ABSTRACT
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.
ABSTRACT
Objective@#To observe content of cytokine in human stromal vascular fraction gel (SVF-GEL) and effect of SVF-GEL on biological behaviors of epidermal and dermal cells in vitro and clinical efficacy of SVF-GEL.@*Methods@#(1) SVF-GEL was prepared using liposuction aspirates harvested from females who received abdomen liposuction in author′s unit. SVF-GEL (1 mL) and high-glucose Dulbecco′s modified eagle medium (DMEM, 1 mL) were respectively cultured for 24 h with high-glucose DMEM containing 10% fetal calf serum, 10 g/L penicillin, and 10 g/L streptomycin, denoted as SVF-GEL group and negative control group, with 6 samples in each group. Content of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in the supernatant was determined by enzyme-linked immunosorbent assay. (2) A number of 5×105 human skin fibroblasts (HSF) and HaCaT cells in logarithmic phase were inoculated and cultured in Transwell chambers for 12 h. All Transwell chambers containing cells were divided into SVF-GEL group (0.5 mL SVF-GEL was added for co-culture) and control group (0.5 mL high-glucose DMEM was added for co-culture), with 9 samples in each group for HSF and HaCaT cells. Scratch assay was performed after culture for 24 h, and residual scratch width was observed at post scratch hour (PSH) 0 (immediately), 24, and 48. Cell migration distance was measured at PSH 24 and 48. After culture for 24, 48, and 72 h, the number of living cell was counted using cell counter. (3) From June 2018 to June 2019, SVF-GEL was applied clinically to treat 15 patients with depressed scars on face, including 2 males and 13 females, aged 19 to 42 years. Survival condition of SVF-GEL and whether complications or not were observed 6 months after surgery. Before surgery and 6 months after surgery, depressed degree, color, and pliability of scar were compared. Vancouver Scar Scale (VSS) was employed to access color, vascularity, and pliability before surgery and 6 months after surgery, and total score was calculated. The number of patients with complete satisfaction or satisfaction was counted six months after surgery. Data were processed with analysis of variance of factorial design, paired samples t test, and Wilcoxon rank sum test.@*Results@#(1) The content of EGF in SVF-GEL group and negative control group was (316.6±12.8) and (3.4±0.6) pg/mL, and the content of VEGF in SVF-GEL group and negative control group was (568.67±12.19) and (4.93±0.16) pg/mL, with statistically significant differences between the two groups (t=48.777, 92.485, P<0.01). (2) Residual scratch widths of HSF and HaCaT in SVF-GEL group and control group were decreased gradually along with time elapse, in which those in SVF-GEL group at PSH 24 and 48 were less than those in control group. At PSH 24 and 48, cell migration distances of HSF and HaCaT in SVF-GEL group were more than those in control group (tHSF=-20.304, -43.516, tHaCaT=-15.060, -8.684, P<0.01). After culture for 24, 48, and 72 h, the number of living cell of HSF and HaCaT in SVF-GEL group was significantly more than that in control group (tHSF=-3.374, -6.809, -18.036, tHaCaT=-4.793, -6.028, -8.141, P<0.05 or P<0.01). (3) Six months after surgery, SVF-GEL grafted into patients survived well without complications, and depressed degree of scar ameliorated obviously with lightened pigmentation and softer texture as compared with before surgery. Compared with those before surgery, VSS scores of color, vascularity, and pliability, and total score of 15 patients with depressed scars on face were obviously decreased 6 months after surgery (Z=-2.06, -2.07, -2.07, t=-15.811, P<0.05 or P<0.01). One patient was satisfied with the clinical outcome, and the rest 14 patients were completely satisfied with the clinical outcomes.@*Conclusions@#SVF-GEL contains cytokines EGF and VEGF, which can enhance cell migration ability and proliferation ability of HSF and HaCaT cells and have obvious effects on depressed scars for clinical application.
ABSTRACT
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.
