The Combined Effect of Lumenato and Ceramide in the Protection of Collagen Damage Induced by Neutrophils in Normal Human Dermal Fibroblasts

Introduction: Collagen is the primary structural protein fibroblasts produce in the skin’s extracellular matrix. Infiltration of neutrophils into the epidermis and dermis by exposure to UV causes collagen damage and contributes to photoaging. Methods: To study the combined effect of Lumenato and ceramide in preventing collagen-1 damage induced by phagocytes, we used co-cultures of normal human dermal fibroblasts (fibroblasts) and activated human neutrophils. The present study aimed to determine the protective effect of the combination of Lumenato and ceramide on fibroblast collagen-1 damage induced by neutrophils. Results: Lumenato (in the range of 6.5 - 208 μg/ml) or ceramide (in the range of 0.1 - 50 μM) inhibited the production of superoxides and MPO by TNFα-stimulated neutrophils, as well as the production of NO by LPS-stimulated macrophages in a dose-dependent manner. The combinations of Lumenato and ceramide, in low concentrations, caused synergistic prevention of fibroblasts’ collagen-1 damage induced by TNFα-activated neutrophils, detected by fluorescence immunostaining and WB analysis. MPO activity in the supernatants of the co-cultures was also synergistically inhibited. Adding Lumenato or ceramide singly or in combinations in these low concentrations to the fibroblast cultures did not affect the expression of collagen-1. The combinations of Lumenato or ceramide in these concentrations also caused a synergistic inhibition of NO production by activated macrophages. Conclusions: The results suggest that combining low concentrations of Lumenato and ceramide results in synergistic protection against fibroblasts’ collagen-1 damage induced by neutrophils, thus indicating their possible potential for enhanced skin health.

Chicken collagen hydrolysates differentially mediate anti-inflammatory activity and type I collagen synthesis on human dermal fibroblasts

Collagen is a major extracellular matrix protein.Given the potential anti-inflammatory and antioxidant profiles of these bioactive compounds,there has been increasing interest in using collagen derived peptides and peptide-rich collagen hydrolysates for skin health,due to their immunomodulatory,antioxidant and proliferative effects on dermal fibroblasts.However,all hydrolysates are not equally effective in exerting the beneficial effects;hence,further research is needed to determine the factors that improve the therapeutic applicability of such preparations.We used different enzymatic conditions to generate a number of different collagen hydrolysates with distinct peptide profiles.We found that the use of two rather than one enzyme for hydrolysis generates a greater abundance of low molecular weight peptides with consequent improvement in bioactive properties.Testing these hydrolysates on human dermal fibroblasts showed distinct actions on inflammatory changes,oxidative stress,type I collagen synthesis and cellular proliferation.Our findings suggest that different enzymatic conditions affect the peptide profile of hydrolysates and differentially regulate their biological activities and potential protective responses on dermal fibroblasts.

9-Hydroxy-6,7-dimethoxydalbergiquinol suppresses hydrogen peroxide-induced senescence in human dermal fibroblasts through induction of sirtuin-1 expression

Objective:To investigate the potential anti-aging mechanism of9-hydroxy-6,7-dimethoxydalbergiquinol(HDDQ)on hydrogen peroxide(H2O2)-induced oxidative stress in human dermal fibroblasts(HDFs).Methods:The effect of HDDQ on cell viability was assessed by MTT assay,and the effects of HDDQ on senescence-like phenotypes were determined by senescence-associatedβ-galactosidase(SA-β-gal)staining,Western blotting analysis,and a cell proliferation assay.The expression level and activity of sirtuin-1(SIRT1)induced by HDDQ were also measured.Results:HDDQ reversed senescence-like phenotypes in the oxidant-challenged model,through reducing SA-β-gal activity and promoting cell growth.Meanwhile,decreases in ac-p53,p21Cip1/WAF1,and p16Ink4a and an increase in p Rb were observed.HDDQ induced the expression of SIRT1 in a concentration-and time-dependent manner.Moreover,HDDQ inhibited H2O2-induced phosphorylation of Akt by SIRT1 up-regulation and reduced SA-β-gal staining.Conclusions:HDDQ inhibits H2O2-induced premature senescence and upregulation of SIRT1 expression plays a vital role in the inhibition of the senescence phenotype in HDFs.

