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OBJECTIVE: To construct the RNA interference(RNAi) lentiviral vector of suppressor of variegation 3-9 homolog 1(Suv39 h1) and verify its interfering efficiency by transfecting it to the bone marrow-derived mesenchymal stem cells(BMSCs). METHODS: The oligonucleotides of RNA plasmid were designed and synthesized according to the gene sequence of Suv39 h1 and short hairpin RNA design principles. Three kinds of LV-Suv39 h1-RNAi recombinant plasmids with different lentivirus knockdown targets(KD1, KD2 and KD3) were constructed. After identification by restriction analysis and sequencing, the packaged lentivirus vectors with the three kinds of Suv39 h1 gene were transfected into rat BMSCs at logarithmic growth stage, and were named KD1, KD2 and KD3 transfection groups. The control group was transfected with the negative control virus. After 72 hours transfection, the transfection efficiency was evaluated, and the relative mRNA levels of Suv39 h1 were determined by quantitative real-time polymerase chain reaction(qPCR). RESULTS: Sequencing analysis demonstrated that three kinds of LV-Suv39 h1-RNAi recombinant plasmids were constructed correctly. The results of transfection efficiency evaluation showed that more than 80.00% green fluorescence was expressed in the BMSCs transfected with the three lentiviral vectors with a multiplicity of infection of 20. These results indicated that lentivirus was successfully constructed and transfection efficiency was high. The results of qPCR showed that the relative expression of Suv39 h1 mRNA in BMSCs of KD1, KD2 and KD3 transfection groups was lower than that in the control group(all P<0.05), and the relative expression of Suv39 h1 mRNA in KD1 and KD3 transfection groups was lower than that in KD2 transfection group(both P<0.05). However, there was no significant difference in the relative expression of Suv39 h1 mRNA between KD1 and KD3 transfection groups(P>0.05). CONCLUSION: The constructed lentiviral vector with low expression of Suv39 h1 was constructed successfully. This vector can be expressed in rat BMSCs, which lays a foundation to study the effect of Suv39 h1 gene in acute myeloid leukemia.
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@#AIM: To explore the differentiation of bone marrow-derived mesenchymal stem cells from peripheral blood to the retina and the expression of ciliary neurotrophic factor(CNTF). We also investigate the mechanism by which Bu Shen Yi Jing Fang could treat dry age-related macular degeneration(ARMD). <p>METHODS:C57BL/6 mice were administered with sodium iodate(NaIO3)by tail intravenous injection. One day after modeling, 3×106 green fluorescent protein labeled bone marrow-derived mesenchymal stem cells(GFP-BMSCs)were injected into the tail vein. The injected mice were randomly divided into distilled water group and Bu Shen Yi Jing Fang group according to random number table, and 12 mice in each group. The mice were intragastrical administrated with either Bu Shen Yi Jing Fang solution or distilled water every day. Twelve healthy C57BL/6 mice were fed regularly as the normal group. At 14d after the treatment, the differentiation of GFP-BMSCs in retina was determined by immunofluorescence, and the expression of CNTF in the retina was detected by immunofluorescence and quantitative real-time PCR.<p>RESULTS: Immunofluorescence staining showed that there were more glial fibrillary acidic protein(GFAP)and GFP double-stained positive cells in the Bu Shen Yi Jing Fang group than in the distilled water group(<i>P</i><0.01), and the positive rate of retinal pigment epithelium 65(RPE65)was not significantly different between two groups(<i>P</i>>0.05). There were no Rhodopsin and GFP double-stained positive cells in the two groups. Immunofluorescence and quantitative real-time PCR showed that the expression of CNTF in the Bu Shen Yi Jing Fang group was higher than which in the distilled water group(<i>P</i><0.05). <p>CONCLUSION: Bu Shen Yi Jing Fang facilitated the differentiation of peripheral blood stem cells into glial cells in the retina and the expression of CNTF, which might be one of the mechanisms of Bu Shen Yi Jing Fang in the treatment of dry ARMD.
