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Objective@#To investigate the effects of PRX-2 gene on phenotype changes in epidermal stem cells differentiating into sweat gland cells.@*Methods@#Epidermal stem cells and sweat gland cells separated and cultured from healthy foreskin and adult full-thick skin respectively, were identified by immunofluorescence staining. Lentiviral vector-mediated overexpression and knockdown of PRX-2 gene in epidermal stem cells were performed respectively, with empty vector-mediated epidermal stem cells as a control group. Overexpression、blank control and knowdown group′s PRX-2 expressions in gene and protein levels were detected using RT-PCR and Western blot technology. The ESCs of each group were co-cultured with sweat gland cells through transwell plate, and the expressions of CEA and β1 integrin in epidermal stem cells were determined by flow cytometry before and after co-culturing.@*Results@#Epidermal stem cells and sweat gland cells were in line with their respective specific antigens. Before co-cultured, epidermal stem cells highly expressed β1 integrin (98.69±0.67)%, hardly expressed CEA (6.20±3.15)%. After co-cultured, β1 integrin expression levels were showed as knockdown group (19.30±0.53)%<blank control group (65.77±2.32)% <overexpress group (92.63±10.97)%, and CEA expression levels as knockdown (95.43±2.36)%> blank control group (51.20±0.79)%> overexpress group (45.91±0.93)%. There had significant differences between those of each two groups.@*Conclusions@#PRX-2 gene can inhibit the phenotypic change of Epidermal Stem Cells differentiating into Sweat Gland Cells and improve the ability to maintain their own specific antigens.
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Objective To construct a recombinant lentivirus containing human beta defensins -3 ( hBD3 ) , connective tissue growth factor gene (CTGF) and enhanced green fluorescent protein (EGFP), and to detect its translation in rabbit bone marrow mesenchymal stem cells (BMSC).Methods The lentivirus containing hBD3, CTGF and EGFP genes was constructed in vitro.The titer of lentivirus was tested with end-paint dilution assay .Rabbit BMSCs were transfected with recombinant virus.The best value of multiplicity of infection (MOI) was tested.The expression condition, transfection efficacy and genetic stability of the target genes were evaluated by using fluorescence microscopy and flow cytometry . Western blotting was used to detect the expression of the target protein .Results Recombinant lentivirus vectors: Lenti-CTGF-hBD3-EGFP, Lenti-hBD3-EGFP, and Lenti-EGFP, were successfully obtained . The titer of the recombinant lentiviruses was 3.21 ×108, 5.80 ×108, and 1.16 ×109, respectively.The best MOI value to transfect BMSCs was 150. The transfection efficacy of these lentivirus vectors was high , reaching 79.72%as assessed by flow cytometry , and it could be stably inherited .Western blotting displayed that target protein expression was successful .Conclusion The construction of recombinant lentiviruses carrying hBD3 and CTGF genes is successful and can be effectively transfected into BMSCs .
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Tissue-engineered skin plays an important role in clinical applications,and even the rapid development of science and technology promotes the research about it.Choosing an appropriate animal model for wound repair is the prerequisite for the objective evaluation of the object of study.In this paper,the research progress of animal models of wound repair was introduced from several aspects,such as selection of experimental animals,making of wound models,skin-related cells and materials,wound healing evaluation indexes,etc.,hoping to provide reference for later research work.
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Objective To probe the periodontal ligament regeneration following the implantation of bone marrow mesenchymal cells ( BMSCs ) sheet-collagen membrane-BMSCs sheet sandwich complex.Methods BMSCs cell sheet-collagen membrane-BMSCs sheet complexes were compounded on the root surface of teeth of Beagle dogs.All the dogs were killed on 4 and 12 weeks after implantation.Periodontal ligament regeneration was observed by radiological means, HE staining and Sirus-red staining.Results Compared with collagen membrane group and blank control group, there was a clearly periodontal ligament like tissue and Sharpey′s like fibres formation in test group only.Conclustion Cell sheet-collagen membrane-cell sheet sandwich complex can effectively improve the periodontal ligament regeneration.
