丹皮酚对TNF-α诱导真皮成纤维细胞MMP-9 mRNA及细胞因子表达的影响

目的通过研究丹皮酚对TNF-α诱导的真皮成纤维细胞MMP-9 mRNA及细胞因子表达的影响,探讨丹皮酚抗皮肤炎症的可能机制。方法采用RT-PCR法考察正常人真皮成纤维细胞中MMP-9 mRNA表达情况以及丹皮酚对TNF-α诱导的真皮成纤维细胞MMP-9 mRNA表达的影响。采用ELISA法测定丹皮酚对TNF-α诱导的真皮成纤维细胞表达IL-1β、IL-6及IL-8的影响。结果MMP-9在正常真皮成纤维细胞上表达极弱,TNF-α可以诱导MMP-9 mRNA的表达,丹皮酚可以抑制TNF-α诱导引起的MMP-9 mRNA表达的上调。成纤维细胞可分泌少量的IL-1β、IL-6及IL-8,TNF-α可以明显增加成纤维细胞IL-1β和IL-8的分泌量,丹皮酚可以抑制TNF-α诱导的IL-1β和IL-8的产生,但对IL-6的分泌无影响。结论丹皮酚可以抑制TNF-α诱导真皮成纤维细胞引起的MMP-9 mRNA及炎症因子IL-1β和IL-8表达水平的上调。

同一个体成骨细胞和牙周韧带成纤维细胞的原代培养

目的 :探索用简单 ,高效的方法对同一个体的成骨细胞和牙周韧带成纤维细胞进行原代培养 ,以便迅速建立相关的体外研究模型 ,为进一步研究两种细胞的相互作用奠定基础。方法 :实验用酶消化法结合组织块培养法获取大鼠牙周韧带成纤维细胞 ,用组织块移行生长法培养成骨细胞 ,并对细胞培养中取材的对象和部位、酶消化时间、污染的控制等进行探讨。结果 :发现所获得的细胞符合成骨细胞和成纤维细胞的形态特征和生物学特性 ,且生长良好。结论 :用酶消化法结合组织块培养法培养大鼠牙周韧带成纤维细胞 ,其方法可行。

皮肤组织工程

目的 综述近阶段皮肤组织工程研究领域中的进展。方法 广泛查阅国内外近期有关皮肤组织工程研究的文献 ,着重阐述表皮替代物 ,真皮替代物 ,培养的表皮真皮复合皮片的研究进展和重要问题。结果 多数学者认为理想的皮肤替代物应及时重建已丧失的表皮和真皮结构。目前的研究主要集中在如何尽早移植表皮细胞 ,并保持移植后细胞的活性和功能 ;以及研制能更有效地促进细胞功能 ,诱导创床血管长入 ,移植后可降解 ,无毒性 ,无病原携带风险的细胞外基质。结论 尽早移植表皮细胞 ,缩短创面愈合时间 。

肥大细胞在瘢痕疙瘩发病机理中作用的研究

为探讨肥大细胞在瘢痕疙瘩发病机理中的作用，用透射电镜观察活跃增生瘢痕疙瘩的超微结构。用组胺分别处理体外培养正常皮肤与瘢痕疙瘩成纤维细胞，采用3H-脯氨酸掺入、胃蛋白酶消化法检测其胶原合成量。结果：瘢痕疙瘩中有大量含有高度发达粗面内质网的成纤维细胞，还可见有脱颗粒的肥大细胞。瘢痕疙瘩成纤维细胞在胶原合成量已有增加的基础上，经一定浓度组胺作用，其胶原合成量仍有显著增加。结果示肥大细胞可有助于瘢痕疙瘩成纤维细胞胶原合成功能的活化及其活化表型的维持。

Molecular determinants of the profibrogenic effects of endothelin-1 in pancreatic stellate cells