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ObjectiveExosomes secreted by BMSC overexpressing GATA-4 gene (BMSCGATA-4-exosome) can promote the differentiation of BMSC into cardiomyocyte-like cells, thereby improve cardiac function after myocardial infarction. However, the molecular mechanism of BMSCGATA-4-exosome in cardiomyocyte-like cell differentiation is unknown. The effect of the secretion of BMSCGATA-4 exosome from bone marrow mesenchymal stem cells (BMSC) in the differentiation of stem cells into cardiomyocytes was determined in miRNA-673-5p/Tsc-1 axis dependent manner.MethodsMouse models of myocardial infarction were established and divided into seven groups. Simulation group (BMSCmiR-673-5p-mimic exosome), inhibition group (BMSCmiR-673-5p-inhibitor exosome), GATA-4 group (BMSCGATA-4 exosome), empty vector group (BMSCempty vector exosome）, and BMSC group (BMSC exosome) were injected into the tail vein for 48 h, and the untreated and normal mice were used as the control group. Cardiac ultrasound was used to detect cardiac function in each group. miRNA-673-5p expression in myocardial infarction was detected using real-time polymerase chain reaction (RT-PCR). The myocardial tissues were extracted from the same myocardial infarction site. Myocardial-specific molecules, such as α-actin, Desmin, cTnT, and Cx43, were detected using RT-PCR. Western blot was used to determine the expression of the corresponding target gene of miRNA-673-5p, Tsc-1, Erk1/2, and Mef2c proteins.ResultsThe simulation group wan shown the most significantly improved myocardial function (P<0.05) with an expression peak of miRNA-673-5p in cardiomyocytes (P<0.05). The highest content of myocardial-specific molecules including α-actin, Desmin, cTnT, and Cx43 was found in the simulation group. The simulation group had the lowest expression of Tsc-1 in cardiomyocytes (P<0.05).ConclusionOverexpressed BMSCGATA-4 exosomes inhibit Tsc-1 expression through miRNA-673-5p to improve cardiac function during myocardial infarction.
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Objective Exosomes secreted from mouse bone marrow mesenchymal stem cells (BMSC) overexpressing the cardiomyocyte transcription factor GATA-4 (BMSCGATA-4-exosome) may play a key role in repairing myocardial injury. This study aimed to investigate the molecular regulatory network of BMSCGATA-4-exosome for inhibiting the apoptosis of cardiomyocytes. Methods Exosomes extracted from GATA-4-overexpressing BMSCs of the mouse cultured with miR-330-3p-mimic were cultured with myocardial cells under hypoxic and serum-free conditions for 24 hours (the experimental group), the overexpressed GATA-4, empty vector and BMSCs were taken as the confounding factor control (CFC), the myocardial cells cultured under hypoxic and serum-free conditions for 24 hours were used as the positive control, and those cultured under the normal condition for 24 hours as the negative control. The apoptosis rates of myocardial cells in different groups were measured by flow cytometry, the expression levels of miR-330-3p in the myocardial cells determined by RT-PCR, and those of the corresponding miR-330-3p target gene Ap2m1 and transcriptional protein Cnot4 detected by Western blot. Results CD29 was expressed in 99.71% of the mouse BMSCs, CD44 in 97.28%, SCA-1 in 99.40%, and CD11b overexpressed in only 0.1%. The early apoptosis rate of myocardial cells was significantly higher in the experimental than in the negative control group ([7.90 ± 0.34]% vs [2.30 ± 0.09]%, P < 0.05) but lower than in the positive control ([51.48 ± 0.40]%), BMSC ([18.32 ± 3.03]%), empty vector ([16.99 ± 2.93]%) and overexpressed GATA-4 groups ([10.22 ± 0.35]%) (P < 0.05). The expression of miR-330-3p in the myocardial cells was markedly higher in the experimental ([396.10 ± 1.02]%) than in the negative control ([1.37 ± 0.33]%), positive control ([0.26±0.32]%), BMSC ([1.40 ± 0.42]%), empty vector ([1.41 ± 0.27]%) and overexpressed GATA-4 groups ([3.80 ± 0.62]%) (P < 0.05). The expressions of Ap2m1 and Cont4 in the myocardial cells were remarkably decreased in the experimental group compared with those in the other five groups (P < 0.05). Conclusion Overexpressed BMSCGATA-4-exosomes suppress the apoptosis of myocardial cells by inhibiting the expression of the Ap2m1 protein via miR-330-3p.
