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Objective:To explore the role of an exosomal long non-coding RNA zinc finger E-box-bingding homeobox 1 antisense 1(ZEB1-AS1) from adipose-derived MSC(adMSC) and its action mechanism in DN.Methods:DN rat model and high glucose(HG)-induced glomerular mesangial cell(GMCs) model treated with exosomal ZEB1-AS1 from adMSC were used for biochemical analysis and inflammation/oxidative stress assessment. The binding relationships among ZEB1-AS1, microRNA(miR)-142-5p, and phosphatase and tensin homolog deleted on chromosome 10(PTEN) were confirmed by dual luciferase reporter assay. Western blotting was used for the measurement of PTEN protein level.Results:adMSC-secreted exosomal ZEB1-AS1 reduced the levels of blood glucose, serum creatinine, 24 h urinary protein, kidney weight, fibrosis, and inflammatory cell infiltrations in DN rats. Meanwhile, both in DN rats and HG-induced GMCs, the levels of tumor necrosis factor(TNF)-α, interleukin(IL)-6, and IL-1β were inhibited, but glutathione peroxidase(GPx), superoxide dismutase(SOD), catalase(CAT), and glutathione(GSH) were promoted by exosomal ZEB1-AS1 treatment. Additionally, miR-142-5p was identified to bind to ZEB1-AS1, while miR-142-5p further targeted PTEN. miR-142-5p overexpression or PTEN silencing reversed the inhibiting effects of exosomal ZEB1-AS1 on inflammatory cytokine levels and the promoting effects on the concentrations of antioxidant enzymes.Conclusion:Exosomal ZEB1-AS1 from adMSC prevents DN progression by controlling inflammation and oxidative stress via the miR-142-5p/PTEN pathway, suggesting that ZEB1-AS1 may serve as a potential and effective therapeutic target for treating DN.
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BACKGROUND: Skin transplantation is one of the most effective methods for treating large-area burns. How to effectively suppress the immune rejection after allogeneic skin transplantation is a problem that needs to be solved urgently. OBJECTIVE: To investigate the effect of human adipose derived mesenchymal stem cells (hADSCs) on the immunoregulation of skin grafts in different strains of mice. METHODS: Isolated hADSCs were cultured to the 3rd generation. Sixty ICR neonatal mice, 2-4 days of age, were randomly divided into four groups (n=15). The skin tissues of ICR neonatal mice were transplanted into adult C57BL/6 mice to establish a different strain of mouse skin graft immune rejection model. PBS and low dose (5×104), medium dose (10×104), high dose (20×104) hADSCs were injected into the model mice through tail vein, and the survival time of transplanted skin in each group was recorded. On the 7th day after operation, five mice from each group were randomly selected to remove their spleen and serum, and the expression of immune factors interleukin-10, tumor necrosis factor-α and interferon-γ were detected by RT-PCR and ELISA respectively. The transplanted part of the skin was taken to make pathological sections for observing the infiltration of lymphocytes. RESULTS AND CONCLUSION: Compared with the PBS group, the survival time of the skin was prolonged in the low dose hADSCs group; however, there was no significant difference between the two groups (P > 0.05). Compared with the PBS and low dose hADSCs groups, the survival time of the skin was significantly increased in the medium and high dose groups (P 0.05). Compared with the PBS group, the relative expression of tumor necrosis factor-α and interferon-γ in the spleen and serum was significantly decreased in the low, medium and high dose hADSCs groups (P < 0.05), whereas the level of interleukin-10 was significantly elevated in the medium and high dose hADSCs groups (P < 0.05). To conclude, the appropriate dose of hADSCs can significantly prolong the survival time of transplanted skin between different strains of mice, by regulating the expression of related immune factors in the recipient mice.