AIM： To gain molecular insights into the expression and functions of endothelin-1 （ET-1） in pancreatic stellate cells （PSC）.METHODS： PSCs were isolated from rat pancreas tissue, cultured, and stimulated with ET-1 or other extracellular mediators. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2＇-deoxyuridine into DNA and cell migration was studied in a transwell chamber assay. Gene expression at the level of mRNA was quantified by real-time Polymerase chain reaction. Expression and phosphorylation of proteins were monitored by immunoblotting, applying an infrared imaging technology. ET-1 levels in cell culture supernatants were determined by an enzyme immunometric assay. To study DNA binding of individual transcription factors, electrophoretic mobility shift assays were performed.RESULTS： Among several mediators tested, transforming growth factor-β1 and tumour necrosis factor-α displayed the strongest stimulatory effects on ET-1 secretion. The cytokines induced binding of Smad3 and NF-κB, respectively, to oligonucleotides derived from the ET-1 promoter, implicating both transcription factors in the induction of ET-1 gene expression. In accordance with previous studies, ET-1 was found to stimulate migration but not proliferation of PSC. Stimulation of ET-1 receptors led to the activation of two distinct rnitogen-activated protein kinases, p38 and extracellular signal-regulated kinases （ERK）1/2, as well as the transcription factor activator protein-1. At the mRNA level, enhanced expression of the PSC activation marker, α-smooth muscle actin and two proinflammatory cytokines, interleukin （IL）-1β and IL-6, was observed. CONCLUSION： This study provides novel lines of evidence for profibrogenic and proinflammatory actions of ET-1 in the pancreas, encouraging further studies with ET-1 inhibitors in chronic pancreatitis.

Silk Fibroin-modified Poly(butadiene) urethane Films and Their Effects on Fibroblast Viability

Surface-modified poly(butadiene)urethane (PBTU) films with silk fibroin (SF) were prepared by simple chemical method under the normal temperature. The physical properties and biological behaviour of the SF-modified PBTU film were evaluated. The results showed that the SF-modified PBTU films kept the tenacity and pliability very well, and could overcome rigid and brittle weaks of silk fibroin films. The morphology of SF in the PBTU film was dendritic aggregations, and the water-contact angle measurement indicated that the surface hydrophilicity of modified films was apparently enhanced. The biocompatibility of PBTU films was improved due to the change of surface components. The degree of platelet adhesion and the cell viability of rat embryo dermal fibroblasts seeded on PBTU films, SF films, and SF-modified PBTU films were measured by counting platelets before and after they contacted the films and MTT assay, respectively. The results indicated that platelet adhesion resistance and cell viability on the modified film were greatly superior to those on the PBTU film and the compound interface had good stability in the air.

The Polypeptide in Chlamys farreri can protect human dermal fibroblasts from ultraviolet B damage

To investigate the effect of polypeptide from Chlamys farreri (PCF) on NHDF in vitro, we modeled oxidative damage on normal human dermal fibroblasts (NHDF) exposed to ultraviolet B (UVB). In this study, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydro-genase (LDH) were tested to measure cell viability. Enzymes including superoxide dismutase (SOD), glu-tathione peroxidase (GSH-PX), catalase (CAT) and xanthine oxidase (XOD) were determined biochemically. Total antioxidative capacity (T-AOC) and anti-superoxide anion capacity (A-SAC) were also determined. Ultrastructure of fibroblasts was observed under transmission electron microscope. The results showed that: UVB (1.176×10-4 J/cm2) suppressed the growth of fibroblasts and the introduction of PCF (0.25%-l%) before UVB reduced the suppression in a concentration-dependent manner. PCF could enhance the activities of SOD, GSH-PX and T-AOC as well as A-SAC. Also PCF could inhibit XOD activity, while it did not affect CAT activity. Ultrastructure of fibroblasts were damaged after UVB irradiation, concentration-dependent PCF reduced the destructive effect of UVB on cells. These results indicated that PCF can protect human dermal fibroblasts from being harmed by UVB irradiation via its antioxidant pro-erty.

Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or aftermalignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper wedemonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression inairway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targetinghuman IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-α, IL-1β orheat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reducedby shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels ofIκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for thetreatment of inflammatory diseases.

The protective effect of Dendrobium officinale polysaccharides on photoaging fibroblasts by scavenging reactive oxygen species and promoting the expression of TGF-β1

The purpose of this study was to explore the protective effect of Dendrobium officinale polysaccharides （DOP） onphotoaging human skin fibroblasts and its specific mechanism of action. The photoaging fibroblast model wasestablished by ultraviolet B （UVB） irradiation. The toxic effects of different concentrations of DOP were detected usingMTT. Senescent cells were detected using a β-galactosidase kit. Reactive oxygen species （ROS） in cells were detectedusing a flow cytometer. The expression of matrix metalloproteinase-1 （MMP-1）, type I collagen C-terminal peptide（CICP）, and transforming growth factor β-1 （TGF-β1） in spent culture medium was detected by ELISA. The resultsshowed that the low concentration of DOP （20, 40, 80 μg/mL） had no cytotoxicity on fibroblasts. After 60 mJ/cm2UVBirradiation, the number of aging β-gal-positive cells increased, the levels of CICP and TGF-β1 in spent culture mediumdecreased, while the levels of MMP-1 and ROS increased. After administration of DOP on photoaging fibroblasts, thenumber of aging β-gal-positive cells decreased, the levels of ROS and MMP-1 decreased, and the levels of TGF-β1 andCICP increased. This experiment suggests that DOP has the effect of removing ROS induced by UVB, regulating thebalance of collagen production and degradation, and protecting photoaging human skin fibroblasts.

A suppressed gene in integument cells of a fiberless seed mutant in upland cotton

A fiberless seed mutant (fl) was identified in a commercial cotton ( Gossypium hirsutum L.) variety Xu Zhou 142 (FL). This phenotype is associated with lack of fiber cell initiation in the outer integument of the ovule, as was characterized by analysis of genes related to fiber differentiation and development. Two genes, fl E6 and FL E6, were cloned from fl integument cells and FL fiber or integument cells, respectively. Compared with FL E6, fl E6 showed a dramatic change in nucleotide sequence: (1) FL E6 contained a tandem repetitive sequence in which GGCTCA (Gly Ser) is repeated five times between the 82nd and the 93rd codon from the first ATG codon, while in fl E6 the same sequence is repeated four times; (2) The fl E6 gene encodes a polypeptide of 241 amino acids but lacks two codons between the 90th and 93rd codon and three between the 171st and 174th relative to FL E6; (3) There are also 12 nucleotide substitutions which would result in 7 amino acid differences between fl E6 and FL E6. Analysis of RT PCR and Northern Blot showed that expression of the fl E6 gene is suppressed in the fl integument cells, but highly expressed in FL fiber cells. The difference between fl E6 and FL E6 may be associated with lower expression of fl E6 in the fl integument cells. Searches of protein databases with the FL E6 gene sequence showed similarity to the protein backbones of two arabinogalactan proteins (AGPs), one from the filtrate of suspension cultured cells of Pyrus communis (AGPPc2) and the other from Nicotiana alata (AGPNa2). Although the function of the FL E6 protein in differentiation and development of cotton fiber cells is not known, the data indicate that the mutation of fl E6 gene from FL E6 gene may inhibit the fiber cell initiation from epidermal cells of the outer integument of the ovule.

