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1.
    
GAO Li  HU Cheng-hu  JIN Yan 《园艺学报》2013,29(4):707-712
AIM：To investigate the different functions of bone marrow mesenchymal stem cells (BMMSCs) in age-related osteoporosis. METHODS：The senescence-accelerated mice (SAMP6) were used in the experiment. The BMMSCs were isolated from femora and tibiae by flushing. Flow cytometric analysis was performed with MSCs-related monoclonal antibodies. The expression of differentiation genes was tested by real-time RT-PCR. RESULTS：In the progress of age-related osteoporosis, BMMSCs exhibited a decrease in osteogenesis and an increase in adipogenesis. Transforming growth factor β(TGF-β) signaling was significantly changed along with aging in SAMP6 mice. CONCLUSION：The functional changes of BMMSCs may play an important role in senile osteoporosis. The alteration of TGF-β-related gene expression may be the molecular mechanism of dysfunction in BMMSCs.  相似文献   

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AIM: To explore the effect of xanthosine (Xs) on proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: Xs was directly added to the culture system and the effects of Xs on proliferation and differentiation of BMSCs were observed. RESULTS: In the presence of Xs, the growth rate of the 10th passage cells was almost similar to that of the 4th passage cells and no downtrend was observed. However, in control group (without Xs), the growth rate of the 10th passage cells was obviously declined. The BMSCs promoted by Xs still kept up a vigorous capability of differentiation into hepatocytes. CONCLUSION: As an inhibitor of asymmetric cell kinetics, Xs can promote the conversion of BMSCs from asymmetric cell kinetics to symmetric cell kinetics, and keeps synchronously the ability of differentiation from BMSCs into hepatocytes as well. It is helpful for enhancing the proliferation efficiency of BMSCs in vitro, and will have extensive application of clinical practice.  相似文献   

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AIM: This study was performed to investigate the feasibility and efficiency of exogenous mesenchymal stem cells (MSCs) transplantation on post-infarction ventricular remodeling and heart function in rats and compare the effects between adult rat MSCs and neonate rat MSCs transplantation. METHODS: 1-2 hours after left coronary artery ligation, MSCs cultured in ex vivo, marked with BrdU, were injected directly into the border of infarcts in exogenous rats. 6 weeks after transplantation, rat heart function, ventricular remodeling and pathological results were measured. RESULTS: MSCs transplantation decreased LV end-diastolic diameter and end-systolic diameter, limited LV chamber dilatation and reduced collagen content significantly. The numbers of blood vessels and cardiomyocytes were increased. BrdU-labelled MSCs with oval nucleus were widely distributed. There were no significant difference between adult rat MSCs and neonate rat MSCs transplanted groups. CONCLUSION: MSCs can survive and home in exogenous host infarct hearts without addition of any immunosuppressant. MSCs transplantation has benificial effects on remodeling processes and contributes to improvement of cardiac function, which may be related with the reduction of the amount of the collagen, promotion of myogenesis and angiogenesis.  相似文献   

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AIM: To investigate the basic biological characteristics of adult rat bone marrow mesenchymal stem cells(rBMMSCs), and compare to that of human BMMSCs (hBMMSCs). METHODS: rBMMSC and hBMMSCs were separated from bone marrow with the difference of adherence and Ficoll-Paque reagent, and expanded in culture medium in vitro, respectively. The proliferation and growth characteristics of the primary and different passage culture of rBMMSCs and hBMMSCs were analysed. The neural differentiation capacity of rBMMSCs with passages were observed. To detect the surface antigens of rBMMSCs, the labeled cells were analysed on a FACScan flow cytometer. The karyotype of rBMMSCs were detected by blocking cellular fission with colchicines. RESULTS: rBMMSCs and hBMMSCs have a strong self-renewal capacity. Approximately (4-8)×1012 and (3-4)×1012 cells were obtained after passage 15 in vitro, respectively. The ability of proliferation, CFU-Fs, and neural differentiation of rBMMSCs and hBMMSCs were decreased gradually with passages, but the ability of proliferation and CFU-Fs of rBMMSCs were higher than that of hBMMSCs at different passage. FACScan result showed rBMMSCs were uniformly positive for CD29 and CD44, and negative for CD11b, CD45, CD61, CD71, CD80, CD86,MHCⅠ and MHCⅡ. rBMMSCs had an normal karyotype, which had an average of 37.0±4.0 to 40.5±2.5 chromosomes. CONCLUSION: Adult rBMMSCs have strong self-renewal and neural differentiation capacity, and have an normal karyotype. So rBMMSCs can be used as the seed cells for tissue engineering.  相似文献   