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BACKGROUND: Previous studies have found that hypoxia has different effects on the proliferation and differentiation of mesenchymal stem cells and secretion of cytokines, but the effect of hypoxia on canine adipose-derived mesenchymal stem cells has not been seen. OBJECTIVE: To investigate the effect of hypoxic environment on the biological characteristics of canine adipose-derived mesenchymal stem cells. METHODS: Canine adipose-derived mesenchymal stem cells were isolated and cultured by enzyme digestion. Passage 2 adipose-derived mesenchymal stem cells could be divided into normoxia group (21% oxygen volume fraction) and hypoxia group (5% oxygen volume fraction). Morphological characteristics, proliferation speed, cell surface marker expression, differentiation capacity, and cytokine secretion level were compared between the two groups. RESULTS AND CONCLUSION: (1) The growth of adipose-derived mesenchymal stem cells cultured in hypoxic environment was good. The cell morphology was fusiform and polyhedral, the same as that of the normoxia group. (2) The proliferation rate of cells in the hypoxia group was accelerated, and the cell doubling time was shorter than that in the normoxia group (all P < 0.05). The differentiation time of lipogenesis and osteogenesis was shortened. (3) The expression of CD90, CD44 and CD105 was high in both normoxia group and hypoxia group, while the expression of CD45 was low. (4) The mRNA expression levels of brain-derived neurotrophic factor and vascular endothelial growth factor in the hypoxia group were 2.3 times (P < 0.05) and 3.0 times (P < 0.05) higher than those in the normoxia group. (5) The results indicated that the hypoxic environment had no significant effect on the morphology and surface marker of adipose-derived mesenchymal stem cells, but promoted cell proliferation, differentiation and cytokine secretion.
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Objective To investigate the therapeutic effect of SPK1 gene transfected adipose derived mesenchymal stem cells(ADMSC)on experimental autoimmune encephalomyelitis mice and the effect on T helper cell 17(Th17)/regulatory T(Treg) cells balance. Methods EAE was induced by myelin oligodendrocyte glycoprotein 35-55 in mice.Totally 44 mice were randomly divided into four groups:normal control group(NC group),model group(EAE group),ADMSC group,and ADMSC-SPK1 group.Forty days after injection,the pathological changes of brain and spinal cord,Th17/Treg-related inflammatory markers in brain tissue,expressions of interleukin-17A(IL-17A)and forkhead box protein p3(Foxp3)in brain and spinal cord tissue,and flow cytometric results of spleen immune cells were detected. Results Forty days after the injection,serious inflammatory cell infiltration and demyelination occurred in the brain and spinal cord of EAE group,whereas demyelination and axonal injury were improved in ADMSC group and ADMSC-SPK1 group.Compared with EAE group,the ADMSC group and ADMSC-SPK1 group had significantly improved levels of IL-17A(
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Animals , Mice , Adipose Tissue/cytology , Cytokines , Encephalomyelitis, Autoimmune, Experimental/therapy , Interleukin-17 , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Mice, Inbred C57BL , Phosphotransferases (Alcohol Group Acceptor)/genetics , T-Lymphocytes, Regulatory/cytology , Th17 Cells/cytology , TransfectionABSTRACT
BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) secrete various cytokines and growth factors required for bone remodeling, which are considered to be excellent candidate cells for bone regeneration. Bone morphogenetic protein 2 (BMP2) and ADSCs have a synergistic effect on bone regeneration and can significantly enhance the osteogenic differentiation of ADSCs. OBJECTIVE: To explore the efficacy of conditioned medium of ADSCs combined with BMP2 on postmenopausal osteoporosis in a rat model. METHODS: Healthy and female Sprague-Dawley rats aged 8-10 months (n=75) were obtained from the Animal Center of Shenyang Medical College. Ovariectomy was performed to induce postmenopausal osteoporosis in 60 rats, and the remaining 15 rats underwent surgeries without removal of the ovaries (sham group). Ovariectomized rats were randomized into four groups: Osteoporosis group, conditioned medium group, BMP2 group, and conditioned medium+BMP2 group, followed by injection of DMEM medium, ADSCs conditioned medium, BMP2, and ADSCs conditioned medium+BMP2 via the tail vein, respectively. After 12 weeks of treatment, femurs and serum samples of each group were taken. The number and structure of trabecular bone and trabecular spacing were detected histologically. Serum P1NP, ALP, TRAP, OPG, RANKL levels were detected by ELISA. Western blot and real-time PCR were used to detect RANKL and OPG at protein and mRNA levels. Cytokine chip analysis of cytokines in ADSCs conditioned medium was performed. RESULTS AND CONCLUSION: Compared with the sham group, increased trabecular spacing, fewer trabeculae and marked trabecular disconnection were observed in osteoporosis rats. The trabeculae in conditioned medium+BMP2 group appeared to be more complete and continuous with less widened spacing compared with the osteoporosis, conditioned medium and BMP2 groups. Serum levels of P1NP and ALP were dramatically higher, while TRAP level group was decreased significantly in the conditioned medium+BMP2 group compared with the other groups (both P < 0.05). In the conditioned medium+BMP2 group, RANKL/OPG ratio was reduced significantly compared with the other groups (P < 0.01), further promoting bone formation. ADSCs conditioned medium contained a variety of cytokines that were essential for bone formation and remodeling, including bone morphogenetic proteins 4 and 7, leukemia inhibitory factor, brain-derived neurotrophic factor, osteoprotegerin, and insulin-like growth factor 1. These results demonstrate that ADSCs conditioned medium combined with BMP2 can mitigate osteoporosis induced by ovariectomy and it may be an attractive strategy to treat postmenopausal osteoporosis.