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BACKGROUND:Studies have shown that chitosan and other natural polysaccharides have heparin-like anticoagulant function after sulfonated modification. Sulfonated chitosan has good anticoagulant property because the sulfonate group formed by sulfonated chitosan is similar with the active group of heparin. OBJECTIVE: To prepare the anticoagulant chitosan nanoparticles and to detect its morphology, physical and chemical properties and biological security. METHODS: Chitosan nanoparticles were synthesized by emulsion-chemical cross link. Sulfonated chitosan nanoparticles were synthesized by sulfonation reaction. Its morphology was described by transmission electron microscope. The peak-value change of its specific groups was observed by infrared spectroscopy. (1) Coagulation experiment: Heparin, chitosan nanoparticles and 10, 30 and 50 mg of sulfonated chitosan nanoparticles were added into the blood of Spraque-Dawley rats. The coagulation indicators were detected. (2) Hemolysis experiment: deionized water, physiological saline and 10, 30, 50 g/L sulfonated chitosan nanoparticles extracts were added into 2% red blood cel suspension of rabbits. The hemolysis rate was detected. (3) Cytotoxicity experiments: DMEM medium containing fetal bovine serum and 10, 30, 50 g/L sulfonated chitosan nanoparticle extracts were used to culture human umbilical vein endothelial cels. Cel relative growth rate and toxicity grading were detected after 72 hours. RESULTS AND CONCLUSION: Scanning electron microscopy showed that sulfonated chitosan nanoparticles had good morphology, with a diameter of 50 nm. Infrared spectroscopy showed that the sulfonated replacement occurred.In vitro coagulation experiments showed that sulfonated chitosan nanoparticles had significant anticoagulant effects in a dose-dependent manner. Sulfonated chitosan nanoparticles meet the national safety standard for hemolysis rate of less than 5%, non-induced hemolysis property. Cytotoxicity assays showed that sulfonated chitosan nanoparticles extracts had no significant cytotoxicity, and its biological safety was in line with the national standards.
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BACKGROUND:With the aging population, the incidence of lumbar degenerative disease was apparently increased, but how to treatment of degenerative lumbar disease remains controversial. OBJECTIVE:To compare clinical and radiographic results of minimaly invasive posterior lumbar interbody fusion and open posterior lumbar interbody fusion for single-segment degenerative lumbar disease. METHODS: We retrospectively analyzed the clinical data of 97 patients with single-segment degenerative lumbar disease, who were treated in the Huishan District People’s Hospital of Wuxi City from July 2006 to July 2012. These patients were divided into minimal group (minimaly invasive posterior lumbar interbody fusion;n=51) and open group (open posterior lumbar interbody fusion;n=46). These data were compared between the two groups, including operative time, blood loss (intraoperative blood volume+postoperative drainage volume), total blood transfusion, postoperative back pain (visual analogue scale), length of hospital stay, bed time, perioperative complications, clinical function (Oswestry disability index), and radiographic results. RESULTS AND CONCLUSION:Al of 97 patients were folowed up. The duration of folow-up was 28-78 months and 27-76 months in minimal group and open group, respectively. There was no significant difference between the minimal group and open group in term of folowed-up time (P=0.981). Operative time, blood loss, total blood transfusion, bed time, length of hospital stay and visual analogue scale score during final folow-up were significantly lower in the minimal group than in the open group (P 0.05). These results indicate that for the single-segment degenerative lumbar disease, the use of minimaly invasive posterior lumbar interbody fusion or open posterior lumbar interbody fusion can obtain satisfactory clinical function, but the minimaly invasive posterior lumbar interbody fusion has the advantages of a less trauma, shorter length of hospital stay and bed stay, and lighter back pain.
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Objective To study the method of preparation and blood sugar lowering effect of oral chitosan-insulin nanoparticles (INS-NPs) in streptozotocin-induced diabetic Wistar rats. Methods The INS-NPs were prepared by an ionic gelation method. The changes in the morphology and size of the INS-NPs were observed with transmission electron microscope and Zetasizer 3000HS, respectively. The blood sugar lowering effect of the INS-NPs was evaluated by monitoring the blood glucose levels in healthy and streptozotocin-induced diabetic rats. Results INS-NPs were spherical in shape with a mean size of 220.6±15.9nm. Entrapment efficiency of INS-NPs was 75.4%±3.2% and the loading efficiency of INS-NPs was 19.5%±2.6%. In vivo blood sugar lowering study showed that the levels of blood glucose of healthy Wistar rats were significantly reduced from 6h to 12h after oral administration of INS-NPs(25U/kg). The blood glucose level of diabetic rats were significantly reduced at 6h after oral administration of INS-NPs (25U/kg), and this effect was maintained for more than 9h, and the levels of blood glucose were kept in normal range for 7h. Conclusion The INS-NPs prepared by ionic gelation method has the blood glucose lowering effect in streptozotocin-induced diabetic Wistar rats.
