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Tao R.,General Hospital of PLA | Han Y.,First Hospital Affiliated to General Hospital of PLA | Chai J.,First Hospital Affiliated to General Hospital of PLA | Li D.,First Hospital Affiliated to General Hospital of PLA | Sun T.,First Hospital Affiliated to General Hospital of PLA
Cytotechnology | Year: 2010

Human sweat gland epithelial cells (SGECs) have been isolated and grown in vitro, However, slow proliferation makes the culture of these cells extremely difficult. The present study was carried out to explore the modified culture medium for SGECs in vitro. Full-thickness skin samples were minced (1 mm 3) and digested overnight with type II collagenase. The gland coils were removed under an inverted phase-contrast microscope. An adherent culture method was used to isolate and culture SGECs. Staining with hematoxylin and eosin was performed, followed by observation of the morphologic features of these cells. Immunofluorescence staining with antibodies to cytokeratins CK7, CK18, and CK19 and carcinoembryonic antigen (CEA) was performed to verify the presence of SGECs. Growth curves by MTT were created for cells grown in serum-free keratinocyte medium and in modified keratinocyte medium containing 2.5% fetal bovine serum (FBS). One week after culturing, the cells grew well and were polygonal or irregular in shape by inverted phase contrast microscopy. Cell fusion, with a characteristic paving-stone arrangement, reached 100% after approximately 3 weeks in culture. Immunofluorescence staining indicated expression of CK7, CK18, CK19, and CEA. Compared with SGECs grown in serum-free keratinocyte medium, the proliferation of SGECs grown in modified culture medium with low concentration of FBS at days 6, 9, and 12 was significantly accelerated (p < 0.05). This study suggests that keratinocyte medium supplemented with 2.5% FBS is effective and suitable for the culture of SGECs. © 2010 Springer Science+Business Media B.V.


Huang S.,Chinese PLA General Hospital | Yao B.,Nankai University | Xie J.,Tianjin Medical University | Fu X.,First Hospital Affiliated to General Hospital of PLA
Acta Biomaterialia | Year: 2015

Sweat glands perform a vital thermoregulatory function in mammals. Like other skin appendages, they originate from epidermal progenitors. However, they have low regenerative potential in response to injury, and whether adult epidermal progenitors could be specified to differentiate to a sweat gland cell lineage remains largely unexplored. We used bioprinting technology to create a functional in vitro cell-laden 3D extracellular matrix mimics (3D-ECM) with composite hydrogels based on gelatin and sodium alginate because of chemical and structural similarity to ECM components. To achieve specific cell differentiation, mouse plantar dermis and epidermal growth factor were synchronously incorporated into the 3D-ECM mimics to create an inductive niche for epidermal progenitor cells obtained from mice. The biological 3D construct could maintain cell viability, thereby facilitating cell spreading and matrix formation. In vitro data by immunofluorescence and gene expression assay of key cell-surface markers demonstrated that the bioprinted 3D-ECM could effectively create a restrictive niche for epidermal progenitors that ensures unilateral differentiation into sweat gland cells. Furthermore, direct delivery of bioprinted 3D-ECM into burned paws of mice resulted in functional restoration of sweat glands. This study represents the rational design to enhance the specific differentiation of epidermal lineages using 3D bioprinting and may have clinical and translational implications in regenerating sweat glands. Statement of Significance: Sweat gland regeneration after injury is of clinical importance but remains largely unsolved because of low regenerative potential and lack of a definite niche. Some studies have shown sweat gland regeneration with gene-based interventions or cell-based induction via embryonic components, but translation to clinic is challenging. The novelty and significance of the work lies in the fact that we design a 3D bioprinted extracellular matrix that provides the spatial inductive cues for enhancing specific differentiation of epidermal lineages to regenerate sweat glands, which is critical for treating deep burns or other wounds. Our studies are encouraging given the overwhelming advantages of our designed 3D bioprinting construct over other cell delivery technology in maintaining high cell proliferation; another interesting finding is that adult tissue components retain a gland lineage-inductive power as embryonic tissue, which can facilitate translation. © 2015 Acta Materialia Inc.


