showed regenerated ductal elements however concerning apoptotic changes, immunohistochemistry results revealed improvement in both group 3 and 4 over group 2 with no statistical difference between groups 3 and 4. Conclusion: Histological and immunohistochemical features in group 3 and group 4 revealed similar amelioration in regenerative potentials. On the other hand, regenerative features of both experimental groups were statistically significant as compared independently to group 1 (AU)
Objetivo: O carcinoma de cabeça e pescoço é o sexto câncer de maior incidência no mundo sendo a oitava causa de morte por cancer. Relatos recentes revelaram que não apenas a radioterapia, mas também a quimioterapia podem induzir xerostomia. O objetivo desse estudo foi comparar a possivel ação regenerative de BMSCs através de injeção local e sistêmica. Material e Métodos: 52 ratos Albino foram aleatoriamente alocados em 4 grupos: Grupo 1: 10 ratos que receberam 0.5 ml de injeção de PBS. Grupo 2: 14 ratos que receberam injeção intraperitoneal da droga 5-FU. Grupo 3: 14 ratos que foram injetados com a mesma dose de 5-FU e receberam transplante intraglandular de BMSCs ressuspendidas em 0.5mL de PBS no dia 1 após a administração do 5-FU. Grupo 4: 14 ratos que foram injetados com a mesma dose de 5-FU e receberam injeção intravenosa de BMSCs ressuspendidas em 0.5mL de PBS via veia caudal 1 dia após a administração de 5-FU. Resultados: O exame histológico demonstrou que o grupo 2 apresentou alterações degenerativas severas que se agravaram com o tempo. O Grupo 3 mostrou melhora da estrutura ductal ao longo do experimento. Group 4 também mostrou elementos ductais regenerados. Referente a alterações apoptóticas,análise imunohistoquimica mostrou melhora nos grupos 3 e 4 comparados ao grupo 2, sendo que os grupos 3 e 4 foram estatisticamente semelhantes. Conclusão: Análises histológicas e imunohistoquímicas mostram que os grupos 3 e 4 apresentam melhora no potencial regenerativo Por outro lado, os resultados observados para os dois grupos foi estatisticamente semlhante quando comparados independentemente ao grupo 1 (AU)
Subject(s)
Animals , Rats , Salivary Glands , Mesenchymal Stem Cells , Squamous Cell Carcinoma of Head and NeckABSTRACT
With the development of the 3rd-generation high-throughput sequencing technology and tissue engineering, recent studies show that many long-chain non-coding RNAs (LncRNAs) have played an important role in osteogenic differentiation of mesenchymal stem cells (MSCs). LncRNAs, which are involved in the regulation of mechanical regulation, further regulate bone-related cell functions and play a regulatory role at multiple levels, including transcription, post-transcriptional and epigenetic. LncRNAs may be involved in the osteogenic differentiation and bone remodeling of MSCs, the regulation of bone-related cell functions as a mechanical response molecule, as well as the pathological process of skeletal diseases.
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Objective:To investigate the effects of different concentrations of Astragali Radix containing serum on the expression of 24-hydroxylase（CYP24A1），1α-OHase（CYP27B1） mRNA and protein in rat bone marrow mesenchymal stem cells （BMSCs）， and to explore the mechanism of primary osteoporosis （OP）. Method:The experiment was divided into 6 groups，like normal group， model group， low ，middle and high dose group of Astragali Radix containing serum（20%，40%，60%），Vitamin D group. Cell proliferation toxicity assay（CCK-8） was used to detect the effect of different concentrations of Astragali Radix containing serum on survival rate of aging BMSCs.Real-time quantitative PCR（Real-time PCR） and Western blot was used to detect the expression of CYP24A1 CYP27B1 mRNA and protein in senile BMSCs osteogenic differentiation cells by different concentrations of Astragali Radix containing serum. Result:Compared with normal group， the proliferation and survival rate of BMSCs osteoblasts induced by D-galactose in model group was significantly lower than that in normal group （P<0.01）. Compared with model group， medium and high dose groups and Vitamin D group could improve the proliferation and differentiation of aging BMSCs into osteoblasts in different degrees（P<0.01）. The relative expression of CYP27B1 mRNA and protein in model group was significantly lower than that in normal group， while the relative expression of CYP24A1 mRNA and protein in model group was significantly higher than that in normal group. Compared with model group， high dose Astragali Radix containing serum group could increase the relative expression of CYP27B1 mRNA and protein， and decrease the relative expression of CYP24A1 mRNA and protein in a dose-dependent manner（P<0.01）. Conclusion:The mechanism of different concentrations of Astragali Radix containing serum in the treatment of osteoporosis may be related to the regulation of CYP24A1， CYP27B1 mRNA and protein in the osteogenic differentiation of aging BMSCs.