Matrine inhibits proliferation of mouse skin fibroblasts induced by platelet-derived growth factor-BB

Objective:To studythe effectof matrineon proliferationof mouseskinfibroblastsinducedby platelet-derivedgrowthfactor-BB(PDGF-BB).Methods :MouseskinfibroblastswereobtainedfromnewbornI-CRmiceandpropagatedin vitro.Proliferationof cellwasanalyzedby mitochondrialreductionof tetrazoliumsalt MTT andactualcellcount.Re sults:Matrine(50to500μg/ml)causeddose-dependentreductionof serum-stimu-latedcellgrowth.Growthinhibitionwas totallyreversedafterremovalof the drug.Matrinealso inhibited PDGF-BBinducedcellgrowthdose-dependently.Conclusion:Matrineexhibitspotentanti-proliferationeffecton mouseskinfibroblast.Thiseffectappearsto be mediatedby decreaseof PDGF-inducedgrowth.Theseresultssug-gestthatmatrinemighthavepreventiveandtherapeuticimplicationin skinfibrosis.

Preparation and Amplification of Colony of Goat Transgenic Fetal Fibroblast and Mammary Gland Epithelial Cell with Human Lactoferrin Gene

[Objective]The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0%,50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture,neo gene was as screened gene,genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [Result] Compared with non-conditioned culture medium,100% conditioned culture medium could greatly increase survived rate of single colony cells (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%). Compared with control,con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%),confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [Conclusion] The study provides a reliable method for separating transgenic cell,inserting and diagnosing ideal vector,and can save expense and time for transgenic animal production.

Mechanistic Insights of Cells in Porous Scaffolds via Integrated Culture Technologies

This research aimed to combine 3 cell and tissue culture technologies to obtain mechanistic insights of cells in porous scaffolds. When cultivated on 2D （2-dimensional） surfaces, HDFs （human dermal fibroblasts） behaved individually and had no strict requirement on seeding density for proliferation; while HaCat cells relied heavily on initial densities for proliferation and colony formation, which was facilitated when co-cultured with HDFs. Experiments using a 3D CCIS （3-dimensional cell culture and imaging system） indicated that HDFs colonised openpores of varying sizes （125-420 ~tm） on modular substrates via bridge structures; while HaCat cells formed aperture structures and only colonised small pores （125 txm）. When co-cultured, HDFs not only facilitated HaCat attachment on the substrates, but also coordinated with HaCat cells to colonise open pores of varying sizes via bridge and aperture structures. Based on these observations, a 2-stage strategy for the culture of HDFs and HaCat cells on porous scaffolds was proposed and applied successfully on a cellulosic scaffold. This research demonstrated that cell colonisation in scaffolds was dependent on multiple factors; while the integrated 2D＆3D culture technologies and the 3D CCIS was an effective and efficient approach to obtain mechanistic insights of their influences on tissue regeneration.

EFFECTS OF EPCs OR b-FGF INTRAMYOCARDIAL INFUSION ON CARDIAC FUNCTION AND NEOVASCULARIZATION FOR DILATED CARDIOMYOPATHY RATS

Objective To compare the different effects of endothelia progenitor cells （ EPCs ） or basic fibroblast growth factor （b-FGF） intromyocardial infusion on cardiac function and neovascularization for dilated cardiomyopathy（ DCM） rats. Methods Fifty adult female rats received inguinal subcutaneous injections of isoproterenol （ISO, 250 mg/kg） for induction of DCM. Four weeks later, the model rats were randomly divided into EPCs group, b-FGF group and control group. The 2×106 EPCs （ resolved in 100 μL PBS） , 100 μL b-FGF （ lO0 μg/mL ） and 100 μL PBS were evenly transplanted into the myocardium of EPCs group, b-FGF group and control group, respectively. Three months later, echocardiographic examination and regional myocardial blood flow （RMBF） measurement were performed. EPCs were traced by fluorescence in situ hybridization （FISH）. The protein and mRNA expression of b-FGF in each group was measured by ELISA assay and reverse transcription-polymerase chain reaction （ RT-PCR ） . Results Three months after transplantation, sry positive cells were detected only in EPCs group. The cardiac function as well as RMBF was significantly improved in EPCs group compared with b-FGF group or control group. There was higher capillary density in EPCs group. The protein and mRNA expression of b-FGF was stronger than b-FGF group and control group. Conclusion Transplantation of EPCs can improve cardiac function, induce neovascularization and increase RMBF for DCM rats. The treatment with EPCs has better effect than administration of b-FGF alone.

