RESUMO
BACKGROUND: The treatment of spinal cord injury is still a challenge. This study aimed at evaluating the therapeutical effectiveness of neurons derived form mesenchymal stem cells (MSCs) for spinal cord injury. METHODS: In this study, rhesus MSCs were isolated and induced by cryptotanshinone in vitro and then a process of RT-PCR was used to detect the expression of glutamic acid decarboxylase (GAD) gene. The induced MSCs were tagged with Hoechst 33342 and injected into the injury site of rhesus spinal cord made by the modified Allen method. Following that, behavior analysis was made after 1 week, 1 month, 2 months and 3 months. After 3 months, true blue chloride retrograde tracing study was also used to evaluate the re-establishment of axons pathway and the hematoxylin-eosin (HE) staining and immunohistochemistry were performed after the animals had been killed. RESULTS: In this study, the expression of mRNA of GAD gene could be found in the induced MSCs but not in primitive MSCs and immunohistochemistry could also confirm that rhesus MSCs could be induced and differentiated into neurons. Behavior analysis showed that the experimental animals restored the function of spinal cord up to grade 2-3 of Tarlov classification. Retrograde tracing study showed that true blue chollide could be found in the rostral thoracic spinal cords, red nucleus and sensory-motor cortex. CONCLUSIONS: These results suggest that the transplantation is safe and effective.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Neurônios/transplante , Traumatismos da Medula Espinal/terapia , Animais , Glutamato Descarboxilase/genética , Isoenzimas/genética , Macaca mulatta , Masculino , RNA Mensageiro/análise , Traumatismos da Medula Espinal/patologiaRESUMO
OBJECTIVE: To observe gene reversion during differentiation of human adult bone marrow-derived mesenchymal stem cells (MSCs) into neuron-like cells induced by Shenqiye. METHODS: The MSCs were separated, cultured and expanded in the culture medium and induced to differentiate into neuron-like cells with Shenqiye. The expressions of neuron-specific enolase (NSE), neurofilament (NF), and glial fibrillary acidic protein (GFAP) were detected by immunocytochemical method, and the changes of 10 genes of the cells after differentiation were detected by reverse transcriptional PCR. RESULTS: The MSCs exhibited neuronal phenotype when treated with Shenqiye and the neuron-like cells were positive for expressions of NSE and NF but not for glial astrocyte marker GFAP. After withdrawal of Shenqiye from the medium, the neuron-like cells were reversed to MSC, flat or spindal in morphology. The gene expression profiles of the redifferentiated cells were similar to those of undifferentiated MSCs. CONCLUSION: Shenqiye can induce MSC differentiation into neuron-like cells during and after which gene reversion may occur.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Células-Tronco Mesenquimais/citologia , Neurônios/citologia , Animais , Células Cultivadas , Proteína Glial Fibrilar Ácida/biossíntese , Camundongos , Proteínas de Neurofilamentos/biossíntese , Fosfopiruvato Hidratase/biossínteseRESUMO
Mesenchymal stem cells (MSCs) have become a recent focus of experimental and clinical research regarding myocardial regeneration. However, the therapeutic potential of these cells is limited by poor survival. Prostaglandin E1 (PGE1) is known to have antiinflammatory and antiapoptotic effects on the myocardium. The aim of the present study was to determine whether PGE1 could protect MSCs against serum deprivation (SD)induced apoptosis. An SD model was used to induce apoptosis in MSCs in vitro. Apoptotic morphological changes were detected by Hoechst 33258 fluorescent nuclear staining; and Annexin Vfluorescein isothiocyanate/propidium iodide (PI) double staining and flow cytometry was used to quantify the rate of apoptosis. Western blot analysis was used to detect the expression levels of the apoptosisassociated proteins Bcl2, Bax and caspase3. The results of the present study demonstrated that SD induced apoptosis of MSCs, and that treatment with PGE1 attenuated the morphological changes characteristic of apoptosis. Annexin V/PI staining showed that the rate of apoptosis gradually increased with the duration of ischemia. Furthermore, treatment with PGE1 significantly reduced SDinduced apoptosis, decreased the protein expression levels of Bax and caspase3, and increased the expression levels of Bcl2. These data suggest that PGE1 is able to influence the survival of MSCs under certain conditions. These results may aid in improving the therapeutic efficacy of MSC transplantation used to treat chronic ischemic heart disease.