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Supercritical large area or large segmental bone defects are still a clinical problem. Researchers are committed to the development of artificial bone materials, but in order to solve the problem of poor bone formation of artificial bone materials, people are paying more and more attention to application of bone marrow mesenchymal stem cells in bone tissue engineering. In this review, bone marrow mesenchymal stem cells, osteogenic differentiation, osteoblasts cells, clinical application were used as keywords to search CNKI database, Wanfang database, Weipu database and PUBMED database by computer. The isolation and culture of bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cell identification method, osteogenic induction method, osteogenic differentiation identification and clinical applicationt were comprehensively summarized in order to provide a theoretical basis for its use as a seed cell in the treatment of bone tissue diseases. Scholars have preliminarily studied the treatment of bone and cartilage defects, osteoarthritis, femoral head necrosis and other diseases with bone marrow mesenchymal stem cells combined with transplantation, and obtained good clinical efficacy.However, bone marrow mesenchymal stem cells have certain advantages and disadvantages, and further clinical studies and long-term efficacy verification are needed.
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Bone Marrow Cells , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells , OsteogenesisABSTRACT
Vibration represents a micro reciprocating motion of a particle or object along a line or arc relative to a reference position, while the effect of low-magnitude high-frequency vibration (LMHFV) on skeletal system cells is similar to the mechanical stimulation of muscle movement. Bone mesenchymal stem cells (BMSCs), which have been identified as force-sensitive cells, exist in the bone marrows and have the potential of multi-lineage differentiation. Their biological characteristics can change functionally according to the appropriate stimulation in vitro, in order to reach the optimal demand of the stimulation. LMHFV can promote the osteogenic differentiation of BMSCs, therefore, the research on its mechanism can contribute to the application of vibration in the treatment of diseases such as osteoporosis, fracture, osteogenesis imperfecta, obesity as well as the promotion of orthodontic tooth movement. This paper summarizes the recent progress about the effects of vibration on BMSCs stem cells in osteogenesis and the possible mechanisms, so as to provide research ideas and methods for studying the mechanical as well as biological changes of BMSCs under vibration stimulation.
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Extracellular matrix is the main element to provide mechanical clues for cells. The response of stem cells to mechanical signals is mainly achieved through the cytoskeleton. After mechanical signal is transmitted, cytoskeleton can form contractile microfilaments that actively generate tension through reorganization induced by microenvironment changes. The mechanical signals can regulate gene expression through either coupling with the nuclear skeleton directly or being transformed by the second message. Recent studies have proven that cytoskeleton tension has a series of impact on lineage specification, proliferation, differentiation and apoptosis of bone mesenchymal stem cells (BMSCs). BMSCs are of great significance in bone reconstruction and clinical treatment. The possible mechanisms about mechanotransduction and its effects of cytoskeleton tension on osteogenesis of BMSCs after micro-environmental changes were summarized.