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Objective: To explore the homing effect of mouse bone marrow-derived mesenchymal stem cells (mBMSCs) on the tongue squamous cell carcinoma Cal27 cells, and to observe the biological behaviors of tongue squamous cell carcinoma Cal27 cells affected by mBMSCs. Methods: The mBMSCs were isolated and purified by whole bone marrow methods. The cell phenotype was analyzed by flow cytometry to identify the mBMSCs. The mBMSCs were divided into blank control group, Hacat cell control group and Cal27 cell experiment Cal27 cell group. After co-cultured with normal culture medium, Hacat conditioned medium and Cal27 conditioned medium, the number of the migrated Cal27 cells was detected by cell migration assay. The Cal27 cells were divided into single Cal27 cell control group and co-cultured Cal27 and mBMSCs experiment group (co-cultured group). The Cal27 cells in co-cultured group were co-cultured with mBMSCs using Transwell method. Cell proliferation assay and colony formation assay were used to detect the proliferation ability of the co-cultured Cal27cells. Cell migration assay was performed for the detection of the migration ability of the Cal27 cells after co-culture. Results: The inverted microscope results showed that most mBMSCs were short shuttle-like, with abundant cytoplasm, oval or nephron nucleus and irregular cell lengths; the cells arranged well. The results of flow cytometry showed that CD44, CD29, and Sca-1 were positive, and CD45, CDllb were negative in the mBMSCs. Compared with blank control group and Hacat cell control group, the number of mBMSCs homing to Cal27 cells in Cal27 cell group was significantly increased (P<0. 01). Compared with single Cal27 cell control group, the growth rates of Cal27 cells in co-cultured group were significantly increased from the 3rd day after co-culture (P<0. 01), and the number of clones was increased. The cell migration test results showed that the number of transmembrane cells in co-cultured group was significantly increased compared with single Cal27 cell control group (P<0. 01). Conclusion: mBMSCs can home to the tongue squamous cell carcinoma Cal27 cells, which promotes the proliferation and migration of tongue squamous cell carcinoma Cal27 cells and promotes the development of tongue squamous cell carcinoma. So mBMSCs can be used as an anti-tumor target.
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Objective:To compare the ability between bone marrow-derived mesenchymal stem cells (MSCs) (BM-MSCs) and adipose-derived MSCs (AD-MSCs) or umbilical cord-derived MSCs (UC-MSCs) on promotion of vessels formation and vessds stabilization relevant to the functions of EPCs.Methods:In vitro,co-culture blood vessel test was performed to compare the angiogenic ability between BM-MSCs,AD-MSCs or UC-MSCs.In vivo,angiogenic assay dependent on basement membrane matrix Matrigel and immunohistochemistry were performed to compare the ability of vessels formation functions between BM-MSCs and AD-MSCs or UC-MSCs.Results:The lengths and dots of vascular structures formed by EPCs on AD-MSCs layer are greater than those by EPCs on BM-MSCs layer and UC-MSCs layer in angiogenic assay in vitro.The stability of the capillary-like structures formed by EPCs with AD-MSCs on Matrigel was more stable than that by the BM-MSCs,UC-MSCs or EPCs.AD-MSCs and EPCs could form abundant functional vessels with blood perfusion in Matrigelin vivo;UC-MSCs and EPCs could form a few functional vessels with blood perfusion in Matrigelin vivo;BM-MSCs and EPCs could form broken vessels with hemocytes leakage in Matrigel in vivo.Conclusion:AD-MSCs have the stronger ability to promote the angiogenesis and stabilize the vessels compared with BM-MSCs or UC-MSCs ex vivo and in vivo.
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Objective:To explore the therapeutic effects of bone marrow-derived mesenchymal stem cells (BMSCs) on premature ovarian failure (POF) in mice induced by cyclophosphamide (CTX) and the possible mechanisms.Methods:Mouse BMSCs were identified through detection of cell surface markers by flow cytometry.The model of mouse POF was induced by intraperitoneal injection of CTX at a dose of 50 mg/kg,once daily for 15 days.BMSCs were transplanted into POF mice at 2×106 cells/mouse by tail veil.The ovarian tissues were collected for HE staining at 7 days after transplantation to observe the changes of ovarian structure and real-time PCR was performed to detect the folliculogenesis gene expression.Results:BMSCs showed positive expression of CD29 and CD90 while low expression for endothelial and hematopoietic cell markers CD31 and CD34.The numbers of primodial follicle,primary follicle,secondary follicle and antral follicle were significantly decreased,but the numbers of atretic follicle were significantly increased in CTX induced-POF mice (P＜0.05).BMSCs transplantation effectively repaired the structure of damaged ovary.The significant reduction of atretic follicle and significant increase of antral follicle and secondary follicle were observed in ovaries of BMSCs-treated mouse(P＜0.05).BMSCs-transplanted mouse ovaries showed the increased mRNA expression levels ofNano3,Nobox,and Lhx8 (P＜0.05).Conclusion:BMSCs could effectively repair ovarian structure and promote follicle development in CTX-induced POF mouse.
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Postmenopausal osteoporosis is a type of osteoporosis with high bone transformation rate, caused by a decrease of estrogen in the body, which is a systemic bone disease characterized by decreased bone mass and increased risk of fracture. In recent years, as a kind of non-pharmacologic treatment of osteoporosis, defined by whole-body vibration less than 1 ( = 9.81 m/s ), low magnitude whole-body vibration is widely concerned, mainly because of its small side effects, simple operation and relative safety. Studies have shown that low magnitude whole-body vibration can improve bone strength, bone volume and bone density. But a lot of research found that, the therapeutic effects of low magnitude whole-body vibration are different depending on ages and hormone levels of subjects for animal models or human patients. There has been no definite vibration therapy can be applied to each subject so far. Studies of whole-body and cellular level suggest that low magnitude whole-body vibration stimulation is likely to be associated with changes of hormone levels and directed differentiation of stem cells. Based on the analysis of related literature in recent years, this paper made a review from vibration parameters, vibration effects and the mechanisms, to provide scientific basis and clinical guidance for the treatment of postmenopausal osteoporosis with low magnitude whole-body vibration.