Assuntos
Alprostadil/farmacologia , Apoptose/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Anexina A5/metabolismo , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/química , Feminino , Fêmur/citologia , Fêmur/efeitos dos fármacos , Fêmur/metabolismo , Expressão Gênica/efeitos dos fármacos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Cultura Primária de Células , Propídio , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Tíbia/citologia , Tíbia/efeitos dos fármacos , Tíbia/metabolismoRESUMO
BACKGROUND: Mesenchymal stem cell transplantation is a promising method in regenerative medicine. Gene-modified mesenchymal stem cells possess superior characteristics of specific tissue differentiation, resistance to apoptosis, and directional migration. Viral vectors have the disadvantages of potential immunogenicity, carcinogenicity, and complicated synthetic procedures. Polyethylene glycol-grafted polyethylenimine (PEG-PEI) holds promise in gene delivery because of easy preparation and potentially targeting modification. METHODS: A PEG8k-PEI25k graft copolymer was synthesized. Agarose gel retardation assay and dynamic light scattering were used to determine the properties of the nanoparticles. MTT reduction, wound and healing, and differentiation assays were used to test the cytobiological characteristics of rat mesenchymal stem cells, fluorescence microscopy and flow cytometry were used to determine transfection efficiency, and atomic force microscopy was used to evaluate the interaction between PEG-PEI/plasmid nanoparticles and mesenchymal stem cells. RESULTS: After incubation with the copolymer, the bionomics of mesenchymal stem cells showed no significant change. The mesenchymal stem cells still maintained high viability, resettled the wound area, and differentiated into adipocytes and osteoblasts. The PEG-PEI completely packed plasmid and condensed plasmid into stable nanoparticles of 100-150 nm diameter. After optimizing the N/P ratio, the PEG-PEI/plasmid microcapsules delivered plasmid into mesenchymal stem cells and obtained an optimum transfection efficiency of 15%-21%, which was higher than for cationic liposomes. CONCLUSION: These data indicate that PEG-PEI is a valid gene delivery agent and has better transfection efficiency than cationic liposomes in mesenchymal stem cells.
Assuntos
Técnicas de Transferência de Genes , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanopartículas/química , Plasmídeos/química , Polietilenoglicóis/química , Polietilenoimina/análogos & derivados , Animais , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Citometria de Fluxo , Microscopia de Força Atômica , Nanopartículas/administração & dosagem , Ressonância Magnética Nuclear Biomolecular , Plasmídeos/administração & dosagem , Polietilenoglicóis/administração & dosagem , Polietilenoimina/administração & dosagem , Polietilenoimina/química , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) could ameliorate neurological deficits after stroke in the rodent. OBJECTIVE: The purpose of this study was to investigate the potential mechanisms underlying the neuroprotective effects of implanted BMSCs. METHODS: Ischemic stroke was induced by permanent middle cerebral artery occlusion (MCAo) in Sprague-Dawley rats. BMSCs were intravenously transplanted at 24 hours after MCAo. Neurological function was evaluated using modified neurological severity score and Morris water maze test. Immunohistochemistry and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling staining were performed to detect neuronal apoptosis and proliferation. The protein and mRNA levels of vascular endothelial growth factor (VEGF) were determined by ELISA and reverse transcriptase polymerase chain reaction, respectively. RESULTS: Significant improvement of neurological deficits was found in BMSC-treated rats compared with control animals at 14 and 28 days after MCAo (p<0.05). Histological evaluation showed that BMSCs treatment significantly promoted neuronal survival and proliferation in the ischemic boundary area. The expression of VEGF was predominantly increased in the ischemic hemisphere of BMSC-treated rats compared with the other groups. On the other hand, transduction of VEGF RNAi lentivirus partially attenuated the above described beneficial effects of systemically administered BMSCs. CONCLUSION: Our data suggest that intravenously administrated BMSCs facilitate neurological function, reduce neuronal apoptosis and promote neuronal proliferation through the release of VEGF.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proliferação de Células , Transplante de Células-Tronco Mesenquimais/métodos , Neurônios/patologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose/fisiologia , Células da Medula Óssea/metabolismo , Infusões Intravenosas , Masculino , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/cirurgiaRESUMO
AIM: Stem cells hold great promise for brain and spinal cord injuries (SCI), but cell survival following transplantation to adult central nervous system has been poor. Salvianolic acid B (Sal B) has been shown to improve functional recovery in brain-injured rats. The present study was designed to determine whether Sal B could improve transplanted mesenchymal stem cell (MSC) survival in SCI rats. METHODS: SCI rats were treated with Sal B. The Basso-Beatie-Bresnahan (BBB) scale was used to test the functional recovery. Sal B was used to protect MSC from being damaged by TNF-alpha in vitro. Bromodeoxyuridine-labeled MSC were transplanted into SCI rats with Sal B intraperitoneal injection, simultaneously. MSC were examined, and the functional recovery of the SCI rats was tested. RESULTS: Sal B treatment significantly reduced the lesion area from 0.26+/-0.05 mm2 to 0.15+/-0.03 mm2 (P<0.01) and remarkably raised the BBB scores on d 28, post-injury, from 7.3+/-0.9 to 10.5+/-1.3 (P<0.05), compared with the phosphate-buffered saline (PBS) control group. MSC were protected from the damage of TNF-alpha by Sal B. The number of surviving MSC in the MSC plus Sal B groups were 1143.3+/-195.6 and 764.0+/-81.3 on d 7 and 28, post-transplantation, more than those in the MSC group, which was 569.3+/-72.3 and 237.0+/-61.3, respectively (P<0.05). Rats with MSC transplanted and Sal B injected obtained higher BBB scores than those with MSC transplanted alone (P<0.05) and PBS (P<0.01). CONCLUSION: Sal B provides neuroprotection to SCI and promotes the survival of MSC in vitro and after cell transplantation to the injured spinal cord in vivo.