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In order to study the chondrogenic phenotype differentiation of adult sheep bone marrow-derived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering. MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCs were induced with H-DMEM containing TGF-beta3, IGF-I, Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindle-like appearance to a polynal shape, a large amount of endoplasmic reticulum, Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when induced in vitro and can be used as optimal seed cells for cartilage tissue engineering.
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Bone Marrow Cells/cytology , Cell Differentiation , Cell Separation , Cells, Cultured , Chondrocytes/cytology , Chondrogenesis , Mesenchymal Stem Cells/cytology , Phenotype , Sheep , Tissue EngineeringABSTRACT
In order to study the chondrogenic phenotype differentiation of adult sheep bone marrow-derived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering. MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCs were induced with H-DMEM containing TGF-beta3, IGF-I, Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindle-like appearance to a polynal shape, a large amount of endoplasmic reticulum, Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when induced in vitro and can be used as optimal seed cells for cartilage tissue engineering.
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Animals , Bone Marrow Cells , Cell Biology , Cell Differentiation , Cell Separation , Cells, Cultured , Chondrocytes , Cell Biology , Chondrogenesis , Mesenchymal Stem Cells , Cell Biology , Phenotype , Sheep , Tissue EngineeringABSTRACT
Objective To explore the feasibility of the construction and formation of cartilage with immortalized chondrocytes by tissue engineering technology in vitro . Methods Human telomerase reverse transcriptase(hTERT) gene was introduced into rabbit mandibular condylar chondrocytes by eukaryotic vector. After screening with G418, the positive clones were amplified for culture. Normal or immortalized chondrocyte loaded cytoskeleton ? tricalcium phosphate (? TCP) complexes were incubated in vitro for 1～2 d and then implanted into subcutaneous tissue of nude mouse. The complexes of immortalized chondrocyte ? TCP and chondrocyte ? TCP or ? TCP alone were established as the experimental group and control groups respectively. The specimens were harvested within 3 and 6 months after surgical procedure for histological and immunohistochemical observation. Results In experimental groups and control group 1, the complexes packed with cartilage like tissue were found, but there was only a little fiber like tissue formed in control group 2. Immunohistochemistry revealed strong positive staining with safranine O, toluidine blue and collagen type II and obvious formation of cartilage. There was significant difference between the experimental group and the control groups( P
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<p><b>OBJECTIVE</b>To evaluate the feasibility of growing tissue-engineered cartilage using chondrocytes seeded onto a biodegradable porous bioceramic, the beta-tricalcium phosphate (beta-TCP).</p><p><b>METHODS</b>A porous bioceramic template of beta-TCP was created in the shape of a disc. Chondrocytes isolated from rabbit articular cartilage were seeded on the beta-TCP template and then kept in rotatory cell culture system (RCCS) for 1 week prior to subcutaneous transplantation into athymic mice. The three-dimensional structure was well-maintained 16 weeks after implantation. After 4, 8, 16 weeks, the specimens were harvested and examined macroscopically, histologically and immunohistochemically.</p><p><b>RESULTS</b>Gross morphological and histological analysis of the specimens from the chondrocyte-beta-TCP complex demonstrated new cartilage construction. The overall configuration of the experimental specimens closely resembled the structure of beta-TCP template.</p><p><b>CONCLUSION</b>These findings suggest that porous bioceramic (beta-TCP) is a good "matrix" for chondrocyte, and can be used for cartilage engineering.</p>
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Animals , Female , Mice , Calcium Phosphates , Pharmacology , Cartilage , Transplantation , DNA , Glycosaminoglycans , Immunohistochemistry , Mice, Nude , Tissue EngineeringABSTRACT
Objective This paper was conducted to study the preparation methods and the hypoglycemic effects of oral sodium alginate-insulin nanoparticles (INS-NPs) on the blood glucose level in Streptozotocin-induced diabetic Wistar rats. Methods The INS-NPs were prepared by an ionic gelation method. The changes of the morphology and size of the INS-NPs were examined by transmission electron microscope and Zetasizer 3000HS. The hypoglycemic effects of the INS-NPs were evaluated by monitoring the blood glucose levels in streptozotocin-induced diabetic Wistar rats. Results The INS-NPs were spherical or ellipsoidal in shape with a diameter of 236.4?19.3nm. The entrapment efficiency and load efficiency of INS-NPs were 78.5%?6.1% and 22.6%?4.4%, respectively. In vivo hypoglycemic study showed the levels of blood glucose of diabetic Wistar rats declined at 7h after oral administration of INS-NPs (26U/kg). Their hypoglycemic effects were maintained for 12h and the levels of blood glucose were kept with normal range for 6h (less than 7.0mmol/L). Conclusion The INS-NPs have the hypoglycemic effect on the blood glucose level of streptozotocin-induced diabetic rats.