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Vibration represents a micro reciprocating motion of a particle or object along a line or arc relative to a reference position, while the effect of low-magnitude high-frequency vibration (LMHFV) on skeletal system cells is similar to the mechanical stimulation of muscle movement. Bone mesenchymal stem cells (BMSCs), which have been identified as force-sensitive cells, exist in the bone marrows and have the potential of multi-lineage differentiation. Their biological characteristics can change functionally according to the appropriate stimulation in vitro, in order to reach the optimal demand of the stimulation. LMHFV can promote the osteogenic differentiation of BMSCs, therefore, the research on its mechanism can contribute to the application of vibration in the treatment of diseases such as osteoporosis, fracture, osteogenesis imperfecta, obesity as well as the promotion of orthodontic tooth movement. This paper summarizes the recent progress about the effects of vibration on BMSCs stem cells in osteogenesis and the possible mechanisms, so as to provide research ideas and methods for studying the mechanical as well as biological changes of BMSCs under vibration stimulation.
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Objective: To observe the effects of astragaloside IV (AST IV) combined with Panax Notoginseng saponins (PNS) on proliferation, apoptosis, migration and neuronal differentiation of oxygen glucosedeprivation/reoxygenation model rat bone marrow mesenchymal stem cells (BMSCs). Methods: BMSCs were isolated, cultured, amplified and purified by the whole bone marrow adherent method. The positive expression rates of BMSCs surface markers, CD29, CD90, CD34, and CD45 were detected by flow cytometry. The third generation of BMSCs was pretreated with AST IV and PNS doses of high (100 μmol/L + 60 μmol/L), medium (50 μmol/L + 30 μmol/L), and low (25 μmol/L + 15 μmol/L) for 24 h. The model of ischemia-reperfusion injury was established by OGD/R. Meanwhile, the normal group (BMSCs were cultured normally) and the model group (OGD/R was used to establish an ischemia reperfusion injury model) were established. The cell increment rate was detected by CCK-8 assay. Cell apoptosis was detected by flow cytometry. Transwell assay was used to detect the migration of BMSCs. The condition of BMSCs differentiation into neurons and astrocytes was observed by Nestin/NSE and Nestin/GFAP immunofluorescence double labeling. Results: BMSCs were successfully cultured and separated, and the positive rates of CD29 and CD90 detected by flow cytometry were 94.23% and 94.69%, while the positive rates of CD34 and CD45 were 5.76% and 5.31%. Compared with the normal group, the survival rate of the model group was reduced significantly and the apoptosis rate was increased significantly (P < 0.05). Compared with the model group, the combination of different doses of AST IV and PNS could promote the proliferation of BMSCs (P < 0.05, 0.01) and inhibit the apoptosis (P < 0.05, 0.01). Compared with the normal group, the model group and the AST IV and PNS group at different doses could promote the migration of BMSCs (P < 0.05). Compared with the model group, the number of migrated cells in the AST IV and PNS groups at different doses was increased significantly (P < 0.05). Compared with the normal group, the model group and the AST IV and PNS groups at different doses could promote the differentiation of BMSCs into neurons and astrocytes (P < 0.01). Compared with the model group, the positive expression rates of Nestin/NSE and Nestin/GFAP in the AST IV and PNS groups at different doses were increased significantly (P < 0.01). Conclusion: AST IV combined with PNS can promote the proliferation and migration of BMSCs of ischemia-reperfusion model in vitro, inhibit the apoptosis, and induce their directional differentiation into neurons and astrocytes.