Ma L.,First Hospital Affiliated to General Hospital of PLA | Shen C.,First Hospital Affiliated to General Hospital of PLA | Chai J.,First Hospital Affiliated to General Hospital of PLA | Yin H.,First Hospital Affiliated to General Hospital of PLA | And 2 more authors.
Shock | Year: 2015

Background: Testosterone and androgen receptor agonists have been known for a long time to prevent or reverse muscle wasting in burn injury patients, but the exact molecular mechanisms are not clear. Objective: To investigate the underlying molecular mechanisms of testosterone in severely burned rats. Methods: Severe burn injuries were induced by immersing the back of the rat in 100-C water for 12 s. Rats were treated for 14 days with vehicle (burn group) or a physiological replacement dose of testosterone (B + T group) immediately after injury. Gene and protein expressions were assessed by real-time polymerase chain reaction and Western blot. Results: Testosterone improved glucose metabolism, reduced body weight loss, and attenuated tibialis anterior muscle mass loss and muscle protein breakdown. In rat tibialis anterior muscle, testosterone positively regulated the insulin-sensitive glucose transporters Glut3 and Glut4 genes and glycogen synthase 1 protein. These changes would be expected to improve glucose metabolism and nutrient availability in skeletal muscle. Administration of testosterone negatively regulated atrogin 1 (Fbxo32) by increasing total and phosphorylated Foxo3a (forkhead-box transcription factor 3a) levels and positively regulated the expression of the mammalian target of rapamycin (mTOR) and its downstream proteins p70S6 and S6 through mTORYextracellular signalYregulated kinase phosphorylation. Conclusions: Results suggested that testosterone might regulate skeletal muscle glucose and protein metabolism following burn injury in part by affecting extracellular signalYregulated kinaseYmTOR signaling and Foxo3a levels. © 2014 by the Shock Society.


Han Y.,First Hospital Affiliated to General Hospital of PLA
Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery | Year: 2010

To review the latest research progress of full-thickness tissue engineered skin (FTTES), to thoroughly understand its current state of research and application so as to lay a solid foundation for developing new type FTTES and improving the quality of skin substitutes. Domestica and international literature concerning FTTES in recent years was extensively reviewed and comprehensively analyzed. Some progress of FTTES had made in seed cells, scaffold materials and construction, and some therapeutic efficacy had also been achieved in clinical application. But FTTES grafting successful rate was lower, and it had no complete skin structure and had not reached the requirements of clinical application. FTTES is an ideal skin substitute and has great development prospects. However, in seed cells, scaffold materials, construction and applications of FTTES, further studied is still needed.


Du J.D.,First Hospital Affiliated to General Hospital of PLA
Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology | Year: 2011

To explore the effects of percutaneous transhepatic radiofrequency ablation (PRFA) combined with tumor edge of percutaneous absolute ethanol injection (PEI) on liver cancer adjacent to major blood vessels. Seventy five patients with liver cancer adjacent to major blood vessels were randomly divided into two groups: PRFA+PEI therapy group (38 cases) and PRFA control group (37 cases). Tumor necrosis rate, AFP levels, local recurrence rate, median for survival time and cum survival were used as the evaluation index to evaluate the efficacies of the two methods. Tumor necrosis rates of the therapy group and the control group were 84.2% and 54.1% (P < 0.01), respectively; AFP levels of therapy group and control group at 1, 3, 6 and 12 months after treatment were (105.0 ± 35.5) μg/L, (28.4 ± 4.3) μg/L, (58.6 ± 6.7) μg/L, (89.5 ± 12.5) μg/L and (137.2 ± 34.6) μg/L, (84.2 ± 18.4) μg/L, (106.6 ± 20.3) μg/L, (173.7 ± 32.0) μg/L, respectively. The rates of therapy group was significantly lower than of control group. Local recurrence rates of the therapy group and control group were 2.6%, 7.9%, 13.2% and 31.6% vs 10.8%, 21.6% , 40.5% and 62.1% (P < 0.05) at 3, 6, 12 and 24 months after treatment, respectively. Median for survival time of the therapy group and control group were 28.0 ± 2.8 months and 19.0 ± 3.6 months, respectively. Cum survival of the therapy group and control group were 84.2%, 78.9%, 60.5% and 31.6% vs 78.4%, 67.6%, 37.8% and 8.1% (P < 0.05) at 6, 12, 24 and 36 months after treatment, respectively. PEI as a supplementary treatment of PRFA can effectively improve the treatment of liver cancer adjacent to major blood vessels and significantly reduce the local recurrence rate and improve long-term survival rates.


Haijun Z.,First Hospital Affiliated to General Hospital of Pla | Yonghui Y.,First Hospital Affiliated to General Hospital of Pla | Jiake C.,First Hospital Affiliated to General Hospital of Pla | Hongjie D.,First Hospital Affiliated to General Hospital of Pla
Ulusal Travma ve Acil Cerrahi Dergisi | Year: 2015

BACKGROUND: Severe burn injuries are associated with a persistent hypermetabolic response, which causes long-term loss of muscle mass that results in a clinical negative balance of nitrogen and muscle wasting. MicroRNAs (miRNAs) play a critical role in posttranscriptional regulation of gene expression, which negatively regulates gene expression by promoting degradation of target mRNAs or inhibiting their translation. However, the mechanisms of skeletal muscle wasting after severe burn involved in miRNAs still remain unclear. METHODS: In this study, the alterations of miRNAs expression profile in skeletal muscles of thermal rats were detected at an early stage by microarray. All data were presented as mean±SD. Statistical analysis was determined by independent Student’s t-test and one- way ANOVA. The significance was all set at p<0.05, and fold change cut-off was 2.0 for microarray. Significant differentially expressed miRNAs were identified through Volcano Plot filtering. Hierarchical clustering was performed using MEV software (v4.6, TIGR). RESULTS: Thousands of miRNAs could be examined in normal and injured tissues, but only 69 of these were significantly upregulated or down-regulated, which could be used to discriminate skeletal muscles of thermal rats from matched tissues. CONCLUSION: The deregulated miRNAs probably play a potential role in the pathogenesis of skeletal muscle wasting in burn trauma. © 2015 TJTES


Zhu G.,First Hospital Affiliated to General Hospital of PLA | Chai J.,First Hospital Affiliated to General Hospital of PLA | Ma L.,First Hospital Affiliated to General Hospital of PLA | Duan H.,First Hospital Affiliated to General Hospital of PLA | Zhang H.,First Hospital Affiliated to General Hospital of PLA
Biochemical and Biophysical Research Communications | Year: 2013

MicroRNAs regulate a host of physiological and pathological processes in mesenchymal stem cells (MSCs), although no published studies describe changes in microRNA expression or function in MSCs under in vitro hyperglycemic conditions. By using a microRNA microarray approach, we have identified that miRNA-32-5p expression is significantly reduced under hyperglycemic conditions in rat bone marrow-derived MSCs. Expression of miRNA-32-5p targets the 3'-untranslated region of the mRNA encoding phosphatase and tensin homologs deleted on chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Exposure to high glucose levels reduced miR-32-5p expression, induced PTEN expression, and inhibited activation of the PI3K/Akt signaling pathway of MSCs. Conversely, overexpression of miR-32-5p inhibited the expression of PTEN, ameliorated the inhibitory effect of high glucose levels on the PI3K/Akt signaling pathway, and promoted cell cycle progression from G0/G1 to G2/M and S phases. Our study indicates that exposure of MSCs to hyperglycemic conditions reduces miR-32-5p expression and disturbs cell cycle progression through a PTEN-mediated inhibitory effect on the PI3K/Akt signaling pathway. In summary, MiR-32-5p is a potentially important therapeutic agent for preventing MSC dysfunction under hyperglycemic conditions. © 2013 Elsevier Inc.


Han Y.,First Hospital Affiliated to General Hospital of PLA | Chai J.,First Hospital Affiliated to General Hospital of PLA | Sun T.,First Hospital Affiliated to General Hospital of PLA | Li D.,First Hospital Affiliated to General Hospital of PLA | Tao R.,First Hospital Affiliated to General Hospital of PLA
Biochemical and Biophysical Research Communications | Year: 2011

Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18. days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue-engineered dermis. © 2011 Elsevier Inc.


Sun T.,First Hospital Affiliated to General Hospital of PLA | Han Y.,First Hospital Affiliated to General Hospital of PLA | Chai J.,First Hospital Affiliated to General Hospital of PLA | Yang H.,First Hospital Affiliated to General Hospital of PLA
Journal of Burn Care and Research | Year: 2011

Selectively decellularized split-thickness porcine skin (SDSTPS) may be an optimal alternative for allograft. This study was designed to explore the efficacy of microskin autografts overlaid with SDSTPS in the repair of deep burn wounds and to resolve the problem of the shortage and risk of cadaver skin allografts. Full-thickness xenogenic skin was harvested from a healthy ternary pig, and SDSTPS was produced by the glutaraldehyde-trypsin-detergent method. Split-thickness autograft skin was harvested from patients and minced into microskin autografts. The microskin autografts with overlaid SDSTPS were applied to 31 patients with deep burn wounds, 4 to 6 days after injury, and comparisons with cadaver skin allograft were carried out on both sides of the torso and limbs. The cases were followed up for 18 months. The following parameters were investigated: time of rejection and exfoliation, percentage of epithelialized wound area, number of cases with wound ulcer, hypertrophic scars, pain and itching, apparent deformity, and functional impairment. The rejection and exfoliation time of the skin xenograft was 17 ± 3 days and that of the skin allograft was 14 ± 2 days (P < .05), whereas the epithelialized wound area 3 weeks postoperatively for the skin xenograft and allograft was 87 ± 21% and 83 ± 41% (P > .05), respectively. There was no significant difference in skin morphology between the two groups. The satisfactory function was observed in the follow-up visit for 18 months postoperatively. The authors' results indicate that the clinical effect of microskin autografts overlaid with SDSTPS in the repair of deep burn wounds is similar to that of microskin autograft overlaid with frozen cadaver skin, and SDSTPS could be an optimal alternative for allograft. Copyright © 2011 by the American Burn Association.


Li L.,First Hospital Affiliated to General Hospital of PLA | Xia Y.,First Hospital Affiliated to General Hospital of PLA
Annals of Thoracic and Cardiovascular Surgery | Year: 2014

Background: Increasing evidences indicated that adipose-derived mesenchymal stem cells (ADMSCs) can stay survive, then gradually proliferate and differentiate into myocardial cells after transplanted into damaged areas and improve function of heart.Methods: In this article, ADMSCs were isolated from adipose tissue of Wistar rats and cultured. When treated with 5-azacytidine (5-aza), ADMSCs were differentiated into myocardial cells, then we implant these cells into myocardium of rats of DCM to observe cell population and differentiation and compare cardiac function and hemodynamics changes before and after transplantation.Results: The expression of Cardiac-specific markers indicated that ADMSCs which were isolated from adipose tissue of Wistar rats can differentiate into various cell types. Meanwhile, the treatment group displayed a higher level of LVESP, left ventricular intraventricular pressure (+dP/dt max), left ventricular intraventricular pressure (–dP/dt max) and left ventricular EF (%) than the control group. Altogether, these results indicate that heart systolic and diastolic function of rats of DCM was significantly improved meanwhile ventricular dilatation remodeling was inhibited after ADMSCs transplantation.Conclusions: Therefore, this research provides an experimental basis for further clinical application of ADMSCs transplantation for the treatment of DCM and non-ischemic HF. © 2014 The Editorial Committee of Annals of Thoracic and Cardiovascular Surgery. All rights reserved.

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