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AIM：To investigate the effects of recombinant human transforming growth factor β1 (rhTGF-β1) on the ability of proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs), as well as its effects on the expression of bone morphogenetic protein 2 (BMP-2), Smad4 and core binding factor α1 (Cbfa1). METHODS：SD rat MSCs were isolated and purified by the differential time adherent method. MTT assay was used to confirm the optimal concentration of rhTGF-β1 for the proliferation of MSCs. The optimal concentration for differentiation of MSCs into osteoblast was also determined by observing the activity and positive staining of alkaline phosphatase. According to the different induction conditions, MSCs were divided into 4 groups:control group, classic group, rhTGF-β1 group, and rhTGF-β1+classic group. Alkaline phosphatase, type I collagen, bone Gla protein and calcium nodes were detected to evaluate the osteogenic differentiation. BMP-2 was detected by ELISA and the mRNA expression of Smad4 and Cbfa1 was analyzed by real-time quantitative PCR. RESULTS：The optimal concentrations of rhTGF-β1 for the proliferation of MSCs and for the osteogenic differentiation of MSCs were 10 and 5 μg/L, respectively. The MSCs in classical group and rhTGF-β1 group were promoted to osteogenic differentiation, and the mRNA expression of BMP-2, Smad4 and Cbfa1 was increased. rhTGF-β1 induced osteogenic differentiation of MSCs in the early and middle terms. However, in rhTGF-β1+classic group, the osteogenic differentiation of MSCs was more obvious in the late term. CONCLUSION:The induction conditions of classical group, rhTGF-β1 group and rhTGF-β1+ classical group promote the differentiation of MSCs by increasing BMP-2 secretion and starting the TGF-β superfamily/Smads signaling pathway to regulate the differentiation of MSCs.  相似文献   

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AIM: To investigate the mechanism by which human amnion induced mouse embryonic stem (ES) cells to differentiate into epidermal like cells. METHODS: ES- BALB/ c cells were cocultured with human amnionintranswells for 4 - 5 days , andthose cultured alone without amnion were taken as control group. The morphological differentiation were observed . The committed differentiation of EScells into epidermal like cells were detected by integrin-β1 , CK19 , CK15 andinvolucrin immunohistochemistry , respectively .RESULTS: After 4-5 days of coculture, ES cells differentiated into single layer of epidermal like cells, fitted tightly, with polyhedral in shape. The immunohistochemical staining results showed that, most of the cells were integin-β1 positive, only a few cells were CK19 and CK15 positively stained. Most of the cells in control group died, the survived ones were different in morphological shapes, and no integrin-β1, CK19 and CK15 positive cells were found. CONCLUSION: Soluble substances secreted by human amnion may play an important role in inducing the differentiation of mouse ES cells into epidermal like cells.  相似文献   