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Exosomes, a kind of extracellular vesicle, are promising therapeutic agents for spinal cord injury (SCI). This article aimed to investigate effects of exosomes secreted from miRNA-29b-modified bone marrow mesenchymal stem cells (BMSCs) on SCI. Exosomes were extracted from BMSCs transfected with miRNA-29b or negative control (miR NC). SCI rats were injected intravenously with exosomes (control exosomes, miRNA-29b exosomes) and BMSCs (miR NC, miRNA-29b) through the tail vein. The expression of miRNA-29b in spinal cord tissues of SCI rats was detected by qRT-PCR. The hind limb motor function was evaluated by Basso Beattie Bresnahan (BBB) score. The histopathological damage and neuronal regeneration in spinal cord tissues was observed by HE staining and immunohistochemistry, respectively. The injection of miRNA-29b exosomes and miRNA-29b BMSCs both significantly increased the expression of miRNA-29b in spinal cord tissues of SCI rats (P<0.05). Compared with SCI rats, rats in the miRNA-29b exosomes and the miRNA-29b groups exhibited improved SCI, including increased BBB score, NF200 and GAP-43 positive neurons, as well as decreased contractile nerve cell numbers and GFAP positive neurons (all P<0.05). The relieving degree of SCI was significantly higher in the miRNA-29b exosomes group than in the miRNA-29b BMSCs group (P<0.05). Exosomes secreted from miRNA-29b-modified BMSCs were effective in the repair of SCI in rats.
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Animals , Male , Female , Rats , Spinal Cord Injuries/therapy , Transfection , Recovery of Function , MicroRNAs/metabolism , Mesenchymal Stem Cell Transplantation , Exosomes/metabolism , Immunohistochemistry , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Disease Models, AnimalABSTRACT
SUMMARY: Matrigel is a basement membrane matrix extracted from the EHS mouse tumor containing extracellular matrix protein, its main components are laminin, type IV collagen, nestin, heparin sulfate, growth factor and matrix metalloproteinase.At room temperature, Matrigel polymerized to form a three dimensional matrix with biological activity. It can simulate the structure, composition, physical properties and functions of the cell basement membrane in vivo, which is beneficial to the culture and differentiation of the cells in vitro, and can be used for the study of cell morphology, biochemical function, migration, infection and gene expression. In this study, Matrigel three-dimensional culture model of bone marrow mesenchymal stem cells(BMSCs) was established, and its morphology, proliferation and survival were observed. BMSCs were isolated and cultured with whole bone marrow adherence method. The Second generation BMSCs with good growth condition were selected and mixed with Matrigel to form cell gel complexes. The morphology and proliferation of mesenchymal stem cells were observed by phase contrast microscope and HE staining,Live/Dead staining was used to evaluate the cell activity.Phase contrast microscopy showed that BMSCs were reticulated in Matrigel and proliferated well, After 7 days, the matrix gel gradually became soft and collapsed, a few cell reticular crosslinking growth was seen at 14 days; HE staining showed that the cytoplasm of the cells was larger on the fourth day and the cells were elongated and cross-linked on the seventh day; Live/dead staining showed that most cells showed green fluorescence with the prolongation of culture time, on the first, 4 and 7 days, the activity of bone marrow mesenchymal stem cells in Matrigel gradually increased, and the percentages were 92.57 %, 95.54 % and 97.37 %, respectively. Matrigel three-dimensional culture system can maintain the morphology, function and proliferation ability of bone marrow mesenchymal stem cells.
RESUMEN: Matrigel es una matriz de membrana basal extraída del tumor de ratón EHS que contiene proteína de matriz extracelular. Los componentes principales son laminina, el colágeno tipo IV, nestina, sulfato de heparina, factor de crecimiento y metaloproteinasa de matriz. A temperatura ambiente, Matrigel se polimerizó para formar una matriz tridimensional. Es posible simular la estructura, la composición, las propiedades físicas y las funciones de la membrana basal celular in vivo, lo que es beneficioso para el cultivo y la diferenciación de las células in vitro, y se puede utilizar para el estudio de la morfología celular, la función bioquímica, la migración, infección y expresión génica. En este estudio, se estableció el modelo de cultivo tridimensional Matrigel de células madre mesenquimales de médula ósea (BMSC), y se observó su morfología, proliferación y supervivencia. Las BMSC fueron aisladas y cultivadas con el método de adherencia de la médula ósea completa. Se seleccionaron las BMSC de segunda generación con buenas condiciones de crecimiento y se mezclaron con Matrigel para formar complejos de gel de células. La morfología y la proliferación de las células madre mesenquimales se observaron con microscopio de contraste de fase y se tiñó con Hematoxilina-Eosina (HE); para evaluar la actividad celular se usó la tinción Live/Dead. La microscopía de contraste mostró que las BMSC se reticularon en Matrigel y proliferaron bien. Después de 7 días, se observó que el gel de matriz gradualmente se volvió blando y colapsó, y se visualizó un cruce transversal de algunas células reticulares a los 14 días. La tinción mostró que la mayoría de las células mostraron una fluorescencia verde con la prolongación del tiempo de cultivo; en los primeros 4 y 7 días, la actividad de las células madre mesenquimales de la médula ósea en Matrigel aumentó gradualmente y los porcentajes fueron de 92,57 %, 95,54 % y 97,37 %, respectivamente. El sistema de cultivo tridimensional de Matrigel puede mantener la morfología, la función y la capacidad de proliferación de las células madre mesenquimales de la médula ósea.
