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1.
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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AIM: To investigate the effect of transfection of hTERT gene into human mesenchymal stem cells (MSCs) on their telomerase activity and life-span.METHODS: Human MSCs were transfected with a pEGFP-hTERT plasmid by liposome-mediated transfection. Then the hTERT mRNA expression in MSCs was detected by RT-PCR. The activity of telomerase in transfected MSCs was detected by PCR and ELISA. The telomerase-positive MSCs was cultured in vitro and induced into neuron-like cells with EGF and bFGF. Neuron-specific markers (NF-M, MAP2) were detected by RT-PCR.RESULTS: hTERT fragment was identified in the hTERT-transfeced cells but not in the untransfected human bone marrow MSCs. The untransfected human MSCs remained telomerase-negative but the hTERT-transfected cells showed robust telomerase activity. The telomerase-negative MSCs entered a nondividing state and senesced after about 20 to 25 passages. In test group, however, telomerase-positive MSCs to date had undergone 35 passages. RT-PCR analysis showed that telomerase-positive MSCs expressed neuron-specific markers, such as NF-M or MAP2 after induced with EGF and bFGF in vitro. CONCLUSION: Ectopic expression of the hTERT gene in human MSCs reconstitutes telomerase activity. The transfection of hTERT gene into human MSCs extends their replicative life span and maintains their multipotent differentiation capacity.  相似文献   

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AIM:To explore the effects of rat mesenchymal stem cells (MSCs) modified by calcitonin gene-related peptide (CGRP) on the proliferation and phenotype transformation of rat vascular smooth muscle cells (VSMCs) in vitro. METHODS:Rat MSCs and VSMCs were isolated, cultured and identified. The MSCs were infected by lentivirus which carried genes encoding enhanced green fluorescent protein (EGFP) and CGRP. The expression levels of CGRP in CGRP-modified MSCs were detected using real-time PCR and enzyme-linked immunosorbent assay (ELISA). The prolife-ration and migratory abilities of VSMCs were evaluated by MTT assay, Trypan blue staining and scratch test. The expression levels of α-smooth muscle actin (α-SMA) and osteopontin (OPN) were assessed by Western blotting. RESULTS:Compared with MSCs and MSCs-EGFP groups, the expression levels of CGRP in MSCs-CGRP group were markedly increased (P<0.01). The results of MTT assay and scratch test demonstrated that the proliferation and migratory abilities of VSMCs in MSCs-CGRP group were significantly inhibited compared with MSCs and MSCs-EGFP groups (P<0.05). Furthermore, cell viability was >90% shown by Typan blue staining. Western blotting showed that the expression of α-SMA was increased and that of OPN was decreased in MSCs-CGRP group compared with MSCs and MSCs-EGFP groups (P<005). CONCLUSION:CGRP-modified MSCs could secrete CGRP protein and inhibit the proliferation and migration of VSMCs, which may be associated with deterring the phenotype transformation from contractile type to synthetic type. These results lay a foundation for gene therapy in vivo.  相似文献   

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AIM:To construct a recombinant retrovirus vector carrying hTERT for establishing UCBMSCs with hTERT (hTERT-MSCs) to overcome their limited life span and detecting whether telomerized UCBMSCs line maintained long-term self-renewal and differentiation capacity. METHODS:The whole cDNA was generated by PCR amplifications from the plasmid pEGFP-hTERT-C1. The hTERT segments were subcloned into pLNCX2. The target cells were infected with these retroviral particles. The stably transfected cells were selected by neomycin and expanded life span which were designated hTERT-MSCs was observed. The expression of hTERT in mRNA level was detected by RT-PCR and the telomerase activity was measured by TRAP (PCR)-ELISA assay. The hTERT-MSCs were induced with 5-azacytidine to cardiac muscle cells and the specific marker of myocardiocyte was detected. RESULTS:The constructed plasmids were digested with restriction endonucleases (BglⅡand NotⅠ). Two characteristic segments including 6.1 kb and 3.6 kb were obtained. The hTERT-MSCs expressed hTERT in mRNA level. The telomerase activity of hTERT-MSCs was positive. The growth kinetics of hTERT-MSCs was higher than those in UCBMSCs. The hTERT-MSCs were induced to myocardiocyte. CONCLUSION:The hTERT recombinant retrovirus vector has been successfully constructed. The hTERT gene activates the telomerase and prolongs the life-span of cells. No effect of hTERT gene on some type of differentiation potential of MSCs is present.  相似文献   

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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   