Anti-senescence and anti-wrinkle activities of 3-bromo-4,5-dihydroxybenzaldehyde from Polysiphonia morrowii Harvey in human dermal fibroblasts

Objective:To investigate the anti-senescence effect of 3-bromo-4,5-dihydroxybenzaldehyde(BDB)from Polysiphonia morrowii Harvey in human dermal fibroblasts(HDF).Methods:HDF were subjected to treatment of BDB and then treated with hydrogen peroxide(H2O2)to induce premature senescence.Senescence-associatedβ-galactosidase(SA-β-gal)activity in HDF was determined using the SA-β-gal staining method.Intracellular reactive oxygen species(ROS)production was measured using the 2’,7’-dichlorodihydrofluorescein diacetate assay.Western blotting assay was performed to assess the level of antioxidant enzyme glutathione peroxidase 1(GPX1).In addition,intracellular collagen and collagenase contents were analyzed using the respective ELISA kits.Elastase activity in HDF supernatants was measured from p-nitroaniline release and normalized using total protein content.Results:Treatment of HDF with H2O2 increased the activity of SA-β-gal,but BDB pre-treatment resulted in the reduction of SA-β-gal activity.Moreover,BDB significantly reduced H2O2-induced intracellular ROS production.BDB also markedly increased the level of GPX1,which was inhibited by 400μM of H2O2.Furthermore,in in vitro study,BDB significantly increased intracellular collagen content and decreased matrix metalloproteinase-1 and elastase activities in HDF.Conclusions:Our results demonstrate that BDB shows antisenescence and anti-wrinkle activities in vitro.

Effect of a Nutrient Mixture on Fanconi Anemia Fibroblast and Normal Human Dermal Fibroblast: A Comparison

Fanconi anemia (FA) is a fatal heterogeneous autosomal recessive disorder, characterized by progressive bone marrow failure, congenital defect and cancer predisposition. Cell culture from FA fibroblast (FAF) displays certain abnormalities as compared to normal human dermal fibroblast (NHDF). This prompted us to investigate the effect of a specific nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract, which has demonstrated a broad spectrum of pharmacological activities, on FAF compared to NHDF. We investigated the in vitro effect of NM on FAF and NHDF cell proliferation by MTT assay, MMPs secretion by zymography, morphology by H&E staining and apoptosis by green caspase assay. FAF (FA-A: PD20, FA-A: PD220) and NHDF were cultured in modified Dulbecco Eagle media. At near confluence, the cells were treated with different concentrations of NM (0, 50, 100, 250, 500 and 1000 μg/ml) in triplicate. The cells were also treated with PMA to induce MMP-9 activity. NM had no effect on FAF cell viability in both cell lines compared to control. In contrast NM exhibited 20% at 50 and 100, 50% at 250, 60% at 500 and 70% toxicity at 1000 μg/ml on NHDF cells. Zymography demonstrated MMP-2 and MMP-9 on PMA stimulation in FAF and NM inhibited the activity of both MMP-2 and MMP-9 in a dose response fashion with total block at 500 μg/ml. In contrast, NHDF exhibited only MMP-2, both active and inactive forms, and NM inhibited their activities in a dose-dependent manner with total block at 1000 μg/ml. H&E staining did not indicate any morphological changes in FAF nor induced apoptosis at higher concentrations, as seen by caspases assay. However, although no morphological changes in NHDF were noted up to NM 100 μg/ml, progressive changes in cell shrinkage, rounding and nuclear condensation, pertaining to apoptosis, were observed at higher concentrations. These changes were consistent with the results from the green caspases apoptosis assay. Our data demonstrate that NM exhibited different responses toward FAF and NHDF. This may in part be due to elevated chromosomal break, deletion and hypersensitivity to cross linking agents, a DNA repair disorder in FAF that is lacking in NHDF.