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BACKGROUND: The development of regenerative medicine and the appearance of tissue engineering technology provide a new solution for cartilage defect reconstruction. In tissue engineering, mesenchymal stem cells are widely used seed cells. However, as a heterogeneous cell group, stem cells play different roles in different subsets. Therefore, the application of key functional subsets of mesenchymal stem cells in cartilage repair has a broad application prospect. OBJECTIVE: To sort CD146 positive subpopulation cells from human adipose-derived mesenchymal stem cells to verify their biological characteristics and potential as seed cells in cartilage tissue engineering. METHODS: Human adipose-derived mesenchymal stem cells were provided by Zhejiang Jinshidai Biotechnology Co., Ltd. Surface markers of human adipose-derived mesenchymal stem cells were identified by flow cytometry. CD146 positive subpopulation cells were sorted from human adipose-derived mesenchymal stem cells using magnetic-activated cell sorting. Molecular characteristics of two kinds of cells were analyzed by gene chip detection technology and bioinformatics analysis technology. Two kinds of cells were induced to chondrocytes in vitro and histologically examined. Cell viability and apoptosis of two kinds of cells were detected before and after cryopreservation. RESULTS AND CONCLUSION: Human adipose-derived mesenchymal stem cells highly expressed stem cell-associated markers CD73 and CD90, but did not express hematopoietic stem cell-associated markers CD34, CD45, and HLA-DR. Bioinformatics analysis results showed that CD146 positive subpopulation had different functions in inflammatory pathways and musculoskeletal diseases compared with human adipose-derived mesenchymal stem cells. CD146 positive subpopulation could differentiate into cartilage, and its chondrogenic differentiation ability was better than that of human adipose-derived mesenchymal stem cells. CD146 positive subpopulation had better apoptosis and activity than human adipose-derived mesenchymal stem cells after resuscitation. These results suggest that CD146 positive subpopulation has good chondrogenic differentiation potential and is a promising seed cell for cartilage tissue engineering.
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BACKGROUND: Canine kidney injury is characterized by the apoptosis and necrosis of renal tubular epithelial cells. Recent developments in mesenchymal stem cells and their exosomes research have shown great promise for the treatment of kidney injury in humans, rats and mice, but little research has been done on dogs. OBJECTIVE: To investigate the effects of canine adipose-derived mesenchymal stem cells and their exosomes on canine renal tubular epithelial cell injury induced by gentamicin in vitro. METHODS: Canine renal tubular epithelial cells were treated by 5 mmol/L gentamicin sulfate. Subsequently, canine adipose-derived mesenchymal stem cells and their conditional medium and exosomes were co-cultured with damaged canine renal tubular epithelial cells respectively. After 24 and 48 hours, the cell proliferation activity of each group was measured by cell counting kit-8 method, and the apoptosis rate of each group was detected by flow cytometry. Finally, Q-PCR was used to further reveal the effects of canine adipose-derived mesenchymal stem cell exosomes on PCNA, Bcl-2 and Bax genes in these cells. RESULTS AND CONCLUSION: Canine adipose-derived mesenchymal stem cells, their conditioned media, and exosomes could significantly promote proliferation and reduce apoptosis in damaged canine renal tubular epithelial cells (P < 0.05). Among them, canine adipose-derived mesenchymal stem cell exosomes worked best, which could significantly increase the expression of PCNA and Bcl-2 genes in damaged canine renal tubular epithelial cells (P < 0.05). These results suggest that canine adipose-derived mesenchymal stem cells can repair the canine renal tubular epithelial cell damage induced by gentamicin through their exosomes.