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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 explore the effect of annexin A2 (ANXA2) on the proliferation, migration and apoptosis abilities of human cervical cancer HeLa cells. METHODS:Overexpression vectors and siRNA of ANXA2 were constructed, and then transfected into HeLa cells. The HeLa cells were divided into 4 groups:control group, scramble group, ANXA2 overexpression group and ANXA2-siRNA group. The expression of ANXA2 at mRNA and protein levels was examined by real-time PCR and Western blotting. MTT assay, Boyden chamber and flow cytometry were used to determine the effect of ANXA2 on the proliferation, migration and apoptosis abilities of the HeLa cells. RESULTS:The proliferation and migration of HeLa cells were obviously promoted by ANXA2 overexpression. The proliferation and migration of HeLa cells were remarkably inhibited by the transfection of ANXA2-siRNA. ANXA2 had no effect on apoptosis of HeLa cells. CONCLUSION:Silencing of ANXA2 effectively inhibits the proliferation and migration of cervical cancer cells, but has little effect on apoptosis. ANXA2 may play a pivotal role in the occurrence and development of cervical cancer, and may be used as a molecular target for the treatment of cervical cancer.  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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AIM: To study the immunogenicity and biocompatibility of xenogeneic swine corneal stroma as biological carrier for cornea reconstruction and to reconstruct corneal endothelial tissue with this carrier in vitro. METHODS: (1) The lymphocytes from the peripheral blood of F344 rats were immunologically labeled by anti-rat CD25-FITC and anti-rat CD4/CD8-PE, then determined by flow cytometry (FCM) at 12th, 90th day after intracorneal implantation with fresh and dehydrated swine corneal stroma. (2) The fresh and dehydrated grafts made of swine corneal stroma were implanted intralamellarly in corneas of New Zealand rabbits. Clinical examinations were performed monthly and histological examinations were made at 14th, 30th, 60th, 120th and 240th day. (3) The cat corneal endothelial cells were seeded on the Descemet's membrane of the dehydrated swine corneal stroma, then cultured in the medium with epidermal growth factor and laminin for 7 days. The morphology of reconstructed tissue was tested by microscope. RESULTS: (1) Compared to isograft group and negative control, the expression CD4+CD25+, CD8+CD25+ and CD4+/CD8+ of xenograft rat group implantation with swine corneal stroma did not appear significantly different in statistics (P>0.05). (2) In the total 12 rabbits, all the cornea grafts survived without rejection reaction, corneal haze or corneal neovascularization. The fresh grafts got transparent after 2 months, and the dehydrated grafts got transparent after 6 months. Histological study demonstrated both fresh and dehydrated stroma grafts had fused with rabbits'corneal stroma very well without lymphocytes infiltrating. (3) As shown in histological observations, the reconstructed cat corneal endothelial tissue was similar to the nature tissue. Cultured cat endothelial cells connected tightly to each other and attached to the Descemet's membrane closely. CONCLUSION: Swine corneal stroma has low immunogenicity and satisfying biocompatibility,it is an ideal biological carrier for cornea reconstruction in vitro.  相似文献   

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AIM:To investigate the regulatory function of bone marrow-derived mesenchymal stem cells (MSCs) on T helper 17 cells (Th17) and regulatory T cells (Treg) in peripheral blood of severe asthmatic children. METHODS:MSCs were isolated, cultured and identified in vitro. MSCs digested with mitomycin were cocultured with T lymphocytes (TLC) at different ratios (1∶1, 1∶2, 1∶10 and 1∶20) from severe asthmatic children for 72 h. The proliferation of TLC was measured by CCK-8 method. In the coculture system of the 1∶2 ratio and the single TLC system, the supernatant levels of interleukin-17 (IL-17) and transforming growth factor-β (TGF-β) were measured by ELISA. The mRNA expression of retinoic acid-related orphan nuclear receptor C (RORC) and forkhead box protein 3 (Foxp3) in TLC was detected by qRT-PCR. RESULTS:After cocultured with MSCs, the proliferation of TLC decreased significantly in a dose-dependent manner (P<0.05). It also showed decreases in IL-17 (3 799±441 vs 4 890 ±373, P<0.05) and RORC mRNA level (1.21±0.14 vs 3.85±0.48, P<0.05), while an increase in TGF-β level (209±32 vs 117±26, P<0.05) was observed. No influence on the mRNA expression of Foxp3 was found (P>0.05). CONCLUSION:MSCs suppresses Th17 polarization of naive peripheral blood CD4+ T cells and matures Th17 cells secreting IL-17, which may effectively revise Th17/Treg imbalance of asthma.  相似文献   