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BACKGROUND AND OBJECTIVES: Previous studies have shown that integrins alpha5beta1 (ITGA5B1) gene-modified rat bone marrow mesenchymal stem cells (rBMSCs) could prevent cell anoikis and increase the nitric oxide (NO) production. Here we examined the capability of rBMSCs/ITGA5B1 on the phenotype modulation of Human Pulmonary Artery Smooth Muscle Cell (HPASMC) in vitro. METHODS AND RESULTS: The synthetic (dedifferentiated) phenotype of HPASMC was induced by monocrotaline (MCT, 1μM) for 24 h and then co-cultured with rBMSCs/ITGA5B1 in a transwell culture system. The activation of NO/cGMP (nitric oxide/Guanosine-3′, 5′-cyclic monophosphate) signaling was investigated in HPASMC. The changes of pro-inflammatory factors, oxidative stress, vasodilator, vasoconstrictor, contractile and synthetic genes, and the morphological changes of HPASMC were investigated. The results of this study showed that the NO/cGMP signal, endothelial nitric oxide synthase (eNOS) expression, the expression of the vasoprotective genes heme oxygenase-1 (HMOX1) and prostaglandin-endoperoxide synthase 2 (PTGS2) were increased, but the expression of transforming growth factor-β1 (TGF-β1), CCAAT/enhancer-binding proteins delta (Cebpd), Krüppel-like factor 4 (KLF4), and activating transcription factor 4 (ATF4) were reduced in MCT treated HPASMC co-cultured with rBMSCs/ITGA5B1. The synthetic smooth muscle cells (SMCs) phenotype markers thrombospondin-1, epiregulin and the vasoconstrictor endothelin (ET)-1, thromboxane A2 receptor (TbxA2R) were down-regulated, whereas the contractile SMCs phenotype marker transgelin expression was up-regulated by rBMSCs/ITGA5B1. Furthermore, rBMSCs/ITGA5B1 promoted the morphological restoration from synthetic (dedifferentiation) to contractile (differentiation) phenotype in MCT treated HPASMC. CONCLUSIONS: rBMSCs/ITGA5B1 could inhibit inflammation and oxidative stress related genes to promote the HPASMC cell differentiation by activation NO/cGMP signal.
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Animals , Humans , Rats , Activating Transcription Factor 4 , Anoikis , Bone Marrow , Cell Differentiation , Endothelins , Epiregulin , Genes, Synthetic , Heme Oxygenase-1 , In Vitro Techniques , Inflammation , Integrins , Mesenchymal Stem Cells , Monocrotaline , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , Nitric Oxide Synthase Type III , Nitric Oxide , Oxidative Stress , Phenotype , Prostaglandin-Endoperoxide Synthases , Pulmonary Artery , Receptors, Thromboxane A2, Prostaglandin H2ABSTRACT
Bone defect occurs as a consequence of many conditions. Diseased bones don’t heal properly and defects in face area need proper bone reconstruction to avoid psychological and social problems. Tissue engineering is an emerging new modality of treatment. We thought to study different methods to fill skull bone defect in rats in order to find the most safe and effective method. So, this study was designed to evaluate the efficacy of acellular dermal graft (ADM) versus propylene mesh both either loaded or unloaded with bone marrow derived mesenchymal stem cells (BM-MSCs) in healing of skull bone defect of a 5 mm diameter. The study included 36 adult male Wistar albino rats that were divided into three groups according to the way of filling skull bone defect. Group I: Ia (sham control), Ib (negative control). Group II: IIa (unseeded propylene), IIb (seeded propylene) and Group III: IIIa (unseeded ADM), IIIb (seeded ADM). The trephine operation was done on the left parietal bone. Specimens were collected four weeks postoperative and processed for H&E, osteopontin immunohistochemistry and scanning electron microscope. Morphometric and statistical analysis were also performed. After studying the results of the experiment, we found that propylene mesh and ADM were suitable scaffolds that could support new bone formation in clavarial bone defect. Healing of skull bone defect was better in rats that received seeded scaffolds more than rats with unseeded scaffolds. The seeded ADM showed significant increase in bone forming activity as confirmed by histomorphometric and statistical results.