ABSTRACT
OBJECTIVE： To optimize the water extraction technology of Qiwei chanshen formula. METHODS： The content of polysaccharides from Gekko japonicus and Panax quinquefolium was determined by UV spectrophotometry， the contents of camptothecin from Camptotheca acuminata and ginsenoside Rb1 from P. quinquefolium were determined by HPLC. The extraction rate of above three components， dry extractum yield and HPLC fingerprint similarity were used as evaluation indicators； information entropy theory was used to determine the weight of each indicator so as to calculate comprehensive score. L9（34） orthogonal test was used to screen the optimal extraction technology of Qiwei chanshen formula with decoction time， water volume and decoction times as factors. Validation test was also performed. RESULTS： The optimal extraction technology included 10-fold water， decocting for 3 times， 1.0 h each time. The results of validation test showed that the average extraction rate of polysaccharide， camptothecin and ginsenoside Rb1 were 74.306％， 13.860％ and 52.958％， respectively. The average dry extractum yield was 16.150％， the average value of fingerprint similarity was 0.991 （all RSDs＜3.0％， n＝3）. CONCLUSIONS： The optimized extraction technology is reproducible， stable and feasible， providing reference for the subsequent development and industrial production of the formula.
ABSTRACT
OBJECTIVE： To investigate the improvement effects of external application of water extract from Picrasma quassioides on allergic contact dermatitis （ACD） model mice， and to provide reference for its further development and utilization. METHODS： A total of 48 mice were randomly divided into blank control group （normal saline）， model group （normal saline）， hydrocortisone group （positive control， about 0.15 g/ear） and water extract from P. quassioides low-dose， medium-dose and high-dose groups （0.15， 0.3， 0.6 g/ear， calculated by extract）， with 8 mice in each group. The mice were given medicine on ear at 8 a.m. and 4 p.m. for 7 d continuously. Except for blank control group， other groups were given acetone-olive oil solution containing 0.5％ 2，4-dinitrofluorobenzene （DNFB） on the abdomen of mice to induce ACD model. After 7 d medication， modeling group was given acetone-olive oil of 0.25％ DNFB on left ear to induce dermatitis. 24 h later， the pathological changes of left ear tissue were observed by naked eye and skin lesion was scored. The thickness difference and weight difference of left and right ears in mice were measured. The serum contents of IL-6 and IgE were determined. The pathomorphology changes of ear tissue were observed by microscope after HE staining. RESULTS： Compared with blank control group， the skin lesion score of left ear was increased significantly in model group （P＜0.01）； the thickness difference and weight difference of left and right ears， serum contents of IgE and IL-6 were increased significantly （P＜0.01）. Pathological changes were observed in left tissue of mice by microscope， such as lymphocytic infiltration， intercellular edema， erythrocyte exudation. Compared with model group， skin lesion score of left ear in mice was decreased significantly in hydrocortisone group and water extract from P. quassioides high-dose group （P＜0.05 or P＜0.01）； the thickness difference and weight difference of left and right ears in mice were decreased significantly in all administration groups （P＜0.01）. The serum contents of IgE and IL-6 in mice were decreased significantly in hydrocortisone group， water extract from P. quassioides medium-dose and high-dose groups （P＜0.01）； the inflammatory lesions of left ear in mice were alleviated to varying degrees in different administration groups. CONCLUSIONS： External application of water extract from P. quassioides has a good improvement effect on ACD model mice.
ABSTRACT
BACKGROUND: Many studies have shown that cell-assisted lipotransfer promotes the survival of fat implants, but there are few studies exploring stromal vascular fraction (SVF) -assisted fat transplantation for improving facial skin quality. OBJECTIVE: To evaluate the clinical effect of autologous adipose-derived SVF-assisted autologous fat transplantation in the improvement of facial skin quality. METHODS: From September 2016 to June 2018, in the Plastic and Cosmetic Center, the Affiliated Hospital of Xuzhou Medical University, 20 patients were recruited and then randomly divided into experimental group and control group, namely SVF-assisted fat transplantation and simple fat transplantation, respectively. A VISIA skin detector was used to detect facial skin quality of patients preoperatively and 6 months postoperatively, including spots, wrinkles, texture, pores, ultraviolet spots, brown spots, couperose skin and porphyrin. RESULTS AND CONCLUSION: The effect of improvement in wrinkles and texture in the experimental group was better than that of the control group, and the difference was statistically significant (P < 0.05). In terms of spots, pores and couperose skin, the therapeutic effect in the experimental group was better than that in the control group, but the difference was not statistically significant (P> 0.05). There was no significant difference between the two groups in the improvement of ultraviolet spot and brown spots. The porphyrin was not affected by the subjects. To conclude, SVF can promote the effect of fat transplantation in the improvement of facial skin quality.