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AIM:To study the autophagy of prostate cancer PC-3 cells induced by CD147 in vitro. ME-THODS:The method of amino acid starvation to induce autophagy was used. The expression of CD147was detected by Western blotting. To study the functional effects of CD147 on autophagy in prostate cancer PC-3 cells, the down-regulation of CD147expression was induced by the technique of RNAi. The conversion of autophagic marker protein LC3-I to LC3-II was determined by Western blotting. The cell death after starvation-induced autophagy was analyzed by trypan blue exclusion assay. RESULTS:The CD147 expression gradually increased in starvation-induced autophagy. The down-regulation of CD147 significantly increased the expression of autophagy-related protein LC3-II compared with control group. Meanwhile, the cell death rates increased from (19.3±3.1)% and (22.3±3.5)% in control groups to (38.4±3.1)% in silencing the expression of CD147in the PC-3 cells (P<0.05). CONCLUSION:CD147 inhibits starvation-induced autophgy and autophagy death in the prostate cancer PC-3 cells.  相似文献   

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AIM:To investigate the influence of continuous subculturing of human umbilical cord mesenchymal stem cells (hUC-MSCs) on the mRNA expression of all 23 family members of NOD-like receptors (NLRs), and to search for the way of improving the subculture quality of hUC-MSCs and increasing the quantity and safety in the experimental and clinical application. METHODS:Neonatal umbilical cord was collected to isolate and purify the hUC-MSCs with the collagenase II digestion and adherence screening methods. These cells were continuously subcultured. The hUC-MSCs at passage 3 and passage 28 were identified by flow cytometry and induced differentiation. The mRNA expression of NLRs in the passage 3 and passage 28 hUC-MSCs was detected by RT-qPCR. RESULTS:The cell phenotypes of both passage 3 and passage 28 hUC-MSCs were CD29+/CD44+/CD105+/ CD31-/CD34-/CD40-/CD45-/CD106-/HLA-DR-, and both of the cells were induced into osteoblasts and adipocytes, which were conformed to the criteria of International Society for Cellular Therapy to define MSCs. All the NLR family members were expressed in passage 3 hUC-MSCs. NOD1, NLRC4, NLRC5, NLRP1, NLRP3, NLRP10, NAIP, NLRX1 and APAF1 at mRNA levels were highly expressed, and the rest were lowly expressed. When hUC-MSCs were subcultured to passage 28, NLRP10 mRNA was increased, NLRC5 mRNA and NLRX1 mRNA were hardly changed, and all of the rest members were decreased. The difference of NLRP1 mRNA expression between passage 3 and passage 28 hUC-MSCs was observed with statistical significance (P<0.05). CONCLUSION:The effects of subculturing on the expression of NLR family in hUC-MSCs are pleiotropic. It requires further investigation to confirm whether these effects are related to the proliferation, differentiation and immunomodulation of MSCs.  相似文献   

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AIM: To construct a eukaryotic expression vector containing pancreatic duodenal homebox-1 (PDX-1) and to elevate the expression efficiency of exogenous gene in rat bone marrow mesenchymal stem cells (MSCs). METHODS: Recombinant vector containing PDX-1 was constructed. Flow cytometry was used to identify the cell cycle of bone marrow mesenchymal stem cells (MSCs) cultured in vitro. Recombinant vector containing PDX-1 was transfected into bone marrow MSCs using superfect in medium. After being selected by G418, RT-PCR and Western blotting were used to investigate the expression of PDX-1 in MSCs. RESULTS: Restricted enzyme analysis and sequencing showed that PDX-1 gene segment was consistent with that in GenBank. Flow cytometry showed that there were about 85.9% cells at the cell cycle of G0/G1. The whole cells transfected emitted green fluorescence under flow cytometry. The efficiency of transfection was above 40%. RT-PCR and Western blotting demonstrated that there was expression of PDX-1 in transfected bone marrow MSCs. CONCLUSION: Recombinant vector containing PDX-1 was constructed successfully. Superfect mediated expression of exogenous gene in bone marrow MSCs in a high efficiency, and bone marrow MSCs containing exogenous gene are an ideal cells for gene therapy.  相似文献   

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AIM:To study the differentiation of rat marrow-derived mesenchymal stem cells(MSCs) into myoid cells in vitro.METHODS:The MSCs of SD rat were cultured、passaged、induced and differentiated in vitro used by routine culture technique, and evaluated by FACScan flow cytometer, detected by immunohistochemistry and analyzed by Hitachi H-600 transmission electron microscope(TEM).RESULTS:FACScan shows that cells expressed the antigens of CD29 and CD44, not those of CD11b and CD45;cells show positive response of staining with desmin and myoglobin after processing by two compounds of 5-azacytidine and amphotericin B. There were stripform zone of myofilament without any organells beside the edge of membrane in myoid cell of induction and differentiation checked by TEM.CONCLUSION:The passaged cells were MSCs and the MSCs may have specific gene structures that can differentiate into myoid cells. The demethylation or hypomethylation may conduct by compounds of 5-azacytidine and amphotericin B, which could be involved in activating phenotype-specific genes to differentiate MSCs into myoid cells. There are good outlook on clinical treatment of illness of myatrophy using by MSCs.  相似文献   