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Adult , Animals , Humans , Male , Rats , Bone Marrow , Immunohistochemistry , Mesenchymal Stem Cells , Methods , Osteogenesis , Osteopontin , Parietal Bone , Skull , Social Problems , Tissue Engineering , TransplantsABSTRACT
RESUMEN: Las células madre de la línea germinal masculina son factores clave para la espermatogénesis masculina y la fertilidad. Las células sustentaculares (células de Sertoli) como células somáticas juegan un papel fundamental en la creación de un microambiente esencial para la auto-renovación y diferenciación de las células de la línea germinal masculina. Las células madre mesenquimales son reconocidas como células auto-renovables y multipotentes capaces de diferenciarse en múltiples tipos de células. La generación de células germinales masculinas a partir de células madre mesenquimales puede proporcionar un método terapéutico para tratar la infertilidad masculina. En este estudio, las células mesenquimales derivadas de la médula ósea (BMMSCs) se recuperaron de la médula ósea de ratones de 6-8 semanas de edad del Instituto de Investigación Médico Naval (NMRI). En el estudio se aislaron las células sustentaculares y se enrriquecieron usando placas revestidas con lectina. Se obtuvo el medio de condición celular después de diferentes intervalos de tiempo. Posteriormente se cultivaron las BMMSC con diferentes concentraciones de SCCM y medio de Eagle modificado por Dulbecco (DMEM) en diversos momentos. Se evaluaron marcadores específicos de células de línea germinal usando la reacción en cadena de polimerasa transcriptasa inversa (RT-PCR) e inmunocitoquímica. Los resultados mostraron que las BMMSCs cultivadas con SCCM durante 48h exhibieron transcritos específicos de línea germinal (Mvh, Iid4, piwil2) (p <0,05) y marcadores (Mvh, Scp3). Nuestros resultados indican que el cultivo de BMMSCs con SCCM puede conducir a la diferenciación efectiva de BMMSCs en células germinales y proporcionar una estrategia de tratamiento para la infertilidad masculina.
SUMMARY: Male germ line stem cells are key factors for male spermatogenesis and fertility. Sustentacular cells (Sertoli cells) as somatic cells play a pivotal role in creating essential microenvironment for the self-renewal and differentiation of the male germ line cells. Mesenchymal stem cells are recognized as self-renewing and multipotent cells able to differentiate into multiple cell types. The generation of male germ cells from mesenchymal stem cells may provide a therapeutic method to treat male infertility. In this study, Bone marrow derived mesenchymal cells (BMMSCs) were retrieved from the bone marrow of 6-8-week old Naval Medical Research Institute (NMRI) mice. Sustentacular cells (Sertoli cells) were isolated and made rich using lectin coated plates. Sustentacular cell condition medium (SCCM) was collected after different time intervals. Then the BMMSCs were cultured with different concentration of SCCM and Dulbecco's Modified Eagle's medium (DMEM) at various times. Specific markers of Germ line cells were evaluated by using Reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. The results showed that BMMSCs cultured with SCCM for 48h exhibited germ line specific transcripts (Mvh, Iid4, piwil2) (p< 0.05) and markers (Mvh, Scp3). Our findings represent that culturing BMMSCs with SCCM may lead to effective differentiation of BMMSCs into germline cells and provide a treatment strategy for male infertility.
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Animals , Male , Mice , Sertoli Cells/cytology , Mesenchymal Stem Cells/cytology , Sertoli Cells/ultrastructure , Testis/cytology , Bone Marrow , Immunohistochemistry , Cell Differentiation , Culture Media, Conditioned , Reverse Transcriptase Polymerase Chain Reaction , Flow CytometryABSTRACT
OBJECTIVE: To explore the intervening effects of bone marrow-derived mesenchymal stem cells(BMMSCs) for pulmonary fibrosis of rats exposed to silica dust at different stages. METHODS: Specific pathogen free SD rats were randomly divided into model group,2-week group,4-week group and control group with 6 rats in each group(half males and half females). Rats of the first three groups were one-time endotracheally injected with 0. 5 mL aseptic silica suspension at 30 g/L mass concentration. Rats of control group were injected with 0. 5 mL 0. 90% sodium chloride solution. Rats of 2-week group and 4-week group were injected with 0. 5 mL BMMSCs suspension with cell density was 5 × 10~9/L at 2 weeks and 4 weeks respectively after silica dust exposure,while model group and control group were injected with aseptic 0. 90% sodium chloride solution in the same volume. After that all rats were examined by lung computed tomography(CT) scan,pathological sections were observed,lung coefficient were measured,lung tissue hydroxyproline(HYP) content and serum transforming growth factor β1(TGF-β1) concentration were investigated at the 12 th week after silica dust exposure. RESULTS: Lung CT image showed clean lung field and clear pulmonary parenchyma in control group.Multiple and diffused high density granular shadows of different size and streak/reticular fiber shadows in model group;diffused distribution of very small granular shadows in 2-week group; granular shadows and local reticular fiber shadows in 4-week group,and either the size or the area of granular shadows was smaller than model group. The lung CT value,lung coefficient,lung tissue HYP content and serum TGF-β1 concentration of model group,2-week group and 4-week group were higher than those of control group(P < 0. 05). The lung CT value,lung tissue HYP content and serum TGF-β1 concentration of control group,2-week group,4-week group and model group were elevated in turn(P < 0. 05),while the lung coefficient of model group and 4-week group was higher than that of 2-week group respectively(P < 0. 05).CONCLUSION: BMMSCs could delay pulmonary fibrosis caused by silica dust,and the protective effect is better at early stage than later stage of fibrosis.