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AIM：To investigate the changes of Wnt signaling pathway in catalpol-induced proliferation of rat bone marrow mesenchymal stem cells (BMSCs). METHODS：The BMSCs were isolated from SD rats, purified by differential time adherent method and divided into control group and catalpol (1.0 mg/L) group. Flow cytometry was used to detect the proliferation index of BMSCs. The mRNA levels of Wnt3a, Wnt5a, Wnt11 and β-catenin was evaluated by real-time PCR. In addition, the protein expression level of β-catenin was determined by Western blotting. RESULTS：Prolife-ration index was increased from 8.90%±0.46% to 17.93%±1.68% after treatment with catalpol (P<0.01). Compared with control group, the mRNA expression of Wnt5a, Wnt11 and β-catenin was all increased with catalpol treatment. No difference of Wnt3a mRNA expression between control group and catalpol group was observed. Meanwhile, the protein expression of β-catenin was increased in catalpol group compared with control group. CONCLUSION：Catalpol promotes BMSCs going into the cell cycle. Classical and non-classical Wnt signaling pathways are activated in this process.  相似文献   

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AIM: To study the effects of Chinese herbal monomer naringin (NG) on the MAPK signal pathway in bone marrow mesenchymal stem cells (MSCs) derived from SD rats during the differentiation into osteoblasts in vitro . METHODS: The changes of evaluating indicators alkaline phosphatase (ALP), bone gla protein (BGP) and type I collagen (Col I) in MSCs were observed under the conditions of normal, adding p38 pathway inhibitor SB203580, adding extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059, adding c-Jun N-terminal kinase (JNK) pathway inhibitor SP600125, and adding SB203580, PD98059 and SP600125 together. The protein phosphorylation of p38, ERK1/2 and JNK was measured by Western blotting. The mRNA expression levels of transforming growth factor beta 1 (TGF-β1), bone morphogenetic protein 2 (BMP-2) and core binding factor α1 (Cbfα1) were measured by fluorescence quantitative PCR. RESULTS: The most effective concentration of NG to promote the differentiation of MSCs into osteoblasts was 10-7 mol/L. The highest expression levels of both ALP and BGP were observed in NG group (P<0.05), while the expression of Col I did not reveal significant difference (P>0.05). Compared with NG group, the expression levels of ALP, BGP and Col I decreased differently after adding different inhibitors. Compared with control group, the protein phosphorylation of JNK was increased (P<0.05), and the phosphorylation of p38 was decreased (P<0.05), while the phosphorylation of ERK1/2 did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the protein phosphorylation of p38, ERK1/2 and JNK showed fluctuation with some increasing and others decreasing. Compared with control group, the expression of BMP-2 was increased (P<0.05), and the expression of Cbfα1 was decreased(P<0.05), while the expression of TGF-β1 did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the mRNA expression levels of TGF-β1, BMP-2 and Cbfα1 decreased differently after adding different inhibitors. CONCLUSION: Activation of ERK/JNK signaling and up-regulation of BMP-2 expression may be the main mechanism of NG to promote the differentiation of MSCs into osteoblasts. NG has strong impact on p38 pathway to improve the expression of BMP-2 in MSCs.  相似文献   

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AIM: To investigate the effects of azathioprine (AZA) on the proliferation, cell cycle and apoptosis of mesenchymal stem cells (MSCs) from the bone marrow of Sprague-Dawley rats in vitro. METHODS: MSCs were cultured in low-glucose DMEM containing 10% FBS,and treated with AZA at concentrations of 50 mg/L, 100 mg/L, 200 mg/L and 300 mg/L for 24 h, 48 h and 72 h. The effects of AZA on the growth curve and proliferation of MSCs were tested by cell counter under microscope. The apoptosis and cell cycle was determined by flow cytometry. RESULTS: Pure MSCs were gained by 3 times of passages. No significant effect of AZA at concentration of less than 100 mg/L on the proliferation, cell cycle and apoptosis of MSCs was observed (P>0.05). Under the condition of more than 200 mg/L for 72 h, AZA inhibited the growth of MSCs.The inhibitory rate was more than 66%, and the rate of apoptosis was increased (P<0.05). However, at the concentration of 300 mg/L for 72 h, AZA decreased the apoptotic rate and the necrotic rate of MSCs was obviously increased (P<0.05). Using AZA at concentration of more than 200 mg/L, as the action time prolonged, the MSCs in G0/G1 phase were increased, and those in S phase were decreased (P<0.05). CONCLUSION: At some concentrations, AZA significantly affects the proliferation, apoptosis and cell cycle of MSCs. Large dose of AZA may cause MSCs to death.  相似文献   

