Construction of tissue-engineered skin with epidermal stem cells and de-epidermized dermis / 中华皮肤科杂志

Objective To construct tissue-engineered skin via in vitro inoculation of epidermal stem cells(ESCS)onto de-epidermized dermis.Methods Skin tissue was obtained from the foreskin of a healthy 6-year-old child.and keratinocytes were isolated by two-step trypsinization method followed by the collection of ESCS via rapid adhesion by collagen Ⅳ.The ESCS were identified by morphological observation and immunohistochemical staining with K19 and integrin β1.To construct tissue-engineered skin,selected ESCS were seeded onto the surface of de-epidermized dermis followed by a one-week culture immersed in the medium and a subsequent 4-week culture at the air-medium interface.The tissue-engqneered skin was evaluated with haematoxylin & eosin(HE)staining as well as keratin immunohistochemistry.Results Micro scopically,cultured ESCs showed a paving stone-like appearance and grew into colonies.Immunohistochemistry revealed that the ESCs were positive for integrin-β1 and keratin 19.After 5 weeks of culture,3-6 layers of epidermal cell were observed on the dermis with the formation of stratum corneum.Keratin protein was observed in the artificial epidermal skin.Conclusion Tissue-engineered skin is successfully constructed with epidermal stem cells and de-epidermized dermis in vitro.

Effects of uric acid in different concentrations on the proliferation of human umbilical vein endothelial cells in vitro / 中国组织工程研究

BACKGROUND:Recently, there is more and more attention on uric acid (UA), especially the correlation with cardiovascular disease in the filed of epidemiology; however, researches of the effect of UA on endothelial cells are lack based on cytology.OBJECTIVE:To explore the effects of UA in different concentrations on human umbilical vein endothelial cells line (ECV304), malondialdehyde (MDA), nitric oxide (NO) and 3-(4,5-dimethyl thiazole-2)-2, 5-diphenyl thiazolyl blue (MTT)in vitro.DESIGN: Randomized controlled study based on ECV304.5ETTING: Department of Endocrine, Xijing Hospital, the Fourth Military Medical University of Chinese PLA.MATERIALS:ECV304 was provided by Department of Immunology, the Fourth Military Medical University of Chinese PLA; DMEM Iow-glucose medium by Gibco Company, USA; bovine serum by Beijing Yuanheng Shengma Biotechnology Researching Institute; trypsin by Gibco Company, USA; MTT by Huamei Company; dimethyl sulfoxide (DMSO) analytical pure by Tianjin Bodi Chemical Engineering Co. Ltd.; UA by Sigma Company; NO kit and MDA kit by Nanjing Jiancheng Company; ordinary invert microscope and enzyme-linked immune detector IX70 invert microscope by Olympus, Japan;enzyme-linked immune detector by Eastern China Electron Tube Factory.METHODS: The experiment was carried out in Department of Endocrine and Burning, the Fourth Military Medical University of Chinese PLA from December 2003 to April 2004. Resuscitation, culture, regeneration and inoculation of endothelia cells were undertaken Cell Culture published by Situ et al. Endothelia cells were cultured with non-serum DMEM for 24hours so as to maintain synchronization at the phase of G0/G1 and divided into 4 groups, including control group, low-concentration UA group, moderate-concentration UA group and high-concentration UA group. Each group was divided into 3time points, including 24 hours, 48 hours and 72 hours with 8 samples in each time point. Samples were added with 5％serum medium containing 0 mmol/L, 0.1 mmol/L, 0.2 mmol/L and 0.4 mmol/L UA in control group, low-concentration UA group, moderate-concentration UA group and high-concentration UA group, respectively, and incubated in box with the volume fraction of 0.05 CO2 at 37 ℃. Twenty-four hours later, MDA and MTT were detected; additionally, MTT was detected once more after 48 and 72 hours.MAIN OUTCOME MEASURES: Proliferation of ECV304 and content of NO and MDA.RESULTS:With the increasing concentration of UA, content of MDA was decreased to (2.97±0.05),(2.89±0.09),(2.78±0.10) and (2.44±0.03) μmol/L, respectively; content of NO was (6.86±1.41), (12.5±2.7), (18.9±1.8) and (21.1±1.4) μ mol/L. Absorbencies of NO and MTT were increased and proliferation was increased remarkably at 48 hour.CONCLUSION: A which is characterized by anti-oxidation may promote proliferation of vascular endothelia cells and release of NO.