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OBJECTIVE: To explore the effect of silencing of poly( ADP-ribose) polymerase-1( PARP-1) on cell apoptosis induced by hydroquinone( HQ) in rat bone marrow-derived mesenchymal stem cells( BMMSCs). METHODS: i) The RNA expression vectors for PARP-1 gene were transfected into BMMSCs. Neomycin was used to select the transfected cells that stably expressed PARP-1-shRNA. Western blotting was used to examine the gene silencing efficiency. ii) BMMSCs with PARP-1 silencing were sorted as the treated group,while BMMSCs with empty vector were considered as the control group.HQ dissolved in phosphate buffer solution at the concentrations of 0. 0,2. 5,5. 0,10. 0,20. 0,40. 0,80. 0,160. 0 and320. 0 μmol / L were given to both groups for 24 hours. The cell viability was detected by methyl thiazolyl tetrazolium assay,and the concentration of HQ was chosen for the following study based on cell viability. iii) Both groups were treated by HQ at concentrations of 0. 0-20. 0 μmol / L for 24 hours,then the apoptosis of BMMSCs was detected by flow cytometry. The PARP-1 mRNA expression was determined by real-time fluorescent quantitative polymerase chain reaction assay. RESULTS: i) PARP-1 silencing cells and empty vector control cells were successfully screened with a mass concentration of 400 mg / L neomycin,and confirmed by level of protein expression. The interference efficiency of PARP-1 gene and inhibition efficiency was 85. 00%. ii) Based on the result of cell viability,HQ at concentrations of 0. 0-20. 0 μmol / L was chosen for the following study. iii) Compared with the group treated by HQ at concentration of 0. 0 μmol / L,the rate of early apoptosis of control group increased significantly with HQ at concentration of 10. 0 μmol / L while that of treated group was increased significantly at concentration of 5. 0 μmol / L( P < 0. 05). In addition,at concentrations of 0. 0-10. 0 μmol / L,the rates of early apoptosis in both groups increased in a dose-dependent manner( P < 0. 01). Compared with the group treated by HQ at concentrations of 0. 0 μmol / L,the expression of PARP-1 mRNA of both groups increased significantly at the concentration of 5. 0 μmol / L( P < 0. 05). The expression of PARP-1 mRNA of treated group was less than that of control group with HQ at every concentration( P < 0. 05). At concentrations of 0. 0-20. 0 μmol / L,the expression of PARP-1 mRNA of both groups increased in a dose-dependent manner( P < 0. 01). CONCLUSION: Silencing PARP-1 in BMMSCs caused cell apoptosis. PARP-1 may participate in cell apoptosis induced by HQ.
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Objective To explore the effect of neural cell adhesion molecule (NCAM) on adhesion,migration and morphology of mouse bone marrow-derived mesenchymal stem cells (BMSCs).Methods We isolated and cultured BMSCs from wild-type and NCAM gene knockout mice.The expression of NCAM was detected by Western blot and immunofluorescence.Wound healing and adhesion assays were used to detect cell migration and adhesion ability respectively.The morphological changes were observed and the expressings of protein β1 integrin,E-cadherin,β-catenin and N-cadherin were analysed by Western blot.Results The migration and adhesion of BMSCs were significantly reduced after NCAM gene knockout.Meanwhile,the expression of β1 integrin was lower than those in wild-type BMSCs (P<0.01).The morphology of NCAM gene knockout BMSCs changed from irregular to flattened,and expressed epithelial identification marker E-cadherin and β-catenin (P<0.05).However,the expression level of mesenchymal identification marker N-cadherin was decreased (P<0.01).Conclusions NCAM is involved in adhesion and migration of BMSCs via regulating the expression of β1 integrin.Furthermore,NCAMmay negatively regulate the mesenchymal-epithelial transitions of BMSCs.