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Animals , Dogs , Proteoglycans/chemistry , Collagen/chemistry , Laminin/chemistry , Cell Culture Techniques/methods , Mesenchymal Stem Cells/cytology , Tissue Engineering , Drug CombinationsABSTRACT
Objective: To isolate, culture and identify rabbit bone mesenchymal stem cells (BMSCs) so as to explore the optimal conditions for lentiviral vector-mediated enhanced green fluorescent protein (eGFP) infection in rabbit BMSCs and screen stable transfected BMSCs in rabbits. Methods: BMSCs were obtained by whole bone marrow adherence method. The osteogenic, chondrogenic and adipogenic differentiation of BMSCs was made by alizarin red, toluidine blue and oil red O staining, respectively. The expressions of CD44 and CD90 were detected by immunofluorescence. The concentration of puromycin was used to screen the minimum lethal concentration of BMSCs; the lentiviral vector with multiplicity of infection (MOI) of 50, 100, 150 and 200 mediated eGFP BMSCs were infected; the fluorescence expression was observed under an inverted microscope, and the stable transformation system was screened with puromycin. Results: When MOI was 150, lentiviral vector-mediated eGFP infection of rabbit BMSCs was the most efficient. The optimum concentration of puromycin for stable transfection of rabbit BMSCs was 1.0 μg/mL. Conclusion: Rabbit BMSCs were successfully cultured in this experiment. The stem cells were labeled with lentivirus-mediated GFP and stable transfected rabbit BMSCs were screened. A simple and effective stem cell labeling method was established to label BMSCs in vivo.
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Objective: To construct a novel tissue engineering complex, BMSCs sheet-RADA16 scaffold, by combining cell sheet and self-assembled peptides.. Methods: The self-assembled peptide RADA16 scaffold was wrapped with the BMSCs cell sheet. The morphology of the cells and the complex were observed by SEM and confocal laser microscopy, and the proliferation of cells was assessed by CCK-8. The osteogenic differentiation of BMSCs was examined by detection of related gene expression with RT-PCR. Results: Compared with the BMSCs cell sheet, the numbers of cells on RADA16 scaffold growth rapidly at 3 rd-8 th day, and BMSCs were more on the scaffold than those on the cell sheet (P＜0. 05) . RT-PCR results showed that the expression level of osteogenesis-related genes was higher in the complex (P＜0. 05) . Conclusion: BMSCs Sheet-RADA16 Scaffold may promote proliferation and osteogenic differentiation of BMSCs.