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BACKGROUND: Transforming growth factor Β3/polylactic acid-glycolic acid (TGF-Β3/PLGA) sustained-release microspheres can maintain the effective drug concentration at the site of action and provide the feasibility for efficient utilization of growth factors. OBJECTIVE: To optimize the manufacturing process of TGF-Β3/PLGA sustained-release microspheres, and investigate their effects on the proliferation and migration of rabbit adipose-derived mesenchymal stem cells (ADSCs). METHODS: TGF-Β3/PLGA sustained-release microspheres were prepared by emulsification-solvent evaporation method. The morphology, particle size, drug spatial distribution, encapsulation efficiency, drug loading, and sustained release properties of the microspheres were characterized. The TGF-Β3/PLGA sustained-release microspheres were dissolved in phosphate buffered saline. The concentration of TGF-Β3 in the supernatant was detected at the corresponding time points. The microsphere morphology was observed by scanning electron microscopy at the corresponding time point. Adipose-derived mesenchymal stem cells were divided into six groups and then cultured with single culture medium (negative control) or culture medium containing TGF-Β3 or blank PLGA, or culture medium containing 10,100,1 000 g/L TGF-Β3/PLGA microspheres. Cell proliferation was detected by CCK-8 assay at the corresponding time point. Cells in each group were cultured for 24 hours with corresponding medium in a non-contact manner. The number of migratory cells was counted. RESULTS AND CONCLUSION: (1) TGF-Β3/PLGA sustained-release microspheres were spherical with smooth surface, no adhesion, and evenly distributed particle size. The microspheres had a diameter of 2-50 µm, and the protein drugs in the microspheres were evenly distributed, with high encapsulation efficacy and encapsulation dose. (2) The TGF-Β3/PLGA sustained-release microspheres had good degradation properties and were completely degraded after 6 months in vitro. At the same time, these microspheres had good sustained-release performance and released TGF-Β3 slowly for 45 days in vitro. (3) Blank microspheres and the sustained-release microspheres containing TGF-Β3 had no effect on the proliferation of adipose-derived mesenchymal stem cells. (4) Blank microspheres had no effect on the migration of adipose-derived mesenchymal stem cells, and the transforming growth factor 3 and the sustained-release microspheres containing TGF-Β3 promoted the migration of adipose-derived mesenchymal stem cells. There was no significant difference in the migration promotion between different concentrations of TGF-Β3. (5) These findings suggest that the TGF-Β3/PLGA sustained-release microspheres can promote the migration of adipose-derived mesenchymal stem cells without affecting their proliferation.
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Aim of Study: One of the new methods that have promising results is the use of cell-derived microvesicles (MVs) to kill tumor cells. Given that MVs contain apoptotic materials, genes, and proteins, they can interfere with the fate of adjacent cells. Materials and Methods: In the present study, after adipose tissue-derived mesenchymal stem cells (AT-MSCs) isolation and characterization, MVs were derived from AT-MSCs and then characterized morphologically by standard error of the mean and size determination by DLS, and after that, the influence of MVs on human breast cancer cells (MCF-7) was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay and apoptosis-related gene expression. The raw data were analyzed in SPSS.17 software. Results: The results indicated that MVs have a size range of 500–1500 nm, and the viability of MCF-7 was significantly decreased when treated by different concentrations of MVs and it was confirmed when apoptosis-related genes' expression level was measured by real-time reverse transcription polymerase chain reaction whereas demonstrated that apoptosis genes including Bax, P53, P21, and EP300 (2− ΔΔ CT) and ΔCT values were expressed significantly in MCF-7 treated by MVs higher than those nontreated, and decrease of Bcl-2 expression level in MVs-treated MCF-7 was also significant as an antiapoptosis-related gene. Conclusions: Taking together, AT-MSC-derived MVs demonstrated anticancer or antitumoral properties on MCF-7 cells, and it could also be effective for other types of cancer cells
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The present study was carried out to evaluate the potential of adipose-derived mesenchymal stem cell (AD-MSCs) to enhance the rate of healing of full-thickness excisional skin wounds in rabbits. Six healthy adult New Zealand white rabbits and five healthy Swiss Albino mice were used for the study. Two, 2 × 2 cm full-thickness skin (thoracolumabar region) excisional wounds were created; one on each side of the dorsal midline in each animal. Adipose tissue was collected from the abdomen of the mice and processed for isolation of AD-MSCs. The wounds were randomly assigned to either injection of adipose-derived mesenchymal stem cell into the wound margins (AD-MSCs), or topical application of Povidone iodine (5%) solution (PI) as positive control. The wound healing was assessed by evaluation of granulation tissue formation, epithelisation and histomorphological study on 7th, 14th, 21st and 28th postoperative days. Better epithelisation was seen histologically in AD-MSCs treated wounds than in PI-treated wounds. Histomorphological examination of the healing tissue showed early disappearance of inflammatory reaction, significantly more neovascularisation, and more fibroplasias and early lay down and histological maturation of collagen in AD-MSCs treated wounds than in PI treated wounds. Hence the application of xenogenic stem cells can be used for tissue engineering and regenerative medicine in animals
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Objective To investigate the effect of platelet-rich plasma(PRP) on starting of AnxA1 gene and PPARγ gene of adipose-derived stem cells (ADSCs) in rabbit.Methods Epididymal adipose tissue stem cells from New Zealand white rabbits,and the cells identified by morphology and inducing differentiation,the cells were cultured to the fourth generation,PRP and PPP (platelet-poor plasma) were prepared by traditional centrifugal method from abdominal aortic of rabbit;ADSCs were cultured in culture medium containing PRP (experimental group),PPP (control group) and all medium (blank group) for each 5％ for 24 h,48h and 72 h.Cells of each group were dissociated and total RNA extracted.AnxA1 gene and PPARγ gene were detected by RT-PCR.Results Primary ADSCs of rabbit grew in the way of long spindle swirly.The results of oil red O and alizarin red staining of the ADSCs were positive.AnxA1 gene and PPARγ gene of experimental group significantly increased from the result of RT-PCR (P＜0.05).Conclusions PRP can promote proliferation of the ADSCs of rabbit and increase the expression of AnxA1 gene and PPARγ gene significantly.