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Surface characteristics and cellular response to titanium surfaces that had been implanted with calcium and magnesium ions using plasma immersion ion implantation and deposition (PIIID) were evaluated. Three different titanium surfaces were analyzed: a resorbable blast media (RBM) surface (blasted with hydroxyapatite grit), a calcium ionimplanted surface, and a magnesium ion-implanted surface. The surface characteristics were investigated by scanning electron microscopy (SEM), surface roughness testing, X-ray diffraction (XRD), and Auger electron spectroscopy (AES). Human bone marrow derived mesenchymal stem cells were cultured on the 3 different surfaces. Initial cell attachment was evaluated by SEM, and cell proliferation was determined using MTT assay. Real-time polymerase chain reaction (PCR) was used to quantify osteoblastic gene expression (i.e., genes encoding RUNX2, type I collagen, alkaline phosphatase, and osteocalcin). Surface analysis did not reveal any changes in surface topography after ion implantation. AES revealed that magnesium ions were present in deeper layers than calcium ions. The calcium ion- and magnesium ion-implanted surfaces showed greater initial cell attachment. Investigation of cell proliferation revealed no significant difference among the groups. After 6 days of cultivation, the expression of RUNX2 was higher in the magnesium ion-implanted surface and the expression of osteocalcin was lower in the calcium ion-implanted surface. In conclusion, ion implantation using the PIIID technique changed the surface chemistry without changing the topography. Calcium ion- and magnesium ion-implanted surfaces showed greater initial cellular attachment.
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Humans , Alkaline Phosphatase , Bone Marrow , Calcium , Cell Proliferation , Chemistry , Collagen Type I , Durapatite , Gene Expression , Immersion , Ions , Magnesium , Mesenchymal Stem Cells , Microscopy, Electron, Scanning , Osteoblasts , Osteocalcin , Osteogenesis , Plasma , Real-Time Polymerase Chain Reaction , Spectrum Analysis , Titanium , X-Ray DiffractionABSTRACT
Objective To evalutate the safty of hBMSCs transpalntation and to observe their migration and distribution in the brain of young macaca fascicularis. To establish a new technology platform and theoretical basis for the treatment of central nervous system diseases in children. Methods Labelled hBMSCs were transplanted into the striatum of young macaca fascicularis. Brain sections were examined to evalutate the inflammatory reaction and immunological rejection of local injection sites by HE observation and immunohistochemical staining. Migration and distribution of transplanted?hBMSCs was observed by real?time fluorescence quantitative PCR of male DNA and fluorescence microscope. Results The results showed that the direct intracerebral injection of hBMSCs did not cause systemic symptoms in animals. There is no inflammatory reaction and immunological rejection was detected, and degeneration and necrosis of neural cells and proliferation of glial cells were absent in the local injection sites. The transplanted hBMSCs survived, and migrated into the brain after 4 weeks transplantation. Its migration and distribution have certain regularity and were overlapping between transplant recipients. In addtion, hBMSCs tended to extend rostrally into the forebrain and showed preference of migrating toward the blood vessels and below the ependyma. Conculsions Intracerebral transplantation of hBMSCs is safe. And hBMSCs can survive and migrate into the brain.
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BACKGROUND AND OBJECTIVES: Morinda citrifolia (Noni), an important traditional medicinal plant still used in patients with bone fractures or dislocation to promote connective tissue repair and to reduce inflammation. However, the effects of Noni on bone metabolism and whether it influences the osteogenic differentiation is yet to be clarified. In this study, we investigated the effect of Morinda citrifolia (Noni) juice on the proliferation rate of rat bone marrow derived mesenchymal stem cells (BMSC) and the osteoblastic differentiation as shown by alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) mRNA expression in vitro. METHODS AND RESULTS: Treatment with 200 μg/ml Noni juice enhanced the proliferation rate of the BMSC and also upregulated the osteogenic differentiation marker genes ALP and OCN, and Runx2 measured by RTPCR. Consistent with these results collagen scaffolds implanted in vivo, which were loaded with BMSC pre-exposed to Noni, showed increased bone density measured by computed tomography and histological analysis revealed neo-angiogenesis for bone formation. CONCLUSIONS: These results suggest that Noni stimulates osteoblastogenesis and can be used as adjuvant natural medicine for bone diseases such as osteoporosis.
