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1.
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   

2.
AIM: To investigate the effect of zinc finger protein 521 (Zfp521) on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons. METHODS: Rat MSCs were cultured by conventional method in vitro and divided into non-transfection group, transfection group (transfected with Rn-Zfp521-siRNA) and negative control group (transfected with negative control siRNA). MSCs were induced by β-mercaptoethanol (β-ME) to differentiate into neurons. The fluorescence expressed by transfected MSCs was observed under inverted fluorescence microscope. The expression of Zfp521 was detected after transfection by RT-PCR. Immunohistochemistry, RT-PCR and Western blotting were used to detect the expression levels of neuron-specific enolase (NSE),microtubule-associated protein 2(MAP-2) and Zfp521 after induction. RESULTS: The fluorescence of MSCs was mostly displayed 72 h after transfection and the efficiency of transfection was up to 84.1%±2.3%. Meanwhile, the mRNA expression of Zfp521 was decreased (P<0.05). MSCs were induced by β-ME to differentiate into neurons. The differentiation efficiency of MSCs transfected with Rn-Zfp521-siRNA was the highest and the expression of NSE and MAP-2 was significantly increased compared with other groups (P<0.05). Zfp521 was detected in all groups, and the expression level of Zfp521 was significantly decreased after induction (P<0.01). CONCLUSION: Zfp521 may be down-regulated during the differentiation. The inhibition of Zfp521 promotes the neural differentiation of MSCs. Zfp521 may play an important role in regulating MSCs differentiation into neurons.  相似文献   

3.
AIM: To investigate the role of microRNA-9 in inducing bone marrow mesenchymal stem cell(MSCs) differentiation into neurons.METHODS: The lentiviral vector of microRNA-9-1(microRNA-9-1-LV) was constructed and transfected into mouse MSCs. The cells were divided into non-transfected group, transfected group(transfected with microRNA-9-1-LV) and negative control group(transfected with FU-RNAi-NC-LV). MSCs were treated with β-mercaptoethanol(β-ME) as an inducer for triggering the cells to differentiate into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The mRNA expression of microtublin-associated protein 2(MAP-2) was detected by RT-PCR. The expression of neuron-specific markers,neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP), were measured by immunocytochemical method. The viability of MSCs was determined by MTT method. RESULTS: The results of PCR confirmed successful construction of mouse microRNA-9-1-LV. The virus titer was 1×1012 TU/L(TU, transduction unit). The best transfection efficiency(up to 91.3%±4.2%) and survival rate appeared when multiply of infection(MOI)was 20 and on 4th day. β-ME induced MSCs to differentiate into neurons and the best efficiency of the induction was observed in transfected group. The expression levels of NSE and MAP-2 in transfected cells were higher than those in the cells of other group(P<0.05).CONCLUSION: MicroRNA-9-1-LV has high transfection efficiency in mouse MSCs. Higher differentiation rate from mouse MSCs to neurons is induced by β-ME after the cells are transfected with microRNA-9-1-LV.  相似文献   

4.
AIM: To approach the gap junction distribution and communication function of cardiomyocyte-like cells derived from rat bone marrow mesenchymal stem cells (MSCs) in vitro.METHODS: MSCs were isolated by extraction of bone marrow specimens,gradient centrifugation and the adherence of culture plates.MSCs were culture in vitro,treated with 5-azacytidine and incubated for 24 h.The induced MSCs,which had been incubated for 2,3 and 4 weeks,were divided into group Ⅰ,Ⅱ and Ⅲ.In addition,the normal cardiomyocyte cells were used as control group.The distribution of connexin 43(Cx43) and the mean fluorescence redistribution rate were detected in every group with the laser scanning confocal microscope.RESULTS: Cx43 protein grain density in induced MSCs was increased with the lasting of incubation by quantitive analysis of Cx43 distribution.After 4 weeks,the Cx43 protein density in induced MSCs was nonsignificant deviation with control group (63.87±12.43,64.87±12.15,P>0.05).The diversify tendency of the mean fluorescence redistribution rate was approximation with the result of Cx43 in every group.The results showed that groupⅠ was 19.59%±6.08%,groupⅡ was 37.17%±3.84%,groupⅢ was 46.82%±2.69%,and control group was 49.71%±5.53%.CONCLUSION: MSCs can be differentiated to cardiomyocyte-like cells,which have been induced and incubated for 4 weeks in vitro.The communicational function of those MSCs is similar to the normal cardiomyocyte cells.  相似文献   