ABSTRACT
BACKGROUND: Many studies have shown that cell-assisted lipotransfer promotes the survival of fat implants, but there are few studies exploring stromal vascular fraction (SVF) -assisted fat transplantation for improving facial skin quality. OBJECTIVE: To evaluate the clinical effect of autologous adipose-derived SVF-assisted autologous fat transplantation in the improvement of facial skin quality. METHODS: From September 2016 to June 2018, in the Plastic and Cosmetic Center, the Affiliated Hospital of Xuzhou Medical University, 20 patients were recruited and then randomly divided into experimental group and control group, namely SVF-assisted fat transplantation and simple fat transplantation, respectively. A VISIA skin detector was used to detect facial skin quality of patients preoperatively and 6 months postoperatively, including spots, wrinkles, texture, pores, ultraviolet spots, brown spots, couperose skin and porphyrin. RESULTS AND CONCLUSION: The effect of improvement in wrinkles and texture in the experimental group was better than that of the control group, and the difference was statistically significant (P < 0.05). In terms of spots, pores and couperose skin, the therapeutic effect in the experimental group was better than that in the control group, but the difference was not statistically significant (P> 0.05). There was no significant difference between the two groups in the improvement of ultraviolet spot and brown spots. The porphyrin was not affected by the subjects. To conclude, SVF can promote the effect of fat transplantation in the improvement of facial skin quality.
ABSTRACT
Objective@#The purpose is to explore the method and clinical effects of total nasal reconstruction with the assistance of three-dimensional (3D) scanning, 3D printing and monitoring the blood circulation after operation.@*Methods@#3D scanning: Artex Eva 3D scanner was used to record the nose data of 500 volunteers from Xuzhou Medical University and its affiliated hospital from September 2016 to February 2017. A nose database of normal individuals was established, of which male was 138 and female was 362. In addition, 3D facial scanning was performed in patients wish to total nasal reconstruction. 3D printing: The individualized nasal structure was designed, with the assistant of patients′facial characteristics, combined with the normal nose database and the opinion of the patients. Anactual nose model was used as guidance during the operation. Postoperative monitoring: The blood flow and the retraction rate of forehead flap after surgery were measured using Laser Doppler Flowmeter and Geomagic Qualify software. The blood flow values, the temperature and the surface area of the flap were recorded and analyzed.@*Results@#The nasal database of normal people in the Huaihai region successfully established. Overall, the width of the nose takes up a quarter of the width of the faces, and the length is 1/3 of the distance from the hairline to the chin. From February 2017 to June 2018, 7 cases underwent total nasal reconstruction operations were performed by this procedure. The nasal models were all successfully printed out, as the guide of flap taken during the operation. The mean operation time of the cases was (2.45±0.75) h, and the follow-up time was 5-15 months, with an average of 12.5 months. After the operations, the retraction rate of the forehead flap were (21.8±2.72)% in one month, and (29.1±1.82)% in six months. All patients are satisfied with the nasal appearance.@*Conclusions@#Nasal reconstruction with forehead flap based on 3D scanning and 3D printing, provides objective targets for nasal fine-structure in a noninvasive way. The postoperative monitoring of the blood flow promotes the successful completion of the total nose reconstruction.