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.

EXPERIMENTAL STUDY ON INDUCTION OF OSTEOGENETIC POTENTIAL OF DERMAL FIBROBLASTS

Objectiye To eiucidate the inducing factor in osteogenesis piayed by dermai fibroblasts. mthods Fibroblasts and epithelial cells were procured from rabbit and human skins and separated. These two types of cells were then cultured either together or separately and studied histochemically, immunohistochemically and biochemically. Results Bone nodules were found only in the mtxed culture of fibroblasts and epithelial cells. During the period ol exuberant bone nodule formation, there were both more positive keratin- stained cells (epithelial cells) and higher ALP activity. When few or no bone nodules formed, positive keratin- stained cells declined or disappeared and the ALP activity fell off. Conclusion That dermal fibroblasts show their osteogenic potential depends mainly on the inducing effect of the ePithelial cells.

Dermal fibroblast expression of stromal cellderived factor-1 （SDF-1） promotes epidermal keratinocyte proliferation in normal and diseased skin

Stromal cells provide a crucial microenvironment for overlying epithelium. Here we investigated the expression and function of a stromal cell-specific protein, stromal cell-derived factor-1 （SDF-1）, in normal human skin and in the tissues of diseased skin. Immunohistology and laser capture microdissection （LCM）-coupled quantitative real- time RT-PCR revealed that SDF-1 is constitutively and predominantly expressed in dermal stromal cells in nor- mal human skin in vivo. To our surprise, an extremely high level of SDF-1 transcription was observed in the dermis of normal human skin in vivo, evidenced by much higher mRNA expression level than type I collagen, the most abundant and highly expressed protein in human skin. SDF-1 was also upregulated in the tissues of many human skin disorders including psoriasis, basal cell carcinoma （BCC）, and squamous cell carcinoma （SCC）. Double immunostaining for SDF-1 and HSP47 （heat shock protein 47）, a marker of fibroblasts, revealed that fibroblasts were the major source of stroma-cell-derived SDF-1 in both normal and diseased skin. Functionally, SDF-1 activates the ERK （extracellular-signal-regulated kinases） pathway and functions as a mitogen to stimulate epidermalkeratinocyte proliferation. Both overexpression of SDF-1 in dermal fibroblasts and treatment with rhSDF-1 to the skin equivalent cultures significantly increased the number of keratinocyte layers and epidermal thickness. Con- versely, the stimulative function of SDF-1 on keratinocyte proliferation was nearly completely eliminated by inter- fering with CXCR4, a specific receptor of SDF-1, or by knock-down of SDF-1 in fibroblasts. Our data reveal that extremely high levels of SDF-1 provide a crucial microenvironment for epidermal keratinocyte proliferation in both physiologic and pathologic skin conditions.

Comparative study of the effects of gold and silver nanoparticles on the metabolism of human dermal fibroblasts