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BACKGROUND AND OBJECTIVES: In regenerative medicine, mesenchymal stem cells derived from adipose tissues (Ad-MSCs) are a very attractive target to treat many diseases. In relation to nephrology, the aim of the current study is to investigate the effects of Ad-MSCs for the amelioration of acute kidney injury and to explore the mechanism of renal parenchymal changes in response to allogeneic transplantation of Ad-MSCs. METHODS AND RESULTS: The nephrotoxicity was induced by cisplatin (CP) in balb/c mice according to RIFLE Class and AKIN Stage 3. PCR, qRT-PCR and fluorescent labeled cells infusion, histopathology, immunohistochemistry, functional analyses were used for genes and proteins expressions data acquisition respectively. We demonstrated that single intravenous infusion of 2.5×107/kg mAd-MSCs in mice pre-injected with CP recruited to the kidney, restored the renal structure, and function, which resulted in progressive survival of mice. The renal tissue morphology was recovered in terms of diminished necrosis or epithelial cells damage, protein casts formation, infiltration of inflammatory cells, tubular dilatation, and restoration of brush border protein; Megalin and decreased Kim-1 expressions in mAd-MSCs transplanted mice. Significant reduction in serum creatinine with slashed urea and urinary protein levels were observed. Anti-BrdU staining displayed enhanced tubular cells proliferation. Predominantly, downgrade expressions of TNF-α and TGF-β1 were observed post seven days in mAd-MSCs transplanted mice. CONCLUSIONS: Ad-MSCs exerts pro-proliferative, anti-inflammatory, and anti-fibrotic effects. Ad-MSCs transplantation without any chemical or genetic manipulation can provide the evidence of therapeutic strategy for the origin of regeneration and overall an improved survival of the system in functionally deprived failed kidneys.
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Animals , Mice , Acute Kidney Injury , Cisplatin , Creatinine , Dilatation , Epithelial Cells , Immunohistochemistry , Infusions, Intravenous , Kidney , Low Density Lipoprotein Receptor-Related Protein-2 , Mesenchymal Stem Cells , Microvilli , Necrosis , Nephrology , Polymerase Chain Reaction , Regeneration , Regenerative Medicine , Transplantation, Homologous , UreaABSTRACT
Objective@#To investigate the possibility of adipose tissue mesenchymal stem cell-derived microvesicles (ADSC-MVs) to improve the retention volume of fat transplantation.@*Methods@#Human adipose tissue was obtained from 5 healthy female patients aged from 20 to 30 years, who came to the hospital for abdominal liposuction. Adipose tissue mesenchymal stem cells (ADSCs) were acquired by collagenase enzymatic hydrolysis. ADSC-MVs were isolated from the supernatant of cultured ADSCs through ultra-centrifugation, and characterized by transmission electron microscope and PKH26 staining. Sixteen BALB/c-nu nude mice were randomly divided into 2 groups (n=8, mice/group) by random number table method. In EV group, the mice were subcutaneously injected 0.35 ml human fat, with 7 μg ADSC-EVs (final concentration is 20 μg/ml), while 0.35 ml human fat were in blank group. The mice were sacrificed and the grafts were harvested at 1 month and 3 months after transplantation. Weight and volume of transplanted fat were tested. Morphology of adipocytes, fibrosis and necrosis of fat tissue were confirmed by HE staining. Blood vessel density were accessed by CD31 staining.SPSS 13.0 was used for statistical analysis and Student-t test was applied to compare the data from two groups.@*Results@#One month after fat transplantation, the volume of fat grafts in blank group was lower than that in MV group. Besides, lipoliquefaction can be observed in fat grafts of blank group. Fat grafts in MV group had a higher volume and littler necrosis and lipolique faction. Three months after grafting, the volume of fat grafts in blank group [(0.057±0.009) ml] was significantly lower than that in MV group [(0.103±0.015) ml], t=8.117, P<0.001. MV group had an increased number of vessels (29.6±4.0/field) compared with grafts from the Blank group (23.0±2.4/field), t=2.825, P=0.022. Histological analysis revealed that the fat grafts in MV group consisted less fat necrosis and fibrosis than control group.@*Conclusions@#ADSC-MVs can improve the retention volume of fat transplantation.
