Transplantation of human induced pluripotent stem cell derived keratinocytes accelerates deep second-degree burn wound healing

BACKGROUND Current evidence shows that human induced pluripotent stem cells(hiPSCs)can effectively differentiate into keratinocytes(KCs),but its effect on skin burn healing has not been reported.AIM To observe the effects of hiPSCs-derived KCs transplantation on skin burn healing in mice and to preliminarily reveal the underlying mechanisms.METHODS An analysis of differentially expressed genes in burn wounds based on GEO datasets GSE140926,and GSE27186 was established.A differentiation medium containing retinoic acid and bone morphogenetic protein 4 was applied to induce hiPSCs to differentiate into KCs.The expression of KCs marker proteins was detected using immunofluorescence staining.A model of a C57BL/6 mouse with deep cutaneous second-degree burn was created,and then phosphate buffered saline(PBS),hiPSCs-KCs,or hiPSCs-KCs with knockdown of COL7A1 were injected around the wound surface.The wound healing,re-epithelialization,engraftment of hiPSCs-KCs into wounds,proinflammatory factor level,and the NF-κB pathway proteins were assessed by hematoxylin-eosin staining,carboxifluorescein diacetate succinimidyl ester(CFSE)fluorescence staining,enzyme linked immunosorbent assay,and Western blotting on days 3,7,and 14 after the injection,respectively.Moreover,the effects of COL7A1 knockdown on the proliferation and migration of hiPSCs-KCs were confirmed by immunohistochemistry,EdU,Transwell,and damage repair assays.RESULTS HiPSCs-KCs could express the hallmark proteins of KCs.COL7A1 was down-regulated in burn wound tissues and highly expressed in hiPSCs-KCs.Transplantation of hiPSCs-KCs into mice with burn wounds resulted in a significant decrease in wound area,an increase in wound re-epithelialization,a decrease in proinflammatory factors content,and an inhibition of NF-κB pathway activation compared to the PBS group.The in vitro assay showed that COL7A1 knockdown could rescue the inhibition of hiPSCs-KCs proliferation and migration,providing further evidence that COL7A1 speeds up burn wound healing by limiting cell proliferation and migration.CONCLUSION In deep,second-degree burn wounds,COL7A1 can promote KC proliferation and migration while also suppressing the inflammatory response.

Effect of a Thermal Spring Water on Carbohydrate-Protein Interactions in In-Vitro Models Implicating Normal Human Keratinocytes and Recombinant Lectins

Background: Sugar moiety of macromolecules is today very well known for its implications in many biological recognition mechanisms including cell-cell, extracellular matrix-cell and/or bacteria-cell interactions. In this context lectins, which are carbohydrate-binding proteins displaying a high affinity for sugar groups of other molecules, are of a great importance, notably in immune response involving bacteria, viruses and fungi. As protein-carbohydrate interactions are often mediated by ions such as calcium, zinc or magnesium, we were prompted to study the effect of a thermal spring water (which contains this type of component) on interactions existing between: 1) osidic receptors of human normal keratinocytes and 2) two lectins greatly implicated in the immune response mechanisms (i.e. the dectin-1 and the langerin), and their ligands. Materials and Methods: In a first series of experiments, we studied the effect of increasing concentrations of a thermal spring water on interactions existing between glycosylated molecules and the osidic receptors expressed at the normal human keratinocytes surface. In a second step, and in order to better understand the putative effect of our thermal spring water on the immune response, we analyzed its effect on the interactions existing between the dectin-1 (implicated in the recognition of bacteria, viruses and fungi) and the langerin (expressed by Langerhans cells, the immune cells of the cutaneous tissue), and their ligands in a model using recombinant human lectins and appropriate binding molecules. Results: We showed here that our thermal spring water was able to reinforce interactions between keratinocytes osidic receptors and some of their ligands, in a dose-related manner: From 8% to 55% of increase with 10% to 30% (v/v) of thermal spring water. In the second part of our studies, we also showed that our thermal spring water was able to modulate interactions between dectin-1 and langerin and their ligands through a biphasic effect: Interactions were enhanced by more than 40% and 20% respectively with 10% of thermal spring water, and return to their basal level or lower for higher concentrations. Conclusion: The tested thermal spring water, probably due to its ionic composition, could significantly affect interactions of osidic receptors with their ligands. This property could be of a great interest to help immune system to maintain an appropriate “vigilance state” by using the thermal water at up to a concentration of 10%, and by avoiding any runaway reaction in case of aggression, by using concentrations higher than 10%. .

IL-36β Promotes Inflammatory Activity and Inhibits Differentiation of Keratinocytes In Vitro

Objective Psoriasis is an immune-mediated inflammatory disease.Despite advances in the study of its pathogenesis,the exact development mechanism of psoriasis remains to be fully elucidated.Hyperproliferative epidermis plays a crucial role in psoriasis.This study aimed to investigate the effects of interleukin-36β(IL-36β)on keratinocyte dysfunction in vitro.Methods Human keratinocyte cell lines,HaCaT cells,were treated with 0(control),50 or 100 ng/ml IL-36βrespectively for 24 h.Cell viability was determined with a cell counting kit-8 assay.Flow cytometry was used to assess the effects of IL-36βon apoptosis and cell cycle distribution.Expressions of the differentiation markers,such as keratin 10 and involucrin,were evaluated by quantitative real-time polymerase chain reaction(RT-qPCR).Expressions of the inflammatory cytokines,IL-1βand IL-6 were tested by ELISA.Results CCK8 assay showed the survival rate had no significant difference between the control and treated group(P>0.05).Flow cytometry analysis showed cell cycle arrest at S phase in the IL-36β-treated groups compared with the control group(P<0.05).RT-qPCR verified the decreased mRNA expressions of keratin 10 and involucrin in the IL-36β-treated groups compared with the negative control(P<0.01).ELISA showed 100 ng/ml IL-36βenhanced levels of IL-1βand IL-6 in culture supernatants of HaCaT cells compared with the negative control(P<0.05).Conclusion Taken together,these findings suggest that IL-36βcould induce cell cycle arrest at S phase,inhibit keratin 10 and involucrin expressions and promote inflammatory activity in HaCaT cell lines.