The purpose of this article was to explore the effects of gold nanoparticles(GNPs)and silver nano-particles(SNPs)with different cytotoxicities on human dermal fibroblasts(HDFs)at the metabolic level.First,~20 nm of GNPs and SNPs were prepared,and their effects on the proliferation of HDFs were evaluated.Then,a metabolomics technique was used to analyse the effects of GNPs and SNPs on the expression profiles of metabolites in HDFs after 4,8 and 24h of treatment.Furthermore,the key metabolites and key metabolic pathways involved in the interaction of GNPs and SNPs with HDFs were identified through expression pattern analysis and metabolic pathway analysis of differentially expressed metabolites and were finally verified by experiments.The results of the cytotoxicity experiments showed that there was no cytotoxicity after the treatment of GNPs for 72 h,while the cytotoxicity of the SNPs reached grade 1 after 72 h.By using metabolomics analysis,29,30 and 27 metabolites were shown to be differentially expressed in HDFs after GNP treatment,while SNPs induced the differential expression of 13,33 and 22 metabolites after 4,8 and 24h of treatment,respectively.Six and four candidate key metabolites in the GNP and SNP groups were identified by expression pattern analysis and metabolic pathway analysis,respec-tively.The key metabolic pathways in the GNP and SNP groups were identified as the glutathione metabolic pathway(the key metabolite of which was glutathione)and the citrate cycle pathway(the key metabolite of which was malic acid).Based on the experiments used to verify the key metabolites and key metabolic pathways,it was found that the increase in glutathione after GNP treatment might trigger an oxidative stress protection mechanism and thus avoid cytotoxicity.After exposure to SNPs,the citric acid content was increased,mainly through the citrate cycle path-way,thereby inhibiting the synthesis of malic acid to affect the formation of ATP and finally leading to cytotoxicity.

Honey exposure stimulates wound repair of human dermal fibroblasts

Honey is widely used for treating burns, ulcers and wounds, but the mechanisms of action are poorly known and the product is mainlyused as an antimicrobial. We have examined here the wound healing properties of honey on human fi broblasts, using an in vitroscratch wound healing model. Three kinds of widely used monofl oral honeys were used, viz. acacia (Robinia pseudacacia), buckwheat(Fagopyrum sp.), and manuka (Leptospermum scoparium). Data displayed an increased wound healing activity in fi broblasts, butwith diff erent effi ciency and mechanisms of action among honeys. The eff ects of acacia and buckwheat emerged in both scratchwound and chemotaxis assays, while the eff ect of manuka was signifi cant but lower. The use of inhibitors indicated on the wholean essential role of cytosolic calcium, an important role of ERK and p38, and a secondary role of PI3K. Acacia and buckwheat,but not manuka, induced signifi cant increases in the release of interleukin-4 (IL-4), IL-6, and IL-8, indicating a correlation betweeninterleukin upregulation and wound closure effi ciency. This is consistent with our previous fi ndings suggesting a higher ability ofacacia and buckwheat to activate keratinocyte reepithelialization, with respect to manuka honey. In conclusion, our data indicatethat acacia and buckwheat honeys are particularly effi cient in facilitating fi broblast wound closure activities, suggesting newtherapeutic possibilities for this natural product.

UV-induced senescence of human dermal fibroblasts restrained by low-stiffness matrix by inhibiting NF-κB activation

As a hallmark of skin aging,senescent human dermal fibroblasts(HDFs)are known to lose the ability to divide.However,they can still interact with their cellular environment and the surrounding matrix.As the skin ages,the progressive slowing down of HDFs function decreases the skin’s structural integrity,which is more serious than if there is the dermal collagen matrix eroded.This leads to matters of the unbalanced barrier under the skin,skin fragility,inadequate wound healing,as well as other cosmetic issues.It is also well documented that skin aging comes with significant stiffness increases.Therefore,understanding the interactions between HDFs and the surrounding microenvironments during senescence may provide insights into skin aging.Here we aim to inves-tigate matrix stiffness’effect on HDF senescence and elucidate possible mechanisms that make HDFs senescent.In our experiments,HDFs were cultivated on Polydimethylsiloxane(PDMS)with various stiffnesses and exposed to UV light to trigger senescence.Results show that HDFs are significantly affected by senescence when cultured on a matrix with stiffness.However,the cells are not significantly affected when cultured on a low stiffness matrix.The following characterization revealed cells cultured on stiffsubstrates under UV exposure had stimu-lated the nucleus factor kappa-B(NF-κB)activation.In contrast,cells on a matrix of softness only displayed low activation of NF-κB.NF-κB activity suppression with ammonium pyrrolidine dithiocarbamate(PDTC)decreases UV-induced HDFs senescence on stiffsubstrates.Taken together,we demonstrated that soft matrix defends HDFs against ultraviolet-induced senescence by inhibiting the activation of NF-κB.