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Objective:To examine the expression pattern of DSP,DMP1,CBFA1,BMP2 in rat dental pulp stem cells(RDPSCs).Methods:Immunohistochemical staining was carried out using antibodies against DSP,DMP1, CBFA1 ,BMP2 in rat dental pulp stem cells. Mineralization was induced in the RDPSCs and expression of DSP and DMP1 was measured after induction.Results:CBFA1 and BMP2 were positive in RDPSCs. Only a few RDPSCs were stained positive for DMP1. DSP expression was observed in the minority of these cells. However, the majority of the RDPSCs were found strongly positive for DSP and DMP1 after mineralization induction.Conclusion:Positive expression of CBFA1 and BMP2 indicates the premature nature of RDPSCs. The dentin-specific expression of DSP demonstrates that the RDPSCs can differentiate along odontoblastic lineage.
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AIM: To set up a method of inducing mouse embryonic stem cells (mESC) to differentiate into cardiomyocyte after treatment with 5-azacytidine. METHODS: Cytotoxicity of 5-azacytidine was measured by MTT assay. Treatment of mESC with conditioned culture mediums, which were composed of 5-azacytidine alone or combined with retinoic acid, induced the cell differentiation to cardiomyocytes. The cells induced were identified by detecting the expression of cardiac proteins (myosin, desmin, ?-actin and ?-actinin). Gene MLC-2v, a specific gene of ventricular-like cardiomyocyte, was also detected by RT-PCR. RESULTS: The non-cytotoxic dose of 5-azacytidine was 8 ?mol/L, which was able to induce mESC to differentiate into cardiac syncytiums. Cells induced expressed many cardiac proteins and MLC-2v mRNA. However, combined with retinoic acid inhibited mESC differentiation into cardiomyocyte. CONCLUSION: 5-azacytidine is able to promote mESC differentiation into cardiomyocytes. A method of inducing mESC to differentiate into cardiomyocytes in vitro has been established.
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Objective To evaluate the feasibility of reconstruction of artificial bladder tissue using the expanded bladder cells seeded onto both sides of a synthetic PLGA polymer and cultured in the nude mice by tissue engineering technique. Methods The urothelial cells and smooth muscle cells were obtained from young rabbits by mechanical isolation and enzyme digestion method. In vitro cultured and expanded smooth muscle cells and urothelial cells were seeded onto the outer and inner surfaces of each polymer. Then, the constructs were implanted into the subcutaneous pockets of athymic mice. At 4 and 8 weeks after implantation, the specimens were harvested and examined macroscopically, histologically, and immunohistochemically. Results The polymer was covered with urothelial cells inside and smooth muscle cells outside. As polymer degradation was in progress, the urothelial cells and the smooth muscle cells kept proliferation and converged. Conclusion Artificial bladder tissue reconstructed by tissue engineering technique has the similar properties to those of the normal bladder wall. This study has laid solid foundation for further studies of tissue engineering bladder.
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0.05). Islet secretion viabilities in both Matrigel-coated and type Ⅰ and Ⅳ collagen mixture-coated group were better than the control group (P