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Objective · To investigate whether the quality of embryos will result in biochemical pregnancy or arrest of embryo development in the freezing and thawing cycles of in-vitro fertiliazation-embryo transfer (IVF-ET). Methods · The clinical data of patients who accepted IVF-ET in Center of Reproductive Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from January 2015 to June 2016 were retrospectively studied. The data includes 115 cycles of biochemical pregnancy, 64 cycles of arrest of early embryonic development and 871 cycles of ongoing pregnancy after frozen thawed embryo transfer. We compared the embryo score on the third day after embryo transfer (D3), the blastocyst development rate and the blastocyst grade in the three groups. Results · There were no significant differences in the period of infertility, the age of the patients and their spouses, the endometrial thickness, the estrogen and progestogen levels of the day of transplantation among the three groups (P > 0.05). The scores of most frozen thawed embryos on D3 were from 6 to 8, and the scores were not statistically significant among the three groups (P > 0.05). The proportion of transplanted blastocyst on D5 was higher than that on D6 in the three groups, but there was no significant difference among the three groups (P > 0.05). There was no significant difference in the proportion of inner cell mass of blastocysts which were scored as Grade A&B or Grade C among the three groups. Nevertheless, in the arrest of early embryonic development group, the proportion (52.2%) of the trophoblast of blastocysts which were cored as Grade C was significantly higher than the proportion (35%) in biochemical pregnancy group and the proportion (29.3%) in ongoing pregnancy group (P<0.05). Conclusion · The quality of embryos is not necessarily related to biochemical pregnancy, but the score of trophoblastic may be related to the arrest of early embryo growth.
ABSTRACT
Objective To detect the characteristics and in vitro cell compatibility of human acellular dermal matrix (ADM) with the improved method.Methods Cell components of healthy human skins were removed by the improved method and the traditional method respectively.The porosity, degradation time in vitro of the ADM prepared by two methods and the cytotoxicity of the material infiltration liquid with the improved method on the adipose derived stem cells were detected.HE staining was used to detect the residual of the cells, the integrity of collagen and cell biocompatibility.Scanning electron microscopy (SEM) was used to detect the pore size.Results Both the two methods could completely remove the cells, and maintain the integrity of the collagen scaffold;The porosity of ADM with the improved method was higher (93.1±1.02)% than that of traditional method (74.27±2.04)% (P<0.05);There was no significant difference in the cytotoxicity and in vitro degradation time between the two kinds of ADM;While pore diameter of the improved method was significantly higher [(181.21±66.9) μm] than that [(102.38±15.63) μm] in dermal reticular surface with the traditonal method (P<0.05).Conclusions There is no obvious cytotoxicity of the ADM with the improved method, and therefore it is more suitable for cell adhesion growth with higher porosity and larger pore size.
ABSTRACT
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.
ABSTRACT
Sodium humate (HA-Na) has been topically used as a wound healing and anti-inflammatory agent in folk medicine. In the present study, HA-Na was investigated for cutaneous wound healing in Sprague-Dawley rats. HA-Na solution (1.0%, w/v) was topically administered to rats undergoing excision wound models. Healing was assessed with a recombinant bovine basic fibroblast growth factor for external use as positive control. Wound healing rates were calculated on Day 3, 6, 9, 14 and 21 after injury, and tissues were also harvested after the same intervals for histological analysis. In addition, tissue hydroxyproline levels were measured. Furthermore, mRNA levels and protein expressions of transforming growth factor-β1, 2, 3 (TGF-β1, 2, 3) were determined by RT-PCR and western blot. Protein expression levels of Smad-2, -3, -4 and -7 were also detected by western blot. Our study demonstrates that HA-Na has the capacity to promote wound healing in rats via accelerated wound contraction and increased hydroxyproline content. More importantly, these wound healing effects of HA-Na might be mediated through the TGF-β/Smad signaling pathway. HA-Na may be an effective agent for enhanced wound healing.