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Animals , Humans , Rats , Alkaline Phosphatase , Bone Density , Bone Diseases , Bone Marrow , Collagen , Connective Tissue , Joint Dislocations , Fractures, Bone , In Vitro Techniques , Inflammation , Mesenchymal Stem Cells , Metabolism , Morinda , Osteoblasts , Osteocalcin , Osteogenesis , Osteoporosis , Plants, Medicinal , RNA, Messenger , Transcription FactorsABSTRACT
Critical limb ischemia (CLI) is the most severe peripheral artery disease and caused by thrombus formation in blood vessel. The current strategies for treating CLI does not protect limb amputation and reduction in the risk of mortality. Recently, human bone marrow derived mesenchymal stem cells (BD-MSC) were reported to have a paracrine effects on angiogenesis in several ischemic diseases. So, we validate to determine whether BD-MSC protect against ferric chloride treated CLI and induce angiogenesis. To characterized human bone marrow derived stem cell, BD-MSC differentiated to osteocytes and adipocytes and validated stemness using flow cytometry. Endothelial cell induced angiogenesis followed by mesenchymal stem cell cultured medium treatment in HUVEC in vitro. We also mimicked CLI patients condition using FeCl₃ treated CLI mouse and injected one hundred thousand of BD-MSC along the femoral artery to leg muscle. We validated stem cell survival, blood vessel formation, leg muscle condition and fibrosis compared by saline injected mice 28 days later. In this study, BD-MSC cultured medium treatment increased migration and tube formation of HUVEC and BD-MSC injection had an effective blood vessel formation in FeCl₃ treated CLI. As well as blood vessel formation, limb salvage rate also improved and fibrosis area statistically decreased in BD-MSC injected mice. In conclusion, bone marrow derived mesenchymal stem cell improved not only blood vessel formation but also reduction of fibrosis in FeCl₃ treated CLI mice and finally protected limb amputation.
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Animals , Humans , Mice , Adipocytes , Amputation, Surgical , Blood Vessels , Bone Marrow , Endothelial Cells , Extremities , Femoral Artery , Fibrosis , Flow Cytometry , In Vitro Techniques , Ischemia , Leg , Limb Salvage , Mesenchymal Stem Cells , Mortality , Osteocytes , Peripheral Arterial Disease , Stem Cells , ThrombosisABSTRACT
Objective To investigate the role of transcriptional-coactivator with PDZ-binding motif( TAZ) in genistein-induced osteoblastogenic differentiation of mouse bone marrow-derived mesenchymal stem cells ( BMSCs) .Methods Mouse BMSCs were cultured in phenol red-freeα-MEM containing osteogenic supplements for inducing osteogenic differentiation.BMSCs were transfected with siRNA-TAZ and treated with genistein.The temporal sequence of osteoblastic differentiation in BMSCs cultures was assayed by measuring alkaline phosphatase activity (ALP) and calcium deposition.The mRNA expression of bone sialoprotein ( BSP) and osteocalcin ( OC) were detected by reverse transcription-polymerase chain reaction(RT-PCR).The binding interaction between TAZ and cbfa1 was identified by co-immunoprecipitation.Results TAZ expression was detected during the induction of osteogenic differentiation, the ALP activity and calcium deposition were significantly decreased in BMSCs which were transfected with siRNA-TAZ.Genistein(0.01-1 μmol/L) exhibited a dose-dependent effect on TAZ expression in mouse BMSCs cultures.Treatment with genistein ( 1 μmol/L ) resulted in increased ALP avtivity and calcium deposition of BMSC cultures as function of time.Genistein(1μmol/L) also promoted the nuclear localization of TAZ and augmented the interaction between TAZ and cbfa1, and by which upregulated cbfa1-mediated gene expression such as BSP and OC.However, the ALP avtivity and calcium deposition, as well as the expression of BSP and OC were not promoted by genistein in BMSCs transfected with siRNA-TAZ.Conclusion These data suggest that the TAZ plays an important role in genistein-induced osteoblastic differentiation of mouse BMSCs cultures.
ABSTRACT
Objective To study the influence of bFGF gene transfected bone marrow-derived mesenchymal stem cells (BMSCs) on the inflammatory cytokines of COPD rat. Methods The BMSCs were separated from SD rat and cultured and then bFGF gene was imported to BMSCs by liposome transfection method. The samples were prepared into six groups: normal control group, COPD group (A), BMSCs group (B), pcDNA3.1-BMSCs group (C), bFGF-pcDNA3.1-BMSCs group (D), and bFGF group (E). The expressions of TNF-α and IL-1β by QRT-PCR were detected. Results Compared with COPD group, TNF-α and IL-1β genes from groups B to D dropped significantly (P 0.05). Conclusion BFGF transfected BMSCs, sample BMSCs and pcDNA3.1 transfected BMSCs can inhibit the expression of inflammatory cytokines of TNF-α and IL-1β, but there is no obvious advantage in comparison to bFGF transfected BMSCs and sample BMSCs in respect of inhibiting the expression of inflammatory cytokines of TNF-α and IL-1β.