5.
AIM：To compare the biological characteristics, surface markers and multi-differentiation potential of the mesenchymal stem cells (MSCs) derived from the umbilical cord and bone marrow in the green fluorescent protein (GFP) transgenic mice. METHODS：Umbilical cord MSCs (UCMSCs) were isolated by collagen type II enzymatic digestion and bone marrow MSCs (BMSCs) were isolated by density gradient centrifugation. The growth of the 2 types of MSCs was observed under inverted microscope. The cell proliferation was detected by determining the growth curve and MTT assay. The Trypan blue method was performed to analyze the cell viability rate. The cell cycle and cell surface markers were measured by flow cytometry. The differentiation potentials of the 2 types of MSCs were tested by the differentiation kits toward adipocytes and osteoblasts. RESULTS：The UCMSCs attached to the culture surface 1 d after the isolation, and the cells showed spiral shape with notable growth and proliferation after 2 d of culture. After 3 d, the cell arrived sub-confluent and was ready for passage. BMSCs still showed circular shape and started to attach to the surface 4 d after culture. They formed the small colony shape only after 5 d with obvious proliferative potential. The cells became confluent 7 d after the culture. The original generation of cultivating UCMSCs growth curve was shown typically an “S” shape. But the BMSCs growth was slower than the UCMSCs. The cell proliferation was obvious for UC-MSCs in 3~5 d. BMSCs proliferated significantly only after 7 d. The viability rate arrived more than 96% for both types of MSCs. The cell cycle of both MSCs did not show significant difference (G0/G1 phases were above 85%, P>0.05). Both MSCs positively expressed CD44, CD90 and CD105 (60.7%±2.3%) but the expression of CD45, CD19, CD14 and CD79 was negative (less than 25.6%±4.8%, P>0.05). More than 90% of the MSCs from the umbilical cord and bone marrow differentiated towards the adipocytes and osteoblasts without significant difference (P<0.05). CONCLUSION：UCMSCs have stronger ability of proliferation and multi-directional differentiation potentials. UCMSCs in GFP transgenic mice as a high-quality tracer can serve for tracking the stem cells in vivo.  相似文献   

6.
AIM: To investigate the balance of Treg/Th17 in synovium of collagen-induced arthritis (CIA) and the impact of tumor necrosis factor α(TNF-α) blockage therapy. METHODS: Rat CIA model was established by bovine II collagen injection. The pathological score was evaluated by HE staining and toluidine blue staining. The TNF-α level in plasma was measured by ELISA. The expression of Treg/Th17 in synovium was detected by double staining immunofluorescence. RESULTS: The plasma level of TNF-α in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (P<0.01), whereas no significant difference was found between TNFR-Fc treatment group and control group (P>0.05). No significant difference between CIA group and control group in the ratio of CD4+Foxp3+Treg cells/CD4+ cells in synovium (23.12%±4.93% vs 24.66%±5.82%, P>0.05) was observed, whereas the ratio in TNFR-Fc treatment group was significantly increased(33.07%±5.14%). The ratio of CD4+RORγt+Th17 cells/CD4+ cells in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (9.74%±2.23% vs 1.00%±0.59%, 5.63%±1.76%, P<0.01). CONCLUSION: Differentiation disturbance of Treg/Th17 exists in the synovium of CIA rats. TNFR-Fc may restore the balance of Treg/Th17 by inhibiting Th17 cell differentiation and inducing the production or accumulation of Treg.  相似文献   