In vitro antioxidant and wound healing activity of Sargassum polycystum hydroethanolic extract in fibroblasts and keratinocytes

Objective:To investigate the in vitro antioxidant and wound healing properties of the hydroethanolic extract of Sargassum polycystum,and elucidate the mechanism of its wound healing activity.Methods:Human dermal fibroblast and HaCaT cells were used to evaluate the proliferation by sulforhodamine B and dsDNA assay after treatment with Sargassum polycystum extracts.Scratch wound healing and phalloidin-rhodamine staining were employed to observe migratory activity and filopodia formation,respectively.Western blot and real-time RT-PCR assays were performed to determine the protein and gene expressions related to wound healing activities.Results:The phytochemical analysis found a higher level of flavonoid than phenolic compound in Sargassum polycystum extracts.In human dermal fibroblast cells,Sargassum polycystum extracts at 50 and 100μg/mL significantly increased fibroblast proliferation and the gene expressions of hyaluronic acid synthase 1(HAS1),HAS2,HAS3,collagen type 1 alpha 1 chain(COL1A1),collagen type 3 alpha 1 chain(COL3A1),and elastin.The phosphorylation of Akt,ERK1/2,and p38 MAPK was also significantly upregulated after treatment with Sargassum polycystum extracts.Additionally,50 and 100μg/mL of the extracts prominently enhanced the proliferation,migration,and filopodia formation of HaCaT cells,as well as the protein levels of pFAK/FAK,pSrc/Src,pAkt/Akt,pERK1/2/ERK1/2,Rac1 and Cdc42.Conclusions:Sargassum polycystum extracts show promising wound healing activities in human dermal fibroblasts and keratinocytes.

Protective effect of oat bran extracts on human dermal fibroblast injury induced by hydrogen peroxide

Oat contains different components that possess antioxidant properties;no study to date has addressed the antioxidant effect of the extract of oat bran on the cellular level.Therefore,the present study focuses on the investigation of the protective effect of oat bran extract by enzymatic hydrolysates on human dermal fibroblast injury induced by hydrogen peroxide(H2O2).Kjeldahl determination,phenol-sulfuric acid method,and high-performance liquid chromatography(HPLC) analysis indicated that the enzymatic products of oat bran contain a protein amount of 71.93%,of which 97.43% are peptides with a molecular range from 438.56 to 1 301.01 Da.Assays for 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical scavenging activity indicate that oat peptide-rich extract has a direct and concentration-dependent antioxidant activity.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) colorimetric assay and the TdT-mediated digoxigenin-dUTP nick-end labeling(TUNEL) assay for apoptosis showed that administration of H2O2 in human dermal fibroblasts caused cell damage and apoptosis.Pre-incubation of human dermal fibroblasts with the Oatp for 24 h markedly inhibited human dermal fibroblast injury induced by H2O2,but application oat peptides with H2O2 at same time did not.Pre-treatment of human dermal fibroblasts with Oatp significantly reversed the H2O2-induced decrease of superoxide dismutase(SOD) and the inhibition of malondialdehyde(MDA).The results demonstrate that oat peptides possess antioxidant activity and are effective against H2O2-induced human dermal fibroblast injury by the enhanced activity of SOD and decrease in MDA level.Our results suggest that oat bran will have the potential to be further explored as an antioxidant functional food in the prevention of aging-related skin injury.