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OBJECTIVE@#To observe the effect of acupoint injection of bone mesenchymal stem cells (BMSCs) combined with Chinese herbs of benefiting for activating blood circulation for capillary density and arterioles density in skeletal muscle in ischemic hind limb of diabetes mellitus (DM) rats.@*METHODS@#A total of 80 rats were randomized into a normal sham operation group (10 rats) and a model group (70 rats). Disposable intraperitoneal injection of streptozotocin (STZ, 50.0 mg/kg) was used to establish DM model, and the rats in the model group were randomized into 7 subgroups, 10 rats in each one. The subgroups were the DM sham operation group, DM ischemic group, Chinese herb group (intragastric herbs of benefiting for activating blood circulation), local injection group (BMSCs local injection), local injection + Chinese herb group (BMSCs local injection combined with intragastric herbs of benefiting for activating blood circulation), acupoint injection group (BMSCs acupoint injection), acupoint injection + Chinese herb group (BMSCs acupoint injection combined with intragastric herbs of benefiting for activating blood circulation). The local injection was phosphate buffer (PBS) injection at the equidistant 5 points along the line between the ischemic tissue and the normal tissue a time. The acupoints were "Sanyinjiao" (SP 6), "Zhaohai" (KI 6), "Huantiao" (GB 30), "Housanli" (ST 36) and "Yanglingquan" (GB 34). 100 μL BMSCs with 1×10/mL was totally injected at the above acupoints for one rat, 20 μL an acupoint. 1.5 kg/L Chinese herbs were applied by intragastric administration, including 120 g Radix Astragali, 120 g Codonopsis, 48 g Radix Glycyrrhiza, 120 g Angelica sinensis, 120 g Blood Rattan, 48 g Achyranthes bidentata. Intragastric distilled water was used in the other non-Chinese herb groups. The expressions of α-smooth muscle actin (α-actin), latelet endothelial cell adhesion molecule (CD31) and von willebrand factor (vWF) in the skeletal muscle were detected with immunohistochemical SP two-step method.@*RESULTS@#Twenty-one days after intervention, the expressions of α-actin and CD31 on the operation hind limb were higher than those on the healthy hind limb in all the groups, except the Chinese herb group (<0.05<0.01). The vWF expressions on the operation side were lower than those on the healthy side in the Chinese herb group, the local injection group, the local injection + Chinese herb group and the acupoint injection + Chinese herb group (<0.05, <0.01). The α-actin expression on the operation side in the acupoint injection + Chinese herb group was higher than those in the normal sham operation group, DM sham operation group, the DM ischemic group and the local injection group (<0.05, <0.01). The CD31 expressions in the acupoint injection group, the acupoint injection + Chinese herb group, local injection + Chinese herb group were higher than those in the normal sham operation group, DM sham operation group and DM ischemic group (<0.05, <0.01). The CD31 expression in the acupoint injection + Chinese herb group was higher than those in the Chinese herb group and the local injection group (both <0.05). The vWF expressions in the local injection + Chinese herb group, the acupoint injection group and the acupoint injection + Chinese herb group lower than those in the DM sham operation group and the DM ischemic group (<0.05, <0.01).@*CONCLUSION@#schemia increases the expressions of the vascular density related factors of α-actin and CD31. It is more obvious for the increasing expressions of α-actin and CD31, and decreasing expression of vWF with the interventions of simple BMSCs injection and simple Chinese herbs of benefiting for activating blood circulation, especially with the combination of the above tow methods. It is indicated that acupoint injection of BMSCs combined with Chinese herbs of benefiting for activating blood circulation can improve the angiogenesis of ischemic tissue.