7.
AIM: To study the effect of mesenchymal stem cells (MSCs) infusion on hematopoietic recovery after peripheral blood stem cell transplantation in mice. METHODS: BALB/c mice conditioned by high dose chemotherapy/radiotherapy were infused with106 peripheral blood mononuclear cells (PBMC) mobilized by granulocyte colony-stimulating factor (PBSCT group),104 MSCs culture-expanded in vitro and106 PBMC(experimental group1),106 MSCs and106 PBMC(experimental gruop 2). Survival rate within 4 weeks, white blood cell count, bone marrow nucleated cells (BMNC), granulocyte-macrophage colony forming unit(GM-CFU) and fibroblast colony forming unit (F-CFU) were examined. RESULTS: Survival rate, BMNC, GM-CFU, F-CFU were significantly higher in experimental group 2 than that in PBSCT group (P<0.05), WBC recovery was faster (P<0.01) and F-CFU level was higher in experimental group1than that in PBSCT group (P<0.05). CONCLUSION: Mesenchymal stem cells infusion enhanced hematopoietic reconstitution after peripheral blood stem cell transplantation.  相似文献   

8.
AIM: In order to observe the myocardial differentiation capacity of the dedifferentiated fat (DFAT) cells treated with vitamin C in vitro. METHODS: DFAT cells were dedifferentiated from the mature rat adipocytes with ceiling adherent culture. The DFAT cells of passage 3 were used in the study. Vitamin C and/or neonatal rat heart tissue lysate were added into the culture medium to induce myocardial differentiation for 3 weeks. The cell morphology was observed under microscope. The myocardial-specific markers, such as cTnT, GATA-4 and NKx2.5, were examined by the methods of immunofluorescence, PCR and Western blot. RESULTS: Mature rat adipocytes dedifferentiated into fibroblast-like DFAT cells after ceiling adherent culture. The DFAT cells spontaneously differentiated into cardiomyocyte-like cells under normal culture condition with a low incidence. After treated with neonatal rat heart cell lysate, the DFAT cells became cardiomyocyte-like cells that had bigger size, longer shape and myotubule-structure. The expression of cTnT, GATA-4 and NKx2.5 was remarkably increased at both mRNA and protein levels as compared with the normal cultured DFAT cells. The expression of cTnT, GATA-4 and NKx2.5 was further increased in DFAT cells after treating with vitamin C. No spontaneous beating cell was observed. CONCLUSION: Vitamin C enhances the differentiation of DFAT cells into cardiomyocyte-like cells.  相似文献   

9.
AIM: To study the influence of status of stimulator cells on activation of responder T cells in mixed lymphocyte reaction (MLR), so as to provide some basis for clinical transplantation. METHODS: Stimulator cells were pretreated differently before mixed lymphocyte culture (MLC) to change their functional status, fluorescence conjugated antibodies and flow cytometry were used to detect expression of CD69 by responder T cells at several different time points.RESULTS:The expression percentages of CD69 by responder T cells in MLCa group(stimulator cells were pre-activated)were significantly higher than those in MLC group(stimulator cells were not pre-activated)at 24,48and 72 hours of culture,respectively(5.21%±0.24%vs 1.98%±0.33%,29.81%±0.85%vs 20.65%±1.00%and 39.61%±1.62%vs 13.49%±0.60%,P<0.01). CONCLUSION: Our results showed that with pre-activated stimulator cells, expression of CD69 by responder T cells could be significantly elevated in MLR.  相似文献   

10.
AIM: To observe whether reversing methylation of SARI (suppressor of activator protein-1 regulated by interferon) gene by 5-azacytidine (5-Aza) inhibits the proliferation and invasion of human breast cancer MDA-MB-231 cells. METHODS: MDA-MB-231 cells were treated with 5-Aza at 5 and 10 μmol/L. The methylation of SARI gene promoter was detected by methylation-specific PCR (MSP), and the mRNA expression of SARI was detected by RT-PCR. The protein expression of SARI was determined by Western blot. The cell growth was detected by MTT assay and colony formation assay. The cell invasion ability was detected by Transwell invasion assay. RESULTS: The results of MSP detection showed that the methylation levels of SARI promoter were significantly decreased after 5-Aza treatment (P<0.01). The results of RT-PCR and Western blot showed that the expression of SARI were significantly increased after 5-Aza treatment (P<0.05). The results of MTT and colony formation assays showed that the cell proliferation was significantly decreased after 5-Aza treatment (P<0.05). The results of Transwell invasion test showed that the invasive ability of the cells was significantly decreased after 5-Aza treatment (P<0.05). CONCLUSION: 5-Aza reverses the methylation status of SARI promoter in MDA-MB-231 cells, up-regulates the expression of SARI, and restores its ability to inhibit tumor cell growth and invasion.  相似文献   