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Objective@#To investigate the role and the mechanism of adipose-derived mesenchymal stem cells (ADSCs) in inhibiting bleomycin-induced skin fibrosis in nude mice, in order to provide experimental basis for the prevention and treatment of dermatitis, keloid, scleroderma, and other skin fibrosis disorders.@*Methods@#Twenty-four female BALB/c nude mice aged 5-6 weeks were randomly divided into blank control group, transplantation control group, model group, and transplantation group. There were 6 mice in each group. In blank control group, PBS solution was injected subcutaneously on the back of nude mice twice a day, at intervals of 1 h. The transplanted control group was injected with 1×106/ml ADSCs 1 ml and then injected with 1 ml PBS solution at intervals of 1 h. In the model group, 1 mg/ml bleomycin was injected firstly, and then 1 ml PBS was injected at intervals of 1 h. In the transplantation group, 1 ml of bleomycin was injected firstly, and 1 ml of ADSCs were injected at intervals of 1 h. After 4 weeks injection, the skin texture of each group nude mice was grossly observed. The expression of fibrosis-related proteins was detected by Western blot. The expression of fibrosis-associated genes was detected by RT-PCR. The expression of α-smooth muscle actin (α-SMA), an indicator marker of skin fibrosis, was detected by immunohistochemistry staining. The experimental data was analyzed with GraphPad 7.0 software. Two groups were compared by t test, and multiple groups of samples were compared using one-way ANOVA. The P<0.05 was regarded as statistically significant.@*Results@#The results of Western blot showed that the expression of TGF-β1, Notch1 and α-SMA in ADSCs transplantation group was significantly lower than that in the model group, but there was no significant difference between the blank control group and the transplantation control group. Real-time PCR results showed that the expression levels of TGF-β1, type Ⅰ collagen, Notch1, and Smad3 were significantly higher than those in the ADSCs group. The expression of α-SMA mRNA in the control group, transplantation control group, model group and transplantation group was 0.37±0.01, 0.35±0.05, 1.43±0.10, and 0.83±0.03, respectively. Compared with the blank control group and the transplantation control group, the expression level of α-SMA in the model group was significantly increased (F=352.3, P=0.000), while the expression level of α-SMA in the ADSCs transplantation group was significantly lower than that in the model group (t=10.39, P=0.009).@*Conclusions@#The local injection of ADSCs may reduce the expression of TGF-β1, Smad3 and Notch1 by paracrine function, remodel the extracellular matrix, inhibit the proliferation of fibroblasts, and thus inhibit the process of skin fibrosis.
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To investigate the effects of adipose-derived mesenchymal stem cells (AMSCs) from type 2 diabetes mellitus patients on wound healing of pressure ulcers in mice.Methods@#(1) In September 2016, the subcutaneous adipose tissue of a 60-year-old woman with type 2 diabetes mellitus was harvested, and then AMSCs were extracted by collagenase digestion and cultured. The third passage of cells were used for subsequent experiments. The morphology of cells was observed, and their osteogenic, chondrogenic, and adipogenic differentiation abilities were identified. The expressions of cell surface markers CD90, CD105, CD73, and CD34 were detected by flow cytometer (n=3). (2) Sixteen female C57BL/6 wild-type mice aged 6-8 weeks were selected, and one pressure ulcer wound was created on each side of the spine of each mouse by pressing the skin with two magnets. The two wounds of each mouse were paired and divided into diabetic AMSCs group and negative control group, injected with 100 μL phosphate buffer solution (PBS) containing green fluorescent protein-labeled AMSCs (1×106 cells) and 100 μL PBS, respectively. The wound healing status of the two groups within post injection day (PID) 21 was observed, and their wound healing rates on PID 5, 13, and 17 were calculated. Three mice were sacrificed on PID 11 and 21, respectively, and tissue of three wounds was harvested from each group. The skin structure was observed by hematoxylin-eosin staining, the collagen deposition was evaluated by Masson staining, and the positive expression of CD31, i. e., the number of new blood vessels was counted by immunohistochemistry. Wound tissue samples of two groups prepared on PID 21 as above-mentioned were harvested, and the positive cell rate of S100, representing the regeneration of Schwann cells, was detected by immunohistochemistry. Wound tissue samples of diabetic AMSCs group prepared on PID 11 as above-mentioned were harvested, and the colonization of AMSCs was observed by fluorescence tracer method. Data were processed with paired t test and Bonferroni correction.