Subject(s)
Animals , Rats , Acupuncture Points , Diabetes Mellitus , Ischemia , Lower Extremity , Mesenchymal Stem Cells , Rats, Sprague-DawleyABSTRACT
Hypoxia is beneficial for the differentiation of stem cells transplanted for myocardial injury, but mechanisms underlying this benefit remain unsolved. Here, we report the impact of hypoxia-induced Jagged1 expression in cardiomyocytes (CMs) for driving the differentiation of cardiac stem cells (CSCs). Forced hypoxia-inducible factor 1 (HIF-1) expression and physical hypoxia (5% O) treatment could induce Jagged1 expression in neonatal rat CMs. Pharmacological inhibition of HIF-1 by YC-1 attenuated hypoxia-promoted Jagged1 expression in CMs. An ERK inhibitor (PD98059), but not inhibitors of JNK (SP600125), Notch (DAPT), NF-B (PTDC), JAK (AG490), or STAT3 (Stattic) suppressed hypoxia-induced Jagged1 protein expression in CMs. c-Kit CSCs isolated from neonatal rat hearts using a magnetic-activated cell sorting method expressed GATA4, SM22 or vWF, but not Nkx2.5 and cTnI. Moreover, 87.3% of freshly isolated CSCs displayed Notch1 receptor expression. Direct co-culture of CMs with BrdU-labeled CSCs enhanced CSCs differentiation, as evidenced by an increased number of BrdU/Nkx2.5 cells, while intermittent hypoxia for 21 days promoted co-culture-triggered differentiation of CSCs into CM-like cells. Notably, YC-1 and DAPT attenuated hypoxia-induced differentiation. Our results suggest that hypoxia induces Jagged1 expression in CMs primarily through ERK signaling, and facilitates early cardiac lineage differentiation of CSCs in CM/CSC co-cultures HIF-1/Jagged1/Notch signaling.
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Objective To observe whether BMSCs differentiate into TAFs in inflammatory microenvironment simula-ted by IL-6 and TNF-α.Methods The experiment was divided into 9 groups:the BMSCs group,the 50 ng/mL, 100 ng/mL IL-6 intervention group, the 50 ng/mL,100 ng/mL TNF-α intervention group, the 50 ng/mL IL-6+50 ng/mL TNF-α intervention group,the 50 ng/mL IL-6+100 ng/mL TNF-α intervention group,the 100 ng/mL IL-6+50 ng/mL TNF-α intervention group and the 100 ng/mL IL-6+100 ng/mL TNF-α intervention group; After continuous induction for 42 days, BMSCs cell morphology and cycle, TAFs-tagged α-SMA and FAP protein were examined by phase-contrast microscope,flow cytometry and Western blot method. Results Compared with the normal control group, the 100 ng/mL IL-6+50 ng/mL TNF-α intervention group BMSC cell proliferation was significantly promoted;G1phase decreased in proportion and S phase increased;α-SMA and FAP protein expression was signifi-cantly increased (P＜0.05).Conclusions Microenvironment simulated by the 100 ng/mL IL-6+50 ng/mL TNF-α may induce the abnormal change of the BMSCs morphological and proliferative characteristics. TAFs reference mole-cule α-SMA and FAP expression is increased.
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Objective To study the effects of osteopontin (OPN) on the nuclear mechanics of bone marrow-derived mesenchymal stem cells (BMSCs) as well as its involved mechanisms. Methods The BMSC migration was evaluated using the Transwell assay. An atomic force microscope (AFM) was used to determine the elastic modulus of the BMSC nucleus and analyze the changes in the nuclear mechanics of the BMSCs after treatment with OPN. The activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase1/2 (ERK1/2) was measured by Western blot. The role of the FAK-ERK1/2 signaling pathway in mediating the OPN-affected BMSC nuclear mechanics was investigated by employing a specific inhibitor. RT-PCR and Western blot were used to detect the expression of Lamin A/C at mRNA and protein levels in the BMSCs, respectively. Results The elastic modulus of the BMSC nucleus exhibited a significant decrease after OPN treatment compared with that of the control group. OPN could upregulate the phosphorylation level of FAK and ERK1/2, but the inhibitor of FAK or ERK1/2 restored the OPN-decreased elastic modulus of the BMSC nucleus and inhibited the BMSC migration significantly. After treatment with OPN, the expression of Lamin A/C in the BMSCs reduced significantly, and such a reduced expression could be suppressed by the inhibitor of FAK or ERK1/2. Conclusions OPN could probably downregulate the expression of Lamin A/C of the BMSCs via the FAK-ERK1/2 signaling pathway, decrease the stiffness of the BMSC nucleus, and promote the migration of the BMSCs. The research outcomes provide the experimental evidence for further understanding the mechanism of the OPN-regulated BMSC migration and its potential clinical application.