11.
AIM: To investigate the differentiation from rat mesenchymal stem cells (rMSC) into neuron-like cells. METHODS:rMSC were separated from femur marrow and expanded in L-DMEM culture medium supplemented with 10% FSC. rMSC were induced to differentiate into neurons with L-DMEM/adrenaline,L-DMEM/noradrenaline and L-DMEM/isoprenaline, respectively. Meanwhile, rMSC were cultured in L-DMEM in control group. Nestin, neuron-specific enclose (NSE), glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry. RESULTS: rMSC were expanded as undifferentiated cells in culture from 5 to 22 passages, indicating their differentiated capacity. Simple method induced rMSC to exhibit a neuronal phenotype, expressing positive NSE,nestin, and GFAP, at 5 hours in all group. The undifferentiating cells (control group 53.1%±4.3%), and differentiating cells (treated group: adrenaline 74.7%±2.6%; noradrenaline 75.9%±2.4%; isoprenaline 72.1%±4.4%), expressed characteristics of various neuronal cells, from 5 hours to 6 days. There were neuron-like cells in rMSC cultured in L-DMEM/10%FBS from 7 to 13 passage(66.5%±6.4%). CONCLUSION: It suggests that rat neural stem cells (rNSC) exist in bone marrow, rMSC can be differentiated into various neural cells with adrenaline hormones in vitro.  相似文献   

12.
AIM: To investigate the role of AMD3100 (an inhibitor of CXCR4) in dengue virus type 2 (DV2)-induced apoptosis in human umbilical vein endothelial cell line Eahy926. METHODS: The expression of factor Ⅷ in Eahy926 cells was examined by immunohistochemistry staining. The cells were divided into untreated group, DV2 infection group and DV2+AMD3100 group. Flow cytometric analysis was used to detect the expression of CXCR4 in Eahy926 cells 24 h, 36 h, 48 h and 60 h after DV2 infection. In addition, the percentage of apoptotic cells was also analyzed by flow cytometry. Immunofluorescence was performed to detect the phosphatidylserine (PS) on the surface of Eahy926 cells.RESULTS: Eahy926 cells were factor Ⅷ-positive. Compared with untreated group, the expression of CXCR4 increased in DV2 infection group, most markedly 48 h after infection (66.13%±10.30%, P<0.05). The percentage of apoptotic Eahy926 cells after DV2 infection was the highest at 36 h (29.85%±15.78%, P<0.05). The percentage of DV2-induced apoptotic cells in DV2+AMD3100 group was higher than that in DV2 infection group. The green fluorescence-labeled cells in DV2 infection group and DV2+AMD3100 group were more than those in untreated group. CONCLUSION: DV2 infection induces apoptosis and increases the expression of CXCR4 in Eahy926 cells. AMD3100, the inhibitor of CXCR4, may be a promoter of apoptosis in Eahy926 cells after DV2 infection.  相似文献   