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WANG Jing  ZHANG Yuan 《园艺学报》2007,23(3):584-586
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AIM: To study the effect of meglumine cyclic adenylate (MCA) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes in vitro. METHODS: The whole bone marrow adherent culture method was used to isolate, culture and amplify the BMSCs. The surface markers of BMSCs were determined by flow cytometry analysis. MCA at concentrations of 10-2 mol/L, 10-3 mol/L, 10-4 mol/L, 10-5 mol/L, 10-6 mol/L and 10-7 mol/L was added to the culture medium containing the second generation of BMSCs.5-Azacytidine(5-Aza) was used as a positive control. The cell viability was measured by MTT method.The cAMP content in BMSCs was detected by ELISA. The mRNA expression of GATA-4, Cx43 and β-MHC in MCA group and MCA+H89 (a PKA inhibitor) group was measured by SYBR-RT-PCR. The differentiation effects of MCA and 5-Aza were compared by flow cytometry. RESULTS: Most of the BMSCs expressed CD44 and CD71, and did not express CD45. MCA inhibited the viability of BMSCs in a time-and dose-dependent manner, and MCA atthe concentration of 10-2 mol/L showed particularly remarkable effect. MCA significantly increased intracellular cAMP level in BMSCs in a concentration-dependent manner. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group were significantly higher than that in blank group (P<0.05), and the highest effect was under the condition of MCA induction at the concentration of 10-3 mol/L for 3 days. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group was higher than that in 5-Aza group and H89+MCA group (both P<0.05). Differentiation rate in MCA group was slightly higher than that in 5-Aza group (20.24%±1.02% vs 18.39%±0.58%, P<0.05). CONCLUSION: MCA stimulates BMSCs to increase intracellular cAMP production and inhibits the viability of BMSCs, thus promoting the mRNA expression of GATA-4, β-MHC and Cx43 through the cAMP/PKA signaling pathway.  相似文献   

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AIM:To study the roles of extracellular signal-regulated kinase(ERK) signal pathway in the process of osteogenic differentiation in rat mesenchymal stem cells(MSCs) promoted by quercetin(QUE). METHODS:The optimal concentration of QUE for promoting osteogenic differentiation of rat MSCs was determined by MTT and alkaline phosphatase(ALP) detection. The activity of ALP was detected by the ALP detection kit. The expression of bone Gla protein(BGP) and collagen typeⅠ(ColⅠ) was observed by ELISA analysis. MSCs were exposed to QUE at optimal concentration with or without ERK1/2 inhibitor PD98059. Non-phosphorylated and phosphorylated expression of ERK1/2 was analyzed by Western blotting. The mRNA expression of transforming growth factor β1(TGF-β1), bone morphogenetic protein 2(BMP-2) and core binding factor α1(Cbfα1) was measured by fluorescence quantitative PCR. RESULTS:QUE at concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L induced the expression of ALP in MSCs in a dose-dependent manner, and also promoted MSCs proliferation. The expression levels of ALP, BGP and ColⅠwere higher in QUE group, and was lower in PD89059 group than those in control group. Compared with control group, the level of phosphorylated ERK1/2, and the mRNA expression of TGF-β1, BMP-2 and Cbfα1 increased in QUE group. The mRNA expression of TGF-β1, BMP-2 and Cbfα1 in QUE+PD98059 group decreased as compared with QUE group. CONCLUSION:QUE promotes osteogenic differentiation of MSCs by activating ERK signaling pathway.  相似文献   