Human dermal reticular fibroblasts at confluence display a signature micro pattern <i>in vitro</i>

This paper sets out to demonstrate that scraping of the flat dorsal surface of human dermis with a scalpel blade and cell plating without centrifugation can lead to the recognition and identification of the individual packing micro pattern of dermal reticular fibroblasts at confluence. The characteristic alignment of papillary and reticular fibroblasts at right angles to each other led to the positive identification of reticular fibroblasts. A non-enzymatic means of sub-culturing (passaging), which yields fully functional, healthy cells with normal, phenotypic morphology is also described. Implications for published subcutaneous wound healing studies are discussed as well as the confluent reticular fibroblast configuration, interpreted as ananatomic site identity code,which may be the address of a specific fibroblast gene pattern expression.

<i>In Vitro</i>Analysis of VEGF and HGF Production by Fibroblast in Cultured Dermal Substitute Combined with EGF-Incorporating Top Dressing

This study aimed to investigate the potential of cultured dermal substitute (CDS) to release angiogenic growth factors when laminated with a membrane containing epidermal growth factor (EGF) as a top dressing. Membranes were prepared by air-drying a solution of hyaluronic acid (HA) and collagen (Col) with or without EGF. Membranes were designed to contain EGF at concentrations of 0, 0.1, 0.2 or 0.5 μg/cm2. CDS was prepared by incorporating fibroblasts into a collagen gel combined with a cross-linked HA spongy matrix, followed by culturing for 5 days. CDS was designed to contain fibroblasts at a density of 2 × 105 (Group I) or 4 × 105 cells/cm2> (Group II). CDS was elevated at the interface between air and culture medium, on the top of which each membrane was placed. This culture system was employed as a wound surface model. Metabolic activity of the fibroblasts in the CDS cultured for 7 days on a wound surface model was measured by MTT assay. The amounts of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) after 7 days of cultivation were measured by using ELISA. Membranes containing EGF ranging from 0.1 to 0.5 μg/cm2> facilitated production of both VEGF and HGF, as compared with control membranes without EGF. However, a membrane containing EGF at a concentration of 0.5 μg/cm2> failed to facilitate fibroblast cytokine production in Group I. These results demonstrated that the EGF-incorporating membrane was able to stimulate fibroblasts in the CDS to synthesize an increased amount of VEGF and HGF in a dose-dependent manner.

Hyaluronate Increases Polynucleotides Effect on Human Cultured Fibroblasts

The HA is present in almost all vertebrates and plays a critical role in tissue development and cell proliferation, it has been demonstrated to promote wound healing and involved in angiogenesis and inflammation. Also polynucleotydes (PN) have proved to promote the “in vitro” growth and activity of human fibroblasts and osteoblasts, to increase reparation on UVB damaged dermal fibroblasts and seems to promote proliferation of human pre-adipocytes. Several in vivo studies have demonstrated the PN effect also in vivo, inducing an increase of angiogenesis and healing process. In this paper we have evaluated the effect of a mixture of Polynucleotides (PN) and entire Hyaluronic Acid (HA) on cultured human fibroblasts by analyzing cell growth. Different mixture have been tested and it has been demonstrated that the presence of HA even at low concentration (1 mg/ml) determine an increase of PN activity up to 20%. Furthermore, the addition of HA 1 mg/ml to PN 100 μg/ml induces a cell growth rate comparable to that exerted by PN concentration of 12 μg/ml.

Scientific Viewpoints with Emphasis on Dermal Cellular Regeneration in Wound Sites

The human dermis presents an ongoing problem for regenerative medicine. Current medical management uses various acellular dermal matrices on wound sites. The challenge for scientists is to examine, then to question accepted conventional wisdom and to present new concepts. In this paper, Autologous Cell Therapy will be described by using cell culture of autologous dermal fibroblasts and their extracellular matrix as a foundation for rebuilding the dermis in conditioned wound beds. This proposal seems to create a conflict with the medical approach to keeping a wound bed “moist”.