13.
AIM: To investigate the effect of pretreatment of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) on the proliferation and the differentiation of mesenchymal stem cells (MSCs) into cardiomyogenic cells. METHODS: The MSCs, isolated primarily from bone marrow, and purified by passage culture, were obtained from the adult rats of four groups: the rats were pretreated by 5 daily injections of SCF; the rats were pretreated with G-CSF; the rats were pretreated with SCF and G-CSF; the rats were treated without any intervention. The 4th passage of MSCs was labeled by DAPI and cellular cycle analysis was conducted by flow cytometry before co-culture. The neonatal rat cardiomyocytes cultured for 3 days were co-cultured with DAPI-MSCs. The percentage of the differentiation of MSCs into cardiomyogenic cells during the five co-culture days was analyzed. The morphologic changes of MSCs and the proteins expression of cardiac myosin heavy chain (MHC) and troponin T (TnT) were recorded respectively with digital microscope camera system and immunofluorescence technique. The percentage of the differentiation of MSCs into cardiomyogenic cells was also calculated. RESULTS: The percentage of MSCs in G0/G1 phase in SCF/G-CSF group was significantly lower than that in SCF group, G-CSF group and the control group. The percentage of MHC protein-positive MSCs in SCF/G-CSF group was markedly higher than that in SCF group, G-CSF group and the control group, and that in SCF group and G-CSF group was significantly higher than control group. The percentage of TnT protein-positive MSCs in SCF/G-CSF group, SCF group and G-CSF group was significantly higher than that in control group.CONCLUSION: SCF and G-CSF show the ability to stimulate the proliferation of MSCs and induce MSCs to differentiate into cardiomyocytes. The combination of using SCF and G-CSF is more effective than using only SCF or G-CSF.  相似文献   

14.
AIM: To investigate the role of caveolin-1 in bone marrow mesenchymal stem cells (MSCs) differentiation into neurons. METHODS: The MSCs used in the experiment were divided into non-transfected group, transfected group (transfected with Rn-caveolin-1 siRNA), positive control group (transfected with Rn-MAPK-1 control siRNA) and negative control group (transfected with negative control siRNA). The MSCs were induced by β-ME to differentiate into neurons. The fluorescence expressed by transfected MSCs was observed under inverted fluorescence microscope. The mRNA expression of caveolin-1 and MAPK-1 was detected by RT-PCR. NSE, NF-M and GFAP, the neural cell specific markers, were detected by immunocytochemistry staining. The survival ratio of MSCs was detected by MTT method. RESULTS: The fluorescence of MSCs was mostly displayed at 72th h after transfection and the efficiency of transfection was up to 81.5%±2.8%. Meanwhile, the mRNA expression of caveolin-1 in transfected MSCs was decreased (P<0.05). No significant difference of the survival MSCs ratio between transfected group and other groups was observed by MTT method. β-ME induced MSCs into neural cells. The differentiation efficiency of MSCs transfected with Rn-caveolin-1 siRNA was the highest and the expression of NSE and NF-M was increased significantly compared to the other groups (P<0.01). The expression of caveolin-1 was increased persistently with time and the highest expression was observed 6 d after induction. Furthermore, there was significant difference between before induction and 6 d after induction. CONCLUSION: Lipid raft labeled with careolin as marker protein has important role in regulating MSCs differentiation into neurons.  相似文献   

15.
AIM: To investigate the role of Oct3/4 in inducing differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons in vitro. METHODS: Lentivirus (LV) vector containing Oct3/4 gene was constructed and transfected into rat bone marrow MSCs. The MSCs were divided into non-transfection group, transfection group (transfected with Oct3/4 -LV) and negative control group (transfected with FU-PCG-NC-LV). β-mercaptoethanol (β-ME) was used to induce differentiation of MSCs into neurons. Morphological changes and the fluorescence in transfected MSCs were observed under inverted fluorescence microscope. The expression of Oct3/4 and microtubulin-associated protein 2(MAP-2) at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of Oct3/4 and the neural cell specific markers neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP) were determined by immunocytochemical method. The viability of the MSCs was analyzed by MTT assay. RESULTS: The results of PCR confirmed that the Oct3/4 -LV was successfully constructed and the virus titer was 2×1011 TU/L. The best transfection efficiency and survival rate appeared when multiply of infection(MOI) was 10 and at 48 h, and the fluorescence of MSCs was mostly displayed. The efficiency of transfection was up to 83.4%±2.2%. The shape of the MSCs was changed in transfection group, and the survival rate of the MSCs in transfection group was significant lower than that in other groups (P<0.05). MSCs were induced by β-ME to differentiate into neurons and the best efficiency of induction was observed in transfection group. The typical neuronal morphology was observed in transfection group after induction and the expression levels of NSE and MAP-2 were higher than those in other groups (P<0.05). Compared with other groups, the expression of Oct3/4 in transfection group was significantly increased (P<0.01). Furthermore, the expression of Oct3/4 was time-dependently decreased and there was significant difference between before induction and 5 h after induction (P<0.05). CONCLUSION: Oct3/4 may have an important role in regulating the differentiation of rat MSCs into neurons.  相似文献   