18.
WANG Yue-chun  ZHANG Yuan 《园艺学报》2007,23(11):2205-2209
AIM: To separate and identify the mesenchymal stem cells (MSCs) from human fetal bone and to study their differentiation to hepatocyte like cells under the action of chemical induction.METHODS: The MSCs from human fetal bone were isolated and purified according to the different growth characteristic of attaching to the wall of cell culture flask.The cell cycle and surface markers of MSCs were identified using flow cytometry.The MSCs were pre-induced by adding DMSO,β-Me and 5-aza for 24 h,then adding the inductive medium of H-DMEM and rh-HGF to induce their differentiation to hepatocyte like cells (HLCs).HLCs were identified by the typical morphological change and the expression of special protein with the method of immunocytochemistry.RESULTS: The MSCs derived from human fetal bone expressed adhesion molecules CD29+,CD44+,but not antigens of hematopoietic CD34,CD45,and not antigens related to GVHD,such as HLA-DR,CD80 and CD86.Exposure of these cells to above-mentioned inductive agents resulted in obvious morphological change and an increase in expression of AFP and ALB.CONCLUSION: The results suggest the existence of plentiful MSCs in human fetal bone.MSCs derived from human fetal bone can easily differentiate to HLCs,and they have a lower immunogenic nature,which may provide the ideal source for tissue engineering (bioartificial liver) for cellular therapeutics.  相似文献   

19.
AIM: To investigate the effect of anti-Sonic hedgehog(Shh) blocking antibody on the killing effect of peripheral blood mononuclear cells(PBMCs) on cervical carcinoma HeLa cells. METHODS: The expression levels of Shh and Shh signaling molecules in HeLa cells were detected by immunocytochemistry and RT-PCR. PBMCs from health peoples were isolated by the method of Ficoll density gradient centrifugation, and then co-cultured with HeLa cells in vitro. The expression of CD3, CD69 and CD71 was assayed by flew cytometry. The concentrations of IFN-γ, IL-10 and IL-4 in culture supernatants were detected by ELISA. The killing effect of PBMCs on HeLa cells was observed under microscope. RESULTS: Shh and Shh signaling molecules were expressed in HeLa cells. The level of Shh expression didn't change significantly in the 6th passage of HeLa cells. CD3+ cells were increased in the co-culture system. The expression of CD69 and CD 71, and the secretion of IFN-γ were increased, while the secretion of IL-10 was decreased in the co-culture system treated with anti-Shh blocking antibody. Anti-Shh blocking antibody has no effect on the secretion of IL-4. The killing effect of PBMCs on HeLa cells was strengthened by anti-Shh blocking antibody. CONCLUSION: Anti-Shh blocking antibody promotes the activation of PBMCs and enhances the killing effect of PBMCs on cervical carcinoma HeLa cells.  相似文献   

20.
AIM: To investigate the osteoclastogenic effect of conditioned medium of human multiple myeloma RPMI 8226 cells on preosteoclast RAW264.7 cells. METHODS: The protein expression of soluble receptor activator of NF-κB ligand (sRANKL) was detected by Western blotting. The morphological changes of RAW264.7 cells were observed after tartrate-resistant acid phosphatase (TRAP) staining. The mRNA expression of TRAP and cathepsin K was evaluated by RT-PCR. RESULTS: The result of Western blotting showed that conditioned medium of RPMI 8226 cells contained sRANKL. RPMI 8226 cell conditioned medium induced RAW264.7 cells to differentiate into TRAP-positive multinuclear osteoclasts. Human neutralized RANKL monoclonal antibody suppressed the differentiation of preosteoclasts in a dose-dependent manner, which was induced by 30% RPMI 8226 cell conditioned medium. RPMI 8226 cell conditioned medium increased the mRNA expression of TRAP and cathepsin K in RAW264.7 cells. CONCLUSION: The sRANKL in conditioned medium of human multiple myeloma RPMI 8226 cells has the bioactivity to induce preosteoclast RAW264.7 cells to differentiate into TRAP-positive multinuclear osteoclasts. Human neutralized RANKL monoclonal antibody suppresses the differentiation of preosteoclasts induced by sRANKL in a dose-dependent manner.  相似文献   

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