ABSTRACT
Objective To study the effect of different draining way in the surgical treatment of chronic subdural haematoma (CSDH). Methods 126 patients with CSDH diagnosed by CT scanning or MRI were enrolled. The burr-hole was made at the site of the maximal hematoma thickness. A drainage tube was placed towards either the occipital (group A) or frontal ( group B) region as far as possible respectively, and for the latter, a lateral aperture was made about 2 cm far from the burr-hole on the side of the tube. The surgical complication (such as pneumocephalus, brain injury and recurrent hematoma et al.) and therapeutic efficacy of the two treatments were compared and analyzed to evaluate an optimal surgical technique. Results After treatment, complication rate in group A is 30%, the occurrence of complications in group B rate is 2% (P <0.05); operation time and hospitalization time in group B was significantly shorter than that in group A (P <0.05). Conclusion In the surgical treatment of CSDH, improved drainage pathway is safe and effective, which could improve the treatment efficacy and is worthy of clinical promotion.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Objective To investigate the changes and significance of serum anti-CCP (ACCP)antibody,matrix metalloprotein-ase-3 (MMP-3 ),interleukin-17 (IL-17 ),interleukin-18 (IL-18 )in the patients with rheumatoid arthritis (RA).Methods The ELISA method was adopted to detect the in the peripheral blood serum ACCP antibody,MMP-3,IL-17 and IL-18 in 80 patients with RA,32 patients with osteoarthritis (OA)and 32 cases of healthy controls and the detection results were performed the statis-tical analysis.Results The ACCP antibody,MMP-3,IL-17 and IL-18 levels in the RA group were significantly higher than those in the OA group and the healthy control groups;the ACCP antibody,MMP-3,IL-17 and IL-18 levels in the low,middle and high RA activity groups were higher than those in the stable group,the ACCP antibody,MMP-3,IL-17,IL-18,disease activity score (DAS28),C-reactive protein (CRP)and erythrocyte sedimentation rate(ESR)in the RA group were increased;the MMP-3,IL-17, IL-18 and DAS28 in the ACCP antibody positive group were higher than those in the A CCP antibody negative group;the positive correlation existed among the ACCP antibody,MMP-3,IL-17 and IL-18 in the RA group (P <0.05);the ACCP antibody,MMP-3, IL-17 and IL-18 were positively correlated with the monitoring indicators of CRP and DAS28 in the low,middle and high RA activi-ty groups.Conclusion MMP-3,IL-17 and IL-18 participate in the occurrence and development process of RA;The detection of ser-um ACCP antibody,MMP-3,IL-17 and IL-18 has a certain value in the judgment of disease activity,and prevention and treatment in the patients with RA.
ABSTRACT
Objective:To investigate the expression of miR-16,miR-17,miR-30a etc in peripheral blood plasma ,mononuclear cells ( PBMC) and synovial fluid of patients with rheumatoid arthritis ( RA) and its clinical significance ,to provide a theoretical basis for the further research in the mechanism of RA.Methods:Collected 80 cases of RA patients ,32 cases of osteoarthritis ( OA) patients and 32 healthy human peripheral blood ,synovial fluid ,separating the plasma and PBMC;extraction of small RNA ,with specific stem loop primed reverse transcription into cDNA , establishing a mature miRNA-T carrier and making standard curve;stem-loop method Real-time quantitative PCR was adopted to detect the expression of miRNAs in plasma ,PBMC and synovial fluid,and for correlation analysis;and with RA activity monitoring indicators rheumatoid factor (RF),erythrocyte sedimentation rate (ESR),C-reactive protein (CRP) correlation analysis.Results:In RA group,the expression of miR-16,miR-17,miR-30a,miR-106,miR-101 and miR-130 in plasma,PBMC and synovial fluid were upregμlation compared with OA group,the healthy control group (P<0.05),but the expression level of miR-101 in plasma of RA and OA -group difference was not statistically significant.Correlation analysis showed that 6 kinds of miRNA in the plasma ,PBMC and joint fluid have varying degrees of positive correlation ( P<0.05 ).Correlation analysis showed that miRNA in plasma , PBMC and synovial fluid have one or more indicators of a positive correlation with RF , ESR, CRP ( P<0.05 ).Conclusion:The expression of miR-16,miR-17,miR-30a,miR-101,miR-106 and miR-130 is changes significant in the peripheral blood and synovial fluid of RA patients ,indicate the miRNAs may be through some related genes play an important role in the process of the pathogenesis of RA;its expression level can be used as an effective indicator of RA disease activity , and provide new diagnostic markers for RA.