16.
AIM: To verify the role of enhancing or suppressing the expression of glutathione peroxidase 1 (GPx1) in the growth, migration and invasion of glioblastoma multiforme cell lines U87MG and U118MG. METHODS: U87MG and U118MG cell lines were transfected with the vector containing specific siRNA or pcDNA3.1 recombinant plasmid both targeting GPx1. The mRNA and protein expression levels of GPx1 were detected by real-time PCR and Western blotting. MTS assay was applied for determining the cell activity. The abilities of migration and invasion were examined by Transwell assay. RESULTS: Compared with blank control group and negative group, the inhibitory rate of the cell activity in U87MG cells in siRNA group was significantly reduced by 25.9%, 35.7% and 34.8% at 24 h, 48 h and 72 h, respectively (P<0.05). In contrast, the cell activity of U118MG cells in pcDNA3.1-GPx1 group was significantly increased by 22.7%, 45.8% and 39.8% at 24 h, 48 h and 72 h, respectively (P<0.05). In siRNA group, the inhibitory rate of migration in U87MG cells was 41.6%±8.2% and the invasion was 41.6%±8.2% compared with blank control group and negative group (P<0.05). The cell migration and invasion rates of the U118MG cells in pcDNA-GPx1 group were increased by 55.8%±9.8% and 60.8%±9.2%, respectively, compared with blank control group and negative group (P<0.05). CONCLUSION: The down-regulation of GPx1 by specific siRNA reduces the capability of cell growth, migration and invasion of U87MG cells, while up-regulation of GPx1 by pcDNA3.1-GPx1 increases the capability of cell growth, migration and invasion of U118MG cells.  相似文献   

17.
AIM: To explore the effect of transplantation of human receptor activity-modifying protein 1 ( hRAMP1 ) gene-modified bone marrow mesenchymal stem cells (MSCs) on neointima formation after carotid balloon angioplasty in carotid atherosclerosis rabbits. METHODS: MSCs were collected through density gradient centrifugation and adherent culture. MSCs were transfected with adenovirus vector carrying hRAMP1 gene to generate hRAMP1 gene-modified MSCs (hRAMP1-MSCs). All animals with carotid atherosclerosis and balloon angioplasty were randomly divided into hRAMP-MSCs group, MSCs group and control group. After the model was established, MSCs transfected with pAd2-EGFP-hRAMP1 or pAd2-EGFP and PBS were injected to the ear vein,respectively. The injured carotid arteries were harvested to detect the homing of MSCs,reendothelialization and neointima thickness 7 d, 14 d and 28 d after cell transplantation. The plasma samples were collected for detecting vascular endothelial growth factor (VEGF) by ELISA. The expression of endothelial nitric oxide synthase (eNOS) in injured carotid arteries was measured by Western blotting. RESULTS: The expression of CD31 and EGFP was observed in the neointima at different time points in hRAMP1-MSCs group and MSCs group. Compared to control group, the reendothelialization of carotid significantly increased in both hRAMP1-MSCs group and MSCs group at different time points (P<0.05), and that in hRAMP1-MSCs group showed better than that in MSCs group (P<0.05). The area of neointima and the rate of restenosis were lower in hRAMP1-MSCs group and MSCs group than those in control group, and those in hRAMP1-MSCs group were significantly lower than those in MSCs group. The plasma level of VEGF and the expression of eNOS in the injured carotid arteries were significantly higher in both hRAMP1-MSCs group and MSCs group than those in control group at different time points (P<0.05), and those in hRAMP1-MSCs group were better than those in MSCs group (P<0.05). In the injured carotid arteries, the expression level of proliferating cell nuclear antigen (PCNA) in hRAMP1-MSCs group was the lowest,with the middle level in MSCs group and the highest level in control group. CONCLUSION: The hRAMP1 gene-modified MSCs are better in promoting reendothelialization and attenuating neointima than natural MSCs. The recombinant hRAMP1 adenovirus vectors dont affect the differentiation potential of MSCs into endothelial cells.These findings indicate that the modified stem cells have the potency of more effective reendothelialization to decrease restenosis after angioplasty.  相似文献   