@*Results@#(1) The third passage of cells isolated and cultured from the subcutaneous adipose tissue of a type 2 diabetes mellitus patient grew adherently to the wall in a long spindle and vortex-like manner. After induction, the cells showed osteogenic, chondrogenic, and lipogenic differentiation abilities. The positive expression rates of CD90, CD105, and CD73 on the cell surface were higher than 90.00%, and the expression rate of CD34 was 0.46%. The cells were identified as AMSCs. (2) The mice wounds of diabetic AMSCs group healed quickly, and all the wounds healed completely on PID 17, while the mice wounds in negative control group were not completely closed at this time, and there was still scab on the surface. On PID 5, 13, and 17, the healing rates of mice wounds of diabetic AMSCs group were (35.6±6.5)%, (87.1±2.5)%, and 100.0%, respectively, significantly higher than (19.8±7.2)%, (66.2±5.2)%, and (86.9±5.3)% of negative control group (t=6.49, 14.31, 9.73, P<0.05). Compared with that of negative control group, the inflammatory cell infiltration was reduced in mice wounds tissue of diabetic AMSCs group on PID 11, and thicker epidermis and dermis as well as regenerated skin appendages were observed on PID 21. On PID 11 and 21, the collagen percentages of mice wounds tissue in diabetic AMSCs group was (48.3±1.3)% and (54.1±1.7)%, respectively, significantly higher than (41.4±1.7)% and (50.3±1.2)% of negative control group (t=6.98, 3.99, P<0.01). On PID 11 and 21, the numbers of new blood vessels in mice wounds tissue of diabetic AMSCs group were 17.2±1.3 and 18.0±2.1, respectively, significantly more than 8.0±1.4 and 14.0±1.5 of negative control group (t=10.69, 3.38, P<0.01). On PID 21, the S100 positive cell percentage in mice wounds tissue of diabetic AMSCs group was (1.76±0.12)%, significantly higher than (0.55±0.03)% of negative control group (t=21.68, P<0.001). On PID 11, the colonization of AMSCs in mice wounds tissue of diabetic AMSCs group was observed.@*Conclusions@#Transplantation of AMSCs from type 2 diabetic mellitus patients can accelerate wound healing of pressure ulcers in mice by promoting angiogenesis, collagen deposition, and Schwann cell regeneration.
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Objective@#To observe the repairing effect of adipose mesenchymal stem cells (ADSCs) on lung injury induced by silica in rats.@*Methods@#Primary ADSCs-GFP was obtained from rats. ADSCs-GFP was injected into tail vein of silicosis model rats. The expression of green fluorescence in lungs was observed regularly to determine the homing ability of ADSCs. Primary ADSCs of rats were obtained and randomly divided into control group, exposure group, vehicle group and ADSCs group. Silicosis rat model was established by non-exposed tracheal drip method. 24 hours after silica exposure, rats in ADSCs group were injected with ADSCs of 1×106/kg body weight through tail vein, and the pathological changes of lung tissue were observed and evaluated 28 days after intervention. To explore the early intervention mechanism of ADSCs on pulmonary fibrosis in silicosis model rats, apoptosis-related proteins were detected by immunohistochemistry.@*Results@#28 days after exposure to silica, rats in the exposure group showed obvious pulmonary fibrosis. Compared with exposure group and vehicle group, ADSCs group showed less pulmonary inflammation, less silica nodules and less collagen deposition area. Immunohistochemical results showed that the expression of Caspase-3 and cytochrome C protein decreased and Bcl-2 protein increased after ADSCs transplantation.@*Conclusion@#ADSCs infusion has an obvious intervention effect on postponing early silicosis fibrosis in rats exposed to silica, and its mechanism is related to the regulation of apoptotic process.