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AIM：To study the effect of calcitonin gene-related peptide (CGRP) gene transfection mediated by lentivirus on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to endothelial cells. METHODS：Rat bone marrow MSCs were isolated by density gradient centrifugation combined with adherence method. Recombinant lentivirus vector carrying CGRP gene (Lenti-CGRP) was transfected into the MSCs. The secretion of CGRP in culture supernatants of the transfected MSCs was detected using ELISA method. The cells at passage 3 were divided into three groups: CGRP group (MSCs transfected with Lenti-CGRP), CGRP+CGRP8-37 (an antagonist of CGRP receptor) group and control group (MSCs transfected with PBS). The differentiation of the MSCs was detected by immunocytochemical staining for CD31 and factor Ⅷ-related antigen. The proliferation of the cells was measured by cell counting, and the angiogenic ability of the cells was analyzed using Matrigel assay. RESULTS：The proportion of CD31-and factor Ⅷ-related antigen-positive cells in CGRP and CGRP+CGRP8-37 groups was larger than that in control group (P<0.05). The numbers of the cells in CGRP and CGRP+CGRP8-37 groups were significantly increased compared with control group (P<0.05). Lumen-like structures were observed in CGRP and CGRP+CGRP8-37 groups. The above indexes in CGRP+CGRP8-37 group were reduced compared with CGRP group. CONCLUSION: Transfection with CGRP gene induces rat bone marrow MSCs to differentiate into endothelial cells and enhances their proliferation, suggesting that CGRP may play a role in the regulation of angiogenesis.  相似文献   

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AIM:To investigate the effects of sinapine, an effective monomer of Chinese medicine, on hydrogen peroxide (H2O2)-induced adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).METHODS:The undifferentiated rat BMSCs were identified and screened by flow cytometry. The adipogenic differentiation of BMSCs was induced by H2O2, and the toxicity of sinapine on BMSCs was tested by CCK-8 assay. After the modeling method and the concentration range of sinapine were determined, the lipid droplets in the cells were detected by Oil Red O semi-quantitative assay, and the optimal drug concentration was selected. Finally, Oil Red O assay was observed 24 h after drug intervention, and the expression of adipogenic differentiation-related proteins, adipocyte protein 2 (aP2), peroxisome proliferator-activated receptor γ (PPARγ) and glucose transporter 4 (Glut4), at mRNA and protein levels in the BMSCs was determined by qPCR and Western blot.RESULTS:Treatment with H2O2 at 200 μmol/L for 1 h induced BMSCs to differentiate into adipocytes. Below the concentration of 40 μmol/L, sinapine had no toxicity to BMSCs. The best inhibitory concentration of sinapine on adipogenic differentiation was at 15 μmol/L. The number of lipid droplets in sinapine (15 μmol/L) group was significantly lower than that in model group. In sinapine group, the expression of aP2, PPARγ and Glut4 at mRNA and protein levels was lower than that in model group (P<0.01).CONCLUSION:Sinapine inhibits H2O2-induced adipogenic differentiation of rat BMSCs. The mechanism may be related to the PPARγ/AMPK signaling pathway.  相似文献   

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AIM: To investigate the therapeutic effects of recombinant meglumine cycle adenylate phosphate (MCA) and bone marrow mesenchymal stem cells (BMSCs) on the enhancement of the cell survival and improvement of the cardiac functions in the rat model of adriamycin-induced cardiomyopathic heart failure. METHODS: BMSCs were isolated and expanded using the pre-plating method. Doxorubicin was used by intraperitoneal injection into the Wistar rats to establish the model of cardiomyopathic heart failure. The model animals randomly received the injection of PBS, MCA, BMSCs or MCA+BMSCs respectively, and normal controls were without any treatment. Four weeks after injection, the cardiac functions were determined by echocardiography and multichannel physiological recorder. The levels of brain natriuretic peptide (BNP) were measured by ELISA. The positive rate of BrdU-labeled BMSCs in the myocardium was analyzed by the method of immunohistochemistry. The expression of myocardium-specific protein, GATA-4, connexin 43(Cx43) and cardiac troponin 1(cTNI), was detected by Western blotting. Myocardial fibrosis was observed with Masson's staining. RESULTS: Compared with other groups, the results of echocardiography and hemodynamic showed that the left ventricular functions in BMSCs+MCA group improved significantly (P<0.05). The BMSCs numbers in the myocardium in BMSCs+MCA group were significantly higher than those in BMSCs group (P<0.05). The level of BNP was significantly lower in BMSCs+MCA group than that in BMSCs group (P<0.05). Compared with other groups, the expression of GATA-4, Cx43 and cTNI was significantly increased in BMSCs+MCA group. CONCLUSION: Combination of MCA with BMSCs transplantation improves the cardiac functions, possibly due to the enhancement of BMSCs survival and the increase in the protein expression of GATA-4.  相似文献   