18.
AIM: To investigate the effects of cotransplantation of mesenchymal stem cells (MSCs) and umbilical cord blood (UCB) by intra-bone marrow (IBM) injection on the hematopoietic reconstitution and recovery of bone marrow MSCs in the recipients. METHODS: Wistar female rats were transplanted with fetal and neonatal peripheral blood (FNPB) and BrdU-labeled MSCs separated from BMNCs of F344 rats. The MSCs were infused by IBM injection in bilateral tibiae or intravenous injection (IV), while the FNPB was all via IBM route. The survival rate, reconstitution of hematopoietic and immunological function, engraftment level of HSCs and recovery of bone marrow (BM)-MSCs in recipients were monitored. The origins of BM-MSCs of recipients were examined by immunofluorescence assay. RESULTS: (1)The survival rate in the two cotransplantation groups was 100% at day 60, while that in FNPB group was only 66.7%. (2)The counts of peripheral blood cells and BM hematopoietic stem/progenitor cell colonies of the recipients were better in cotransplantation groups than those in FNPB group, especially in the FNPB (IBM)+MSC (IBM) group. (3)No significant difference between of engraftment level of HSCs in the two cotransplantation groups was observed. The percentage of RT1A1 cells subset in FNPB (IBM)+MSC (IBM) group was much higher than that in FNPB group (P<0.05). (4)At day 30, the growth characteristic of recipient BM-MSCs was still below normal, but that in FNPB (IBM)+MSC (IBM) group was the best of all the experiment groups (P<0.05). (5)The donor MSCs coexisted with host MSCs in only a few recipient rats. CONCLUSION: The cotransplantation of MSCs and FNPB can accelerate the recovery of recipient BM-MSCs and hematopoietic reconstitution, promote the engraftment level of HSCs. Cotransplantation by IBM route is safe and has better effects on hematopoietic reconstitution than by IV route.  相似文献   

19.
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   

20.
AIM：To estimate the neural differentiation efficiency of bone marrow mesenchymal stem cells (MSCs) derived from amyloid precursor protein (APP) transgenic mice and to investigate the correlation with Notch1 signaling and the autophagy activity during the differentiation. METHODS：The MSCs were divided into APP group (MSCs from APP transgenic mice) and WT group (MSCs from wild-type mice). MSCs were treated with β-mercaptoethanol as an inducer for differentiating into neurons. The levels of Aβ40 and Aβ42 were measured using enzyme-linked immunosorbent assay kits. The expression of neural cell-specific markers, neuron-specific enolase (NSE) and microtubule-associated protein 2 (MAP-2), was measured by immunocytochemistry and Western blotting. The expression levels of Notch1, Notch intracellular domain (NICD), Hes5, LC3 and p62 (a selective substrate of autophagy) were also detected by Western blotting. RESULTS：The neural differentiation capacity and the Aβ expression level of the MSCs in APP group were higher than those in WT group, and stronger inhibition of Notch1 signaling pathway in the MSCs from APP group was observed. However, the process of autophagy, which is essential for the survival and function of the neural cells, was impaired in the neural differentiated counterpart of the MSCs in APP group. CONCLUSION:Over-expression of APP might contribute to the high neural differentiation capacity of MSCs by inhibiting Notch1 signaling pathway in vitro. However, autophagy is impaired in the differentiated MSCs from APP transgenic mice.  相似文献   

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