Lubiprostone:Clinical applications beyond constipation

In comparison to polyethylene glycol,lubiprostone offers other advantages and is increasingly being used as an adjunctive agent in diagnostic as well as management strategies not only in gastroenterology, but in other fields.For instance,lubiprostone exerts beneficial effects in cystic fibrosis tissues.It augmernts the chloride secretion in these cells by activating non-cystic fibrosis transmembrane regulator(CFTR) secretion of chloride by afflicted respiratory epithelia. Lubiprostone also seems to improve visualization of the gastrointestinal tract during procedures such as colonoscopy.This is especially true if the lubiprostone is administered prior to bowel cleansing with agents such as polyethylene glycol electrolyte(PEG-E). Lubiprostone also enhances and stimulates contraction in colonic as well as gastric muscles and may thus further contribute as a prokinetic agent.Besides these effects,lubiprostone also causes hyperpolarization in other tissues such as uterine muscle cells.This may prove to be of significant clinical benefit in the management of uterine pathologies in the near future.

OVER-EXPRESSION OF A TRUNCATED TYPE I TGF-P RECEPTOR IN NORMAL DERMAL FIBROBLASTS DECREASES TGF-β1 AUTOPRODUCTION

Objective To determine over-expression of a truncated type ⅡTGF-β receptor in down-regulating TGF-β1 auto production in normal dermal fibroblasts. Methods In vitro cultured dermal fibroblasts were treated with rhTGF-β1 (5ng/ml) or recombinant adenovirus containing α truncated type Ⅱ TGF-β receptor gene (50 pfu/cell). Their effects on regulating gene expression of TGF-β1 were observed with Northern Blot. Results rh TGF-β1 up-regulated the gene expression of TGF-β1, (34 %-150%) and type Ⅰ pro-collagen( 13 %- 190%). Overexpression of a truncated receptor Ⅱ decreased the gene expression of TGF-β1 (53%-66%). Conclusion Over-expression of the truncated TGF-β receptor Ⅱdown-regulated TGF-β1 autoproduction via blocking signal transduction of TGF-β. This study may provide a new strategy for scar gene therapy.

Sonic hedgehog signaling in kidney fibrosis: a master communicator

The hedgehog signaling cascade is an evolutionarily conserved pathway that regulates multiple aspects of embryonic development and plays a decisive role in tissue homeostasis. As the best studied member of three hedgehog ligands, sonic hedgehog(Shh) is known to be associated with kidney development and tissue repair after various insults. Recent studies uncover an intrinsic link between dysregulated Shh signaling and renal fibrogenesis. In various types of chronic kidney disease(CKD), Shh is upregulated specifically in renal tubular epithelium but targets interstitial fibroblasts, thereby mediating a dynamic epithelialmesenchymal communication(EMC). Tubule-derived Shh acts as a growth factor for interstitial fibroblasts and controls a hierarchy of fibrosis-related genes, which lead to the excessive deposition of extracellular matrix in renal interstitium. In this review, we recapitulate the principle of Shh signaling, its activation and regulation in a variety of kidney diseases. We also discuss the potential mechanisms by which Shh promotes renal fibrosis and assess the efficacy of blocking this signaling in preclinical settings. Continuing these lines of investigations will provide novel opportunities for designing effective therapies to improve CKD prognosis in patients.

Criculating fibrocytes： a potent cell population in antigen-presenting and wound healing

Fibrocytes are bone marrow-derived mesenchymal progenitors that co-express hematopoietic cell antigens and markers of monocytic lineage as well as fibroblast products. During wound healing, fibrocytes have been found to possess the ability of antigen-presentation to naive T cells in the inflammatory phase. Moreover, they can promote the endothelial cell proliferation, migration and angiogenesis by secreting several proteins. Fibrocytes can further differentiate into mature mesenchymocyte lineage, such as fibroblasts, myofibroblasts and adipocytes, and they may represent the systemic source of myofibroblasts that exert a contractile force required to close tissue wounds. A deep understanding of the mechanism involved in fibrocyte migration and differentiation may lead to the development of a novel theory of normal physiology and pathology.