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AIM: To investigate the effect of tert-butylhydroquinone(tBHQ) on the replicative senescence of bone marrow mesenchymal stem cells(BMSCs).METHODS: Late stage BMSCs were continuously treated with tBHQ at concentration of 30 μmol/L for 4 weeks and the cells were used for the following assays immediately. The proteasomal activity was determined by chemiluminescence method. The samples were subjected to CCK-8 assay and BrdU incorporation as well as flow cytometry analysis for analyzing the cell vitality and proliferation. Percentage of senescent cells was detected by senescence-associated β-galactosidase(SA-β-Gal) staining. The expression of P53 was measured by Western blot.RESULTS: After the continuous treatment of tBHQ(30 μmol/L) for 4 weeks, the proteasomal activity of late stage BMSCs increased by 21.96%±1.98%(P<0.05). The cell vitality and survival were significantly increased with the increases in tBHQ doses till 40 μmol/L, and no cytotoxicity reaction with the increased dose of tBHQ till 120 μmol/L was observed. BrdU-positive cells, which represented the cell proliferation, were significantly increased(P<0.05). The proliferation index was also significantly increased by flow cytometry analysis(P<0.05). The SA-β-Gal positive cells and the expression of P53 were decreased(P<0.05).CONCLUSION: tBHQ delays the proteasome dysfunction associated senescence progress of BMSCs by increasing the proteasomal activity.  相似文献   

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AIM: To investigate the effect of 18 alpha-glycyrrhetinic acid (18α-GA) on delaying the senescent progress and promoting the proliferation in late-passage bone marrow mesenchymal stem cells (BMSCs). METHODS: Late-passage BMSCs were incubated with 2.0 mg /L 18α-GA or the same volume of DMSO for 30 d, and the cells were harvested to determine the proteasome activity. The expression of senescence-related proteins p53, p21 and p16 was detected by senescence-associated β-galactosidase (SA-β-Gal) staining and Western blot. The cell proliferation, the expression level of cell cycle-related proteins and cell cycle distribution of the cells were measured by CCK-8 assay, BrdU incorporation, Western blot and flow cytometry. RESULTS: Compared with DMSO group, the proteasome activity in 18α-GA group increased significantly by about 0.2 times (P<0.01). SA-β-Gal-positive cells in 18α-GA group decreased, and cell staining was lighter. The contents of p53 and p21 in 18α-GA group were decreased (P<0.05). The results of CCK-8 assay showed that the A value in 18α-GA group was 0.3 times higher than that in DMSO group (P<0.01). BrdU incorporation showed the increased proliferation in 18α-GA group compared with DMSO group (P<0.05). The cells in G1 phase in 18α-GA group decreased significantly compared with DMSO group, while the cells in S phase increased significantly (P<0.05). The expression level of cyclin D1 in 18α-GA group was 2.8 times higher than that in DMSO group (P<0.01), and the CDK4 level was 1.4 times higher than that in DMSO group (P<0.05). CONCLUSION: Activation of the proteasome activity by 18α-GA delays the aging process in the BMSCs and promotes the cell proliferation via up-regulation of the cell cycle-related proteins.  相似文献   

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AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.  相似文献   

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