Effect of laminin and type Ⅳ collagen on proliferation of Schwann cells / 中国组织工程研究

BACKGROUND: Schwann cells play an important role in regeneration of peripheral nerves and the extracellular matrix(ECM) is also important to growth of Schwann cells.OBJECTIVE: To explore the effects of laminin and type Ⅳ collagen on the adhesion and proliferation of Schwann cells.DESIGN: A controlled trial in groups with Schwann cell as subject.SETTING: Plastic Surgery Laboratory of the Fourth Military Medical University of Chinese PLA.MATERIALS: The trial was conducted in the Plastic Surgery Laboratory of Xijing Hospital Affiliated to the Fourth Military Medical University of Chinese PLA from January through April in 1995. The Schwann cells were extracted from newborn rabbits.METHODS: The culture plates were coated with laminin and type Ⅳ collagen. While in the control group, the antibody of laminin and collagen Ⅳ,collagen Ⅰ and polylysine were added respectively. The Schwann cells of the same concentration were cultured in plates of each group. The cells attachment rate in each group ,was determined after 2, 6, 24 hours respectively. After 72 hours 3H-TdR were incorporated into culture matrix fluid and double channel liquid scintillation counter was used for measurement of radiactivity count per minute.MAIN OUTCOME MEASURES: ① Attachment rate of Schwann cell. ②3H-TdR counting.RESULTS: The attachment rates in laminin and collagen Ⅳ groups (66%and 59% ) were higher than those of the type Ⅰ collagen and polylysine groups(45% and 43% ) after 2-hour culturing. Those in laminin antibody and collagen Ⅳ antibody groups were lower. The incorporation value were (10.0±2.7)×103, (1.3±0.3)×103, (10.4±2.4)×103, (1.4±0.5) ×103, (5.8±2.7)×103, (3.3±1.0)×103 per minute respectively in laminin, laminin antibody, collagen Ⅳ, collagen Ⅳ antibody, collagen Ⅰ and polylysine groups. The incorporation values in laminin and collagen Ⅳgroups were higher than controls.CONCLUSION: Laminin and type Ⅳ collagen promote the attachment and proliferation of Schwann cells in vitro. This study provides experimental basis for applying the action of Schwann cell to the nerve tissue engineering.

The manufacturing and clinical application of heterogenous acellular dermal matrix / 中华烧伤杂志

<p><b>OBJECTIVE</b>To lower down the antigenicity of heterogenous swine acellular dermal tissue, and to explore the feasibility of clinical using it as a composite graft for human patients.</p><p><b>METHODS</b>Split-thickness skin was harvested from healthy swines and then processed by two methods. The swine acellular dermal matrix (sADM) was prepared by removing cells from the skin with trypsin and Triton X-100. Then the cross-linked sADM (sADM(1)) and non-cross-linked sADM (sADM(0)) were embedded subcutaneously in rabbits and also transplanted onto the burn wounds of patients. The histological changes and also transplantation results were observed.</p><p><b>RESULTS</b>(1) In animals with sADM(0) embedded subcutaneously, the grafted tissue was invaded immediately by host cells with obvious inflammatory reaction and tissue degradation. But there was less inflammatory reaction, and with no obvious skin degradation and contraction with sADM(1). (2) In ten burn patients with III degree burn wounds and one patient with wound in chest after scar removal, sADM and ultra-thin skin (UTS) composite graft were grafted on the wounds with autologous thin skin (ATS) and autologous razor-thin or UTS as the control. Nineteen pieces of composite skin of sADM with UTS were grafted on the wounds with survival rate of 78.9%, exhibiting no evident difference with that of ATS. When sADM(0) and UTS were grafed, there exhibited remarkable early inflammatory reaction and wound contraction with similar external appearance with that of UTS. Whereas when sADM(1) and UTS were grafted, there appeared less early inflammatory reaction and wound contraction, resulting in an even appearance and soft to touch similar to that with ATS. But ulceration occurred, with exposure of sADM(1), exposure and severe macrophage reaction to foreign body in 6 wounds of 3 cases 12.8 +/- 6.9 weeks after sADM(1) and UTS grafting.</p><p><b>CONCLUSION</b>Grafting of sADM as a dermal substitute of composite skin could alleviate early post-grafting immune reaction and improve UTS grafting results. But the delayed graft rejection couldn't be avoided.</p>

Substance P up-regulates the TGF-beta 1 mRNA expression of human dermal fibroblasts in vitro / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the role of substance P in the formation of hypertrophic scar.</p><p><b>METHODS</b>Dermal fibroblasts derived from human normal skin were cultured with substance P alone or together with selective non-peptide NK-1 tachykinin antagonist, L-703, 606 oxalate salt. The effect of substance P on proliferation of fibroblasts was measured by MTT assay. Furthermore, the TGF-beta 1 mRNA expression in the fibroblasts was determined by in situ hybridization and image analysis.</p><p><b>RESULTS</b>Substance P enhanced fibroblast proliferation dose-dependently, which showed the maximum rate when the concentration of substance P was 25 ng/ml or higher in the culture media. By 48 hours cultured with 25 ng/ml of substance P, the fibroblasts expressed TGF-beta 1 mRNA more significantly than the fibroblasts without substance P. The effects of substance P on both fibroblast proliferation and TGF-beta 1 mRNA expression could be antagonized by L-703, 606 oxalate salt.</p><p><b>CONCLUSION</b>The results suggest that substance P may play an important role in phenotype changes of fibroblasts in skin scarring.</p>

Influential factors on the efficiency in transfecting human keratinocytes with plasmid-liposome complexes / 基础医学与临床

To investigate the optimum transfection condition in transfecting human keratinocytes with plasmid-liposome complexes in vitro,the cultured human keratinocytes at 60% ～ 100% confluences were exposed to the eukaryotic expression plasmid,pCMV*SPORT-β-gal,coated with LipofectAMINE in different DNA/liposome mixing concentration ratios.After cultured for another 48 hours following the ends of 6 ～ 24 hours exposures to the DNA-liposome complexes,the transfected human keratinocytes were visualized by β-galactosidase staining.Then,the transfection efficiency was determined by calculating the rate of β-galactosidase staining positive cells.β-galactosidase expression was showed clearly in human keratinocytes transfected with the DNA-liposome complexes.The highest efficiency was achieved with cultured cells at 80% and 90% confluences,demonstrating by the transfection rates of (31.35±1.35)% and (32.32±2.47)% respectively.Meanwhile,the essential transfection conditions for these efficiencies were in coating pCMV*SPORT-β-gal DNA of 1.5μg/100μL with LipofectAMINE of 12.5μL/100μL,and exposing the cells to the DNA-liposome complexes for 8 hours.These results indicate that LipofectAMINE could effectively transfer eukaryotic expression plasmid into human keratinocytes in vitro,for which the optimization of transfection conditions involve in cells growth state,DNA/liposome mixing concentration ratio,and exposure time of cells to DNA-liposome complexes.

Effects of bFGF on hypertrophic scar in a nude mouse model / 中国普通外科杂志

ObjectiveTo investigate the effects of bFGF on human hypertrophic scar in a nude mouse model. MethodsHuman hypertrophic scars excised in operation for burned patiens were grafted onto the back of nude mice subcutaneously and the mice were randomly divided into: control group, collagenase group, bFGF group, and bFGF plus collagenase group. Two weeks after grafting, preparations were injected into the scars for three times. The size, hardness, and morphological changes of the scars were examined. Biopsies were done 3 weeks after first injection and the histological changes were examined. ResultsIt was found that in bFGF group the size of the grafted scars reduced to 1/2～1/3 of their original size, the majority of the coarse and dense scar collagen bundles resolved and became soft loosed lump. In bFGF plus collagenase group, two thirds of the grafted scar disappeared. ConclusionTopical injection of bFGF into hypertrophic scar degrades scar collagen.

Effects of calcitonin gene related peptide on cultured human umbilical vein endothelial cells following anoxia and reoxygenation / 中国病理生理杂志

AIM: To investigate the effects of calcitonin gene-related peptide (CGRP) on cultured anoxic-reoxygenation human umbilical vein endothelial cells. METHODS:In the present experiment, an anoxic-reoxygenation model was established by using cultured human umbilical vein endothelial cells. The uptake rate of trypan-blue and calcium and magnesium contents of endothelial cells and lactic dehydrogenase (LDH) activity in medium and malondialdhyde (MDA) content of endothelial cells were measured 60,120 and 180 min after anoxia and 30 and 60 min after reoxygenation, and the effects of 1?10 -8 mol/L CGRP on anoxic-reoxygenation endothelial cells were studied. RESULTS: The findings showed that, as anoxia prolonged, the uptake rate of trypan-blue and LDH activity and MDA content gradually elevated and, during reoxygenation, these parameters sharply increased. Calcium and magnesium levels gradually declined as anoxia prolonged, and during reoxygenation calcium content significantly increased. Meanwhile, 1?10 -8 mol/L CGRP might significantly reduce the uptake rate of trypan-blue and LDH activity and MDA content during anoxia and reoxygenation and lessen the increase in calcium content and the loss of magnesium during reoxygenation. CONCLUSION: These results showed that CGRP might have a direct protective function to endothelial cells afflicted with anoxic-reoxygenation injury by inhibiting lipid peroxidation, attenuating calcium overload and loss of magnesium and enzyme.