Cytotoxicity and non-specific cellular uptake of bare and surface-modified upconversion nanoparticles in human skin cells

The cytotoxicity and non-specific cellular uptake of the most popular composition of upconversion nanoparticle （UCNP）, NaYF4：Yb^3＋：Er^3＋, is reported using normal human skin cells, including dermal fibroblasts and immortalized human epidermal linear keratinocytes （HaCaT）. A new hydrophilization reaction of as-synthesized UCNPs based on tetramethylammonium hydroxide （TMAH） enabled evaluation of the intrinsic cytotoxicity of bare UCNPs. The cytotoxicity effects of the UCNP surface-coating and polystyrene host were investigated over the concentration range 62.5-125 μg/mL with 24-h incubation, using a MTT test and optical microscopy. The fibroblast viability was not compromised by UCNPs, whereas the viability of keratinocytes varied from 52% ± 4% to 100% ± 10% than the control group, depending on the surface modification. Bare UCNPs reduced the keratinocyte viability to 76% ± 3%, while exhibiting profound non-specific cellular uptake. Hydrophilic poly（D,L-lactide）- and poly（maleic anhydride-alt-l-octadecene）-coated UCNPs were found to be least cytotoxic among the polymer-coated UCNPs, and were readily internalized by human skin cells. Polystyrene microbeads impregnated with UCNPs remained nontoxic. Surprisingly, no correlation was found between UCNP cytotoxicity and the internalization level in cells, although the latter ranged broadly from 0.03% to 59%, benchmarked against 100% uptake level of TMAH-UCNPs.

Endostatin inhibits hypertrophic scarring in a rabbit ear model

Objective： The present study was designed to use an in vivo rabbit ear scar model to investigate the efficacy of systemic administration of endostatin in inhibiting scar formation. Methods： Eight male New Zealand white rabbits were randomly assigned to two groups. Scar model was established by making six full skin defect wounds in each ear. For the intervention group, intraperitoneal injection of endostatin was performed each day after the wound healed （about 15 d post wounding）. For the control group, equal volume of saline was injected. Thickness of scars in each group was measured by sliding caliper and the scar microcirculatory perfusion was assessed by laser Doppler flowmetry on Days 15, 21, 28, and 35 post wounding. Rabbits were euthanatized and their scars were harvested for histological and proteomic analyses on Day 35 post wounding. Results： Macroscopically, scars of the control group were thicker than those of the intervention group. Significant differences between the two groups were observed on Days 21 and 35 （p〈0.05）. Scar thickness, measured by scar elevation index （SEI） at Day 35 post wounding, was significantly reduced in the intervention group （1.09±0.19） compared with the controls （1.36±0.28）. Microvessel density （MVD） observed in the intervention group （1.73±0.94） was significantly lower than that of the control group （5.63±1.78） on Day 35. The distribution of collagen fibers in scars treated with endostatin was relatively regular, while collagen fibers in untreated controls were thicker and showed disordered alignment. Western blot analysis showed that the expressions of type I collagen and Bcl-2 were depressed by injection of endostatin. Conclusions： Our results from the rabbit ear hypertrophic scar model indicate that systemic application of endostatin could inhibit local hypertrophic scar formation, possibly through reducing scar vascularization and angiogenesis. Our results indicated that endostatin may promote the apoptosis of endothelial cells and block their release of platelet-dedved growth factor （PDGF） and fibroblast growth factor （FGF）, thereby controlling collagen production by fibroblasts. Blood vessel-targeted treatment may be a promising strategy for scar therapy.