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Objective@#To evaluate the feasibility of differentiation of human adipose mesenchymal stem cells (hADSCs) into endothelial-like cells and the safety of induced cells intraocular application.@*Methods@#HADSCs were induced to endothelial-like cells in vitro.The experimental group was added with streptomycin, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and the control group was added with the same amount of phosphate buffered saline (PBS), the expression of von Willebrand factor (vWF), a marker of endothelial cells, was observed in the two groups.Six C57BL/6J mice were randomly divided into experimental group and control group by using the random number method, 3 mice for each group, the induced cells were injected intravitreally into C57BL/6J mice as experimental group, and the same amount of PBS buffer was injected intravitreally into C57BL/6J mice as control group.The changes of retinal cells were observed by electron microscopy.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.@*Results@#After induction of hADSCs in vitro, the endothelial cell marker was expressed.VWF immunofluorescence of the experimental group showed strong green fluorescence, and the color rendering rate was 100%.The control group showed no coloration of vWF immunofluorescence, and the color positive rate was 0.After 1 month of intravitreal injection, the retinal ganglion cells and rod cells were not degraded and necrotic.@*Conclusions@#HADSCs can differentiate into endothelial-like cells in vitro, and there is no retinal toxicity in a short-term.
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Objective@#To evaluate the feasibility of PKH26 marked human adipose derived mesenchymal stem cells (hADSCs) in vitro and intraocular.@*Methods@#HADSCs were cultured in vitro and marked with PKH26.Eighteen C57BL/6J mice were divided into normal control group (6 mice), labeled cell group (9 mice) and unlabeled cell group (3 mice) by using sortition randomization method, labeled cells were injected intravitreally in C57BL/6J mice as labeled cell group, and the unlabeled cells were injected intravitreally in C57BL/6J mice as unlabeled cell group.After 1 month, retinal slab was checked to contrast the results of intraocular labeling.The retina was taken out for hematoxylin-eosin staining and electron microscopy to observe the toxicity.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.@*Results@#In vitro, red fluorescent was found in the cytomembranes after hADSCs were labeled by PKH26.Retinal patch of labeled cell group showed red fluorescence of hADSCs were in front of the retina.Hematoxylin-eosin staining of retinal tissue and optic ganglion cells showed that cell degeneration and proliferation were not found in labeled cell group.The hADSCs in vivo and in vitro marked enhancement results showed that, no fluorescent was found in the unlabeled cell group, the color positive rate were both 0, while red fluorescence was found in the labeled cell group, the color positive rate were both 100%.@*Conclusions@#PKH26 can be used to mark and intraocular trace of hADSCs.Also, it has no morphology toxic reaction.
ABSTRACT
Objective: To investigate the effect of daphnetin (DAP) combined with insulin-like growth factor 1 (IGF-1) gene transfection on chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) in rats. Methods: Rat ADSCs were isolated and amplified by enzymatic digestion. The third generation ADSCs were treated with IGF-1 gene transfection as experimental group and normal ADSCs as control group. The cells of the two groups were treated with different concentrations of DAP (0, 30, 60, 90 μg/mL), respectively. Cell proliferation was detected by cell counting kit 8 (CCK-8) after cultured for 72 hours. After 14 days, real-time fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expressions of chondrocyte markers (collagen type Ⅱ and Aggrecan) in each group; and toluidine blue staining and collagen type Ⅱ immunohistochemical staining were performed. Results: CCK-8 assay showed that with the increase of DAP concentration, the cell absorbance ( A) value of the control group and the experimental group increased gradually ( P0.05). But the mRNA and protein expressions of collagen type Ⅱ and Aggrecan in the experimental group increased gradually, and the 60 and 90 μg/mL DAP concentration groups were significantly higher than 0 μg/mL DAP concentration group ( P<0.05). At the same DAP concentration, the relative mRNA and protein expressions of collagen type Ⅱ and Aggrecan were significantly higher in the experimental group than in the control group ( P<0.05). Toluidine blue staining showed that with the increase of DAP concentration, there was no significant difference in cell staining between the control group and the experimental group. At the same DAP concentration, the cells in the experimental group were slightly darker than those in the control group. Immunohistochemical staining of collagen type Ⅱ showed that with the increase of DAP concentration, there was no significant difference in the cytoplasmic brown-yellow coloring of the cells in the control group. The cytoplasmic brown-yellow coloring of the cells in the experimental group gradually deepened, with 60 and 90 μg/mL DAP concentration groups significantly deeper than 0 μg/mL DAP concentration group. At the same DAP concentration, the color of the cells in the experimental group was significantly deeper than that in the control group. Conclusion: DAP can promote the proliferation of ADSCs in rats. The differentiation of ADSCs into chondrocytes induced by DAP alone was slightly, but DAP combined with IGF-1 gene transfection has obvious synergistic effect to promote chondrogenic differentiation of ADSCs.
ABSTRACT
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.