Photoprotective Effects of Hydroxychloroqine and TCMs on Human Keratinocytes Damaged from Ultraviolet Irradiation

Objective: To investigate damage effects of ultraviolet irradiation on eternal keratinocyte-HaCaT cells and to evaluate photo-protective efficiency of hydroxychloroqine and Traditional Chinese Medicines(epigallocatechingallate[EGCG], baikal skullcap root and szechwan lovge rhizome) on HaCaT cells damaged by middle wave ultraviolet(UVB) irradiation. Methods: Subconfluent HaCaT cells were sham or UVB irradiated and treated with above TCM agents. The damage degree of HaCaT cells was observed by a light microscop. Cell growth was recorded by cell count and cellular activity was detected by MTT method. The secretion amount of IL-6 and TNF-α was measured by ELISA. Results: The irradiation damage of HaCaT cells was depended on the irradiated dosages and cellular activity was reduced by 36%-80%, with a maximum decrease over 90% after 72 h. The intervention of the above drugs may increase the cellular activity by 10%-72%. The photo-protective efficiency was more apparent in EGCG (from 1.19±0.07 to 1.28±0.06, P<0.01) than that in hydroxychloroqine (from 0.43±0.04 to 0.96±0.04, P<0.05). The other two tested drugs also showed photo-protective effect(from 0.44±0.07 to 1.21±0.02, P<0.05). As to cytokine secretion, EGCG could decline the secretion amount of IL-6 and TNF-α apparently. Hydroxychloroqine and baikal skullcap root baikal skullcap root could only reduce the secretion of IL-6. The secretion of IL-6 and TNF-α could not be inhibited by szechwan lovge rhizome. Conclusion: The injury effect of UVB irradiation on cultured keratinocytes is dose-dependent and the tested drugs have photo-protective potency. Inhibition of cytokine secretion may be one of the mechanisms related to reducing the damage effect of UVB irradiation.

Effect of lead on IL-8 production and cell proliferation in human oral keratinocytes

Objective:To investigate the effect of lead on the production of IL-8 and cell proliferation in normal human oral keratinocytes（NHKs）.Methods:NHKs were prepared as outgrowths from normal human buccal mucosa.The cells were treated with three concentrations of lead glutamate(4.5×10-5M,4.5×10-6M and 4.5×10-7M).NHKs grown in glutamic acid were used as control.The amounts of IL-8 secreted in the culture supernatants were evaluated at 12 and 24 h using enzyme-linked immunospecific assay（ELISA）.Cell proliferation was determined by the MTT colorimetric assay.Three cultures were used for each experiment,and three independent experiments were performed.Analysis of variance and Duncan’s multiple range tests were used for statistical analysis.Results:An elevation of IL-8 in culture supernatants of NHKs treated with lead at all concentrations at 12 and 24 h after exposure in a dose-dependent manner was revealed.A significant increase in cell numbers was observed only at 24 h exposed to 4.5×10- 5M lead glutamate.Conclusions:The capacity of NHKs,to secrete IL-8,enhanced by lead glutamate,is demonstrated here.Induction of cell proliferation is revealed only after exposure to high lead concentration.The elevation of secreted IL-8 is a probable initial sign for the acute inflammatory response and may be involved in the pathogenesis of lead stomatitis.

Flow Cytometric Analysis of the Toxicity of Nitrofen in Cultured Keratinocytes

Lactate dehydrogenase (LDH) release test, 3 H-thymidine (3 H-TdR) and 3 H-leucine (3 H-Leu) incoopration tests and flow cytometric analysis (FCM) of cell cycle were empoyed to elucidate cellular and molecular mechanism of nitrofen-induced toxicity in cultured keratinocytes.The results showed that cell morphologic damages were observed after exposure to 1.0 mmol/L and 10.0 mmol/L nitrofen. LDH release increased in a dose- and time-dependent manner. Depressions in 3H -TdR and 3 H-Leu incorpration were found even at 0.01 mmol/L, and increased with the exposure dose. Cell cycle was analyzed from the DNA- histogram with propidium iodde stain. The results showed that there was no pronounced alteration in cell cycle after cells exposed to 0.01 and 0.1 mmol/L nitrofen. At dose of 1.0 mmol/L, S phase cells increased 2 times of that of control. With the increase of dose, G2/M phase cells became to increase about 5 times of that of the control. At 1 .0 mmol/L, time course of cell cycle after exposure was observed. At the beginning of exposure, cells in S phase and G2/M phase were about 8 .7 % and 11 %. Following 24 h incubation with nitrofen, cells in S phase increased to 18.0% with almost no change in G2/M. 72 h after exposure, G2/M phase cells increased to 63 .3%. The forve results demonstrated that S phase and G2/M phase blockage in cultured keratinocytes after exposed to nitrofen seems of importance in the mechanism of nitrofen-induced toxicity.

Effects of RNA Interference Combined with Ultrasonic Irradiation and SonoV ue Microbubbles on Expression of STAT3 Gene in Keratinocytes of Psoriatic Lesions

The most effective sequence of small interfering RNA（si RNA） silencing STAT3 of psoriatic keratinocytes（KCs） was screened out,and the effects of the most effective si RNA combined with ultrasonic irradiation and Sono Vue microbubbles on the expression of STAT3 of KