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Objective:To evaluate the effects of adipose-derived stem cells (ADSCs) and ADM microparticle on diabetic wound healing.Methods:ADSCs was co-cultured with ADM microparticle in vitro. The models of diabetic nude mice were established by intraperitoneal injection of STZ and the full-thickness skin defects were designed on the back. All 24 diabetic mice were randomly divided into 4 group: experimental groups were transplanted with ADSCs and ADM microparticle and the other groups were transplanted with ADSCs, ADM microparticle and blank control group was set up. On the 7th and 14 th days, the wound healing rate of 3 mice randomly selected from each group was calculated, and the thickness between dermis and epidermis was measured by hematoxylin and eosin staining. The density of neovascularization was measured by immunohistochemical staining. The differences were compared between the groups.Results:Compared to the ADSCs groups, the mice of the experimental groups showed higher cell survival rate. The wound healing rate in the experimental groups was (86.0±2.7)% (7 days) and (98.5±1.1)% (14 days), thicker dermis-epidermis distance was (99.1±1.8) μm (7 days) and (124.3±4.3) μm (14 days) ( P<0.05), and higher density of neovascularization was noted. Conclusions:The transplantation with active ADM microparticle can significantly promote neovascularization and wound healing of diabetic wound.
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Objective To study the cell morphology and differentiation efficiency when rabbit bone marrow mesenchymal stem cells (BMSCs) were induced osteogenic differentiation as culturing by autologous platelet-rich plasma (PRP) instead of serum,and to explore a new method of inducing BMSCs osteogenic differentiation.Methods The PRP was prepared by arterial blood of rabbit.Punctured and The bone marrow was sampled from rabbit's iliac bone,and BMSCs were collected,which divided into PRP group,fetal calf serum (FBS) group and serum-free control group,and cultured in 10％ autologous PRP,10％ FBS and serum-free respectively,combined with DMEM-F12 medium.The second generation cells were divided into experimental and control groups.The experimental groups' medium was added dexamethasone,β-sodium glycerophosphate and ascorbic acid,and the control groups went on.The cell morphological difference of each group was Observed between anterior and after inducing differentiation,and compared between each group.Results BMSCs of PRP and FBS groups grew quickly,presented like fusiform form before induction,and increasd in volume,became a triangle,polygonal and round form gradually after osteogenic induction.Cells of PRP and FBS groups aggregated spontaneously and multilayered,and formed calcium nodules and bone-like structure after induced 7 days averagely,which could be stained red by alizarin red S;cells of serum-free groups were induced 14 days averagely,only three samples showed osteogenesis performance.Cells of PRP and FBS groups differentiation efficiency was superior to serum-free groups when inducd 20 days,the difference was statistically significant (P＜0.05),and the difference between efficiency of PRP and FBS groups was not significant (P＞0.05).Conclusions Autologous PRP could be used to proliferate and induce osteogenic differentiation of BMSCs instead of serum.
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Objective A database of normal people's external nose was established through 3D measurement. This database was used to customize the external nose for patients with nasal defects and to assist the operator to carry out the whole nose reconstruction surgery, so as to carry out the postoperative evaluation.Methods 3D scanning of the subject's face, measurement of relevant indexes of the nose and establishment of a database, the operator used normal nose database to customize the customized external nose for 17 patients with nasal defects, assisted them in the whole nose reconstruction surgery, and used independent sample t test for data statistics to evaluate the expected effect of surgery. Results There was no statistically significant differences between the postoperative actual data and the preoperative personalized data (P> 0.05) in right root wing distance, left root wing distance, nose length, nasal base width, nose width, right side vertical bisect nasal line, left side vertical bisect nasal line, nose height, medial malleolus spacing, face width, mouth split width, facial height, nasal width index, nasal width index, interondylar-nasal width index and nasal high index. The actual data of nasal deep was statistically different from preoperative personalized data (P < 0.05). Conclusions Analysis showed no significant difference between the actual data nasal surgery and preoperative customization data. 3D measurement of normal human external nasal establishment database to customize the external nose for patients with nasal defects, can assist the surgeon to perform total nasal reconstruction surgery and improve predictability and make surgery more precise. Postoperative assessments can also be performed to compare preoperative and postoperative outcomes.
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Objective To observe the clinical efficacy of propranolol and 595 nm pulsed dye la ser (PDL) in treatment of infantile hemangioma.Methods 26 infants admitted to our hospital from January 2013 to January 2015 with hemangioma underwent oral propranolol 2 mg/(kg · d) treatment after excluding of taboos.The daily doses were divided equally to two parts,taken on the time of 8:00 and 20:00,when the electrocardiograph and pulse oxygen were monitored and recorded persistently.The patients were discharged from the hospital when it was stable,with review of blood routine examination,fasting blood glucose,liver and kidney function,and the change of size,character and color of hemangioma were recorded,and taken photos every two weeks after discharge.The 595 nm PDL was used to treat the hemangioma faded incompletely when the propranolol was terminated.Results The tension and color of all hemangioma decreased in varying degrees in taking propranolol for 72 hours,and evaluated the efficacy as recovery completely 19 cases;signifivantly effective in 3 cases and partial efficacy in 4 cases;the latter 7 cases were further treated with 595 nm PDL.Followed-up for 6-12 months showed that efficacy of recovery reached 100％.10 cases showed heart rate was mild reversibly slow,with no special treatment.5 cases had diarrhea,and healed with symptomatic treatment.No adverse reactions like liver and kidney dysfunction and so on were found.Conclusions Propranolol and 595 nm PDL can effectively treat infantile hemangioma,and thus it can be used as the recommended treatment of infantile hemangioma.
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Objective To explore the operating methods and its related questions of auricular reconstruction with totally expanded skin in combination with laser hair removal for the treatment of adolescent microtia.Methods From Jan.2013 to Dec.2016,30 adolescent microtia patients were treated with totally expanded skin.At the first stage,the 100 ml kidney-shaped expander was implanted under the skin of mastoid.After expanding capacity of 80 ml,the hair on the expanded skin was depilated once a month with reference to the healthy ear;at the second stage,after expanding capacity of 150 ml,the expander was taken out and the fiber capsule was removed;the tautologous rib cartilage was harvested and the scaffolds were sculptured;the cartilage was implanted and the expanded skin flap was used to cover the frontal surface and back surface of the scaffold;at the third stage,the earlobe transposition,conchal excavation and tragus construction were performed at the same time.Results All the patients were followed up for 3 to 24 months;the results showed 1 case of leakage of expander,4 cases of hematoma,2 case of expanded skin burst,and the complications were treated correctly,all patients were satisfied with the appearance;the color,texture,location,size;and height of ear cranial angle were matched with health ear;there was no obvious scar and auricle subunit structure was clear.Conclusions The laser in combination with the large capacity tissue expander in auricular reconstruction is simple,less trauma and less scarring.
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Objective To study the effect of the lentivirus encoding acidic fibroblast growth factor transfecting human adipose-derived stem cells (ADSCs) on the cell cycle and proliferation of ADSCs.Methods ADSCs were isolated and extracted by enzymatic digestion from the liposuction aspirate.ADSCs were cultured,identified and osteogenic induced reagent was used to induce differentiation of ADSCs towards bone cells.To obtain lentivirus encoding FGF-1,the plasmid PWPXLd FGF-1 was co-transfected with plasmid psPAX2,pMD2.G in 293T cells.ADSCs were infected with lentivirus encoding FGF-1.Expression of green fluorescent protein (GFP) in infected FGF-1 was observed by fluorescence microscope and expression of FGF-1 in ADSCs was verified by Western blot analysis.Flow cytometry was used to detect the cell cycle of ADSCs infected with lentivirus encoding FGF-1.EDU assay was performed to examine cell viability.Results Lentivirus encoding FGF-1 was obtained.After ADSCs being infected green fluorescence was found in about 70％ ADSCs,and overexpression of FGF-1 protein was detected in infceted cells by Western blot analysis.The percentage of G2/M phase cells was significantly increased compared with the control group,and the proliferation of ADSCs infected with lentivirus encoding FGF-1 was promoted as compared with the control group.Conclusions FGF-1 can enhance G2/M phase of ADSCs and promote the proliferation of ADSCs.
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BACKGROUND:Early vascularization is crucial for wound healing. A high-porosity, macrovoid alogeneic skin leads to the rapid vascularization and celular infiltration. OBJECTIVE: To obtain a new alogeneic skin product with high porosity, good cel permeability and good histocompatibility using an improved preparation method of human acelular dermal matrix. METHODS: Cel components of healthy human skins were removed by the improved method and the traditional method, respectively. The improved method was to remove the subcutaneous fat, eliminate the epidermis (1 mol/L NaCl solution at 37℃ for 24 hours) folowed by shaking processing (2% NaOH at 45℃ for 4 hours), and then, the solution was neutralized with PBS rinsing, dried and stored at 4℃ for standby. We detected the porosity and degradation time in vitroof the acelular dermal matrices prepared by two methods and the cytotoxicity of the material infiltration liquid on the adipose-derived stem cels. Hematoxylin-eosin staining was used for the detection of the cel residual, the integrity of colagen and cel biocompatibility. Scanning electron microscopy was used to detect the pore size. RESULTS AND CONCLUSION: Both the two methods could completely remove the cel components, and maintain the integrity of the colagen scaffold. The porosity of acelular dermal matrix with the improved method was (93.22±0.99)%, which was significantly higher than that with the traditional method [(74.28±2.06)%;P 0.05). No obvious cytotoxicity of the acelular dermal matrix prepared with the improved method was detected. At 3-7 days of co-culture, the adipose-derived stem cels cultured on the acelular dermal matrix prepared with the improved method could penetrate the basement membrane to the deep dermis, while there was no obvious cel invasion and growth in the deep dermis prepared by the traditional method. Compared with the traditional method, the improved method is more suitable for cel infiltration and growth with higher porosity and larger pore size.
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Objective To explore the effects of recombinant plasmids of pGPU6/GFP/NeoshRNA-CTGF (shRNA-CTGF) on the type Ⅰ collagen (COL-Ⅰ) protein expression in keloid,through RNA interference on connective tissue growth factor (CTGF) in vivo and in vitro.Methods Recombinant plasmids were designed and constructed by specific shRNA-CTGF; After transfeced human keloid fibroblast with shRNA-CTGF in vitro,RT-PCR was used to detect the CTGF mRNA level,and Western blot to detect the secretion of COL-Ⅰ.After transfected the keloid of nude mice with shRNA-CTGF in vivo,RT-PCR was used to detect the CTGF and COL-Ⅰ mRNA level,and Western blot was used to detect the protein expression of COL-Ⅰ.Results Recombinant plasmids of CTGF were successfully constructed,and the CTGF gene expression was significantly decreased in vivo and in vitro by 86.8％ and 54.1 ％,respectively; Down-regulation of CTGF in vitro significantly inhibited the mRNA and protein level of COL-Ⅰ by 76.8％ and 65.6％,respectively; Down-regulation of CTGF in vivo significantly reduced the COL-Ⅰ mRNA and protein level by 52.7％ and 48.0％,respectively.Conclusions CTGF gene expression is successfully down-regulated by the recombinant plasmid of shRNA-CTGF in vivo and in vitro.shRNA-CTGF significantly reduces the COL-Ⅰ protein level in keloid.It implies that CTGF gene is a potential target in the therapy of pathological scar.
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BACKGROUND:A great amount of mesenchymal stem cels can be successfuly derived from fat tissue and induced to differentiate into osteoblasts, chondrocytes, adipocytes and myocardial cels. OBJECTIVE:To establish the method of isolating, culturing and osteogenic differentiation of adipose-derived stem celsin vitro, and to explore the potential of adipose-derived stem cels as seed cels for bone tissue engineering. METHODS:Colagenase enzymatic digestion was used to isolate adipose-derived stem cels from human fat tissue which were then culturedin vitro. Flow cytometry was used to detect cellsurface markers. cellcounting kit-8 assay was performed to examine cellviability. Adipose-derived stem cels were induced by osteogenic induced reagent to differentiate into bone cels. In addition, we also performed BCIP/NBT method to detect alkaline phosphatase activity. Alizarin red staining was used to detect the formation of calcium node. RT-PCR was performed to examine alkaline phosphatase and osteopontin expression. RESULTS AND CONCLUSION:We successfuly obtained adipose-derived stem cels from fat aspirated liquid. Adipose-derived stem cels obtained could passage stably and proliferate highly. Flow cytometry data showed the expression of stem cellsurface markers. Adipose-derived stem cels showed typical osteoblast morphology after osteogenic differentiation. Alkaline phosphatase staining was positive and alizarin red staining displayed the formation of calcium node. Furthermore, we found that alkaline phosphatase and osteopontin mRNA was expressed after differentiation 0, 3, 7, 14, 21, 28 days. These findings indicate that adipose-derived stem cels can be obtained from fat tissue through enzymatic digestion, differentiate towards bone cels, and express alkaline phosphatase and osteopontin, which can become potential seed cels for bone tissue engineering.
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Objective To investigate the specific silencing of connective tissue growth factor (CTGF) in a nude mouse keloid model,using RNA interference (RNAi) technique,and to provide the basis for gene therapy of keloid.Methods The nude mouse keloid model was established,and then transfected in vivo with well-amplifiating plasmid.The mRNA expression levels of CTGF mRNA and type Ⅰ collagen mRNA were detected with reverse transcription-polymerase chain reaction (RT-PCR).The distribution and protein expression levels of CTGF and type Ⅰ collagen were determined quantitatively using immunohistochemistry.Results The expression of CTGF at mRNA and protein levels was decreased in the experiment group,and the expression of type Ⅰ collagen at mRNA and protein levels was also decreased after transfection,as compared with negative control group and blank group,with significant difference between groups (P＜0.05).Moreover,the expression of type Ⅰ collagen and CTGF was positively correlated (r=0.979).Conclusions Keloid type Ⅰ collagen can be decreased through in vivo inhibiting CTGF expression.The transfection of CTGF gene in vivo may have effects on type Ⅰ collagen generation,and thus inhibit the keloid growth.
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ObjectiveTo investigate the influence of connective tissue growth factor (CTGF) onthe collagen metabolism in human keloid fibroblasts with RNA interference (RNAi).Methods Human keloid fibroblasts (KFB) in vitro were transfected by 3 pairs of specific small interfering RNA (siRNA) CTGF plasmid synthesized for human CTGF,respectively.Reverse transcriptase-polymerase chain reaction (RT-PCR) contributed to the screening of the best siRNA in interfering of CTGF expression in human keloid fibroblasts to construct the plasmids,with the application of RNAi,to test the changes of expression level and collagen content of CTGF in transfected keloid fibroblasts through RT-PCR and Western blotting compared to its control groups.ResultsThe 3rd pair (C3) siRNA- CTGF expression of genes and proteins was remarkably inhibited after being interfered with human keloid fibroblasts,with inhibitory rates of 86.8 ％ and 65.6 ％.ConclusionsKeloid fibroblasts transfected by plasmid siRNA-CTGF effectively inhibite the expression of CTGF and deposition of collagen,and CTGF promotes the collagen synthesis in keloid development.