两种奶牛真皮成纤维细胞分离培养方法的比较

为了比较两种体外分离和培养奶牛真皮成纤维细胞方法,采集奶牛蹄部真皮组织,通过组织块贴壁法和双酶消化法(胰蛋白酶-Ⅰ型胶原酶)分离原代奶牛真皮成纤维细胞,通过传代培养进一步纯化并扩增。利用显微镜观察细胞形态及生长特性,MTT法用于绘制细胞生长曲线,利用细胞免疫荧光技术检测其表面标志物,鉴定细胞纯度。倒置显微镜下观察,贴壁后第6天有细胞爬出,形态多呈长梭形,排列紧密呈漩涡状或束状;双酶消化组第2天有长梭形细胞生长,混杂少量上皮细胞生长。生长曲线图表示,组织块贴壁组细胞生长速度较快。免疫化学荧光检测第四代奶牛真皮成纤维细胞,波形蛋白呈阳性表达,角蛋白-18呈阴性表达,表示细胞纯度较高。两种方法均能分离出奶牛真皮成纤维细胞,与双酶消化法相比,组织块贴壁法分离的细胞纯度更高,活性更佳,增殖周期更短,提示组织块贴壁法是分离培养奶牛真皮成纤维细胞的首选方法。

纳秒脉冲电场刺激促进人真皮成纤维细胞成骨的实验研究

目的 探究超短纳秒脉冲电场(Nanosecond Pulsed Electric Field,nsPEF)是否能够促进成纤维细胞向成骨细胞的表型转化。方法 用0、5 kV/cm的场强刺激成纤维细胞,观察其在普通培养基和三向分化诱导培养基中生长情况,蛋白印迹测定细胞内调控生长及分化Hippo通路下游信号分子水平,并通过体内注射细胞-凝胶复合体进行裸鼠体内成骨实验。结果 5kV/cm nsPEF促进了成纤维细胞的增殖,并促进了成骨、成软骨分化,但对成脂分化影响不显著。低能量脉冲电场刺激抑制了Hippo通路下游各信号分子磷酸化水平,促进了裸鼠体内成骨,灰度分析显示,YAP1及LATS1的磷酸化蛋白与非磷酸化蛋白比值在5 kV/cm的脉冲电场刺激后明显降低,提示HIPPO信号通路受到抑制(P<0.05)。结论 低强度nsPEF刺激有效促进了成纤维细胞的增殖和表型转化,并在裸鼠模型上证实了可行性和有效性。

小鼠毛乳头细胞体外分离培养方法的优化和评价

目的 :探索小鼠毛乳头细胞(DPCs)体外分离培养的改良新方法。方法 :选用雌性C57BL/6小鼠,以精细显微解剖获取毛乳头部位,联合胶原酶消化,并添加10 ng/mL碱性成纤维细胞生长因子(bFGF)以优化培养体系,建立体外分离培养小鼠DPCs的改良新方法;以光镜下观察、细胞计数、碱性磷酸酶(ALP)特异性染色及荧光定量PCR鉴定DPCs的ALP mRNA表达水平,与传统方法比较进行相关评价。结果 :与传统方法比较,改良方法的毛乳头基本全部贴壁,细胞可更快迁出及生长融合,所获得的细胞数量是传统方法的5.85倍;经传代后传统方法的低代次DPCs有聚集性生长现象,随代次增加,细胞形态逐渐变大衰老,增殖明显迟缓,聚集性生长愈发不明显,至第5代聚集性现象消失。而改良方法的DPCs经传代后,第1、2和5代形态仍呈长梭形及多角形的特点,至第5代仍保有聚集性生长的特点;且具有更强的ALP活性,尤其是高代次(第5代)细胞更为明显,而传统方法的高代次细胞则几乎无ALP活性;改良方法的ALP mRNA表达水平与传统方法相比也明显上调,尤其是第5代细胞更加明显。结论 :改良方法可获取更多数量、并具有较好功能的小鼠DPCs,为开展毛囊相关研究提供可靠的细胞来源。