Assuntos
Antioxidantes/farmacologia , Benzofuranos/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Traumatismos da Medula Espinal/terapia , Animais , Comportamento Animal/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/psicologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The study was purposed to investigate the effects and mechanism of bone marrow-derived mesenchymal stem cells (MSCs) on graft-versus-host desease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The model of GVHD in rat had been established by allo-HSCT with donor derived T cells. The occurence of GVHD in recipients was observed in condition with or without donor derived MSC co-transplantation. Effects of MSCs on GVHD were analyzed by model rat survival rate and pathology. Proportions of CD4+CD25+ regulatory T cells were determined by using label spleen lymphocytes and thymocytes with double fluorescent-labeled antibodies and flow cytometry. The results showed that MSCs inhibited the lethal GVHD after HSC co-transplantation and increased the survival rate. The ratio of CD4/CD8 deceased in GVHD group in different levels, as compared with that in the experimental group. The proportion of CD4+CD25+ regulatory T cells of spleen lymphocytes was 31.55 +/- 7.58% and 20.90 +/- 1.90% in experimental and GVHD groups, respectively. Similarly, the proportion of CD4+CD25+ regulatory T cells of thymocytes was 93.20 +/- 2.69% and 57.17 +/- 6.79% in experimental and the GVHD groups, respectively. Meanwhile the proportion of CD4+CD25+ regulatory T cells was higher in experimental group than that in GVHD group. It is concluded that MSCs may prevent the lethal GVHD after allo-HSC co-transplantation and raise the survival rate of model rats by acting on the CD4+CD25+ regulatory T cells in vivo.
Assuntos
Transplante de Medula Óssea , Linfócitos T CD4-Positivos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Células-Tronco Mesenquimais/fisiologia , Linfócitos T Reguladores/imunologia , Animais , Células da Medula Óssea/citologia , Transplante de Medula Óssea/efeitos adversos , Linfócitos T CD8-Positivos/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Células-Tronco Mesenquimais/imunologia , Ratos , Ratos Endogâmicos F344 , Ratos WistarRESUMO
Fanconi anaemia (FA) is an autosomal recessive inherited disorder caused by defects in hematopoietic stem cells. The clinical manifestations of FA are diverse and complicated. FA cells display high hypersensitivity to agents which produce interstrand DNA cross-links such as mitomycin C (MMC) or diepoxybutane (DEB). At least eight complementation groups with defects in eight genes (FANCA, FANCB, FANCC, FANCD(1), FANCD(2), FANCE, FANCF and FANCG) have been identified by gene analysis. Six genes (corresponding to subtypes A, C, D(2), E, F and G) have been coloned, and the encoded FA proteins interact in a common cellular pathway - "FA Pathway", through which modulate DNA repair. The progress of research on FA molecular mechanism provides gene therapy of FA with theory basis. FA cells transduced with the use of retrovirus carring the normal FA gene cDNA manifestate phenotypic correction of hypersensitivity to DNA cross-linking agents, such as MMC. In this review the clinical manifestations and gene composition of FA, and the functions of encoded FA proteins were summarized. The hematopoietic stem cell transplantation and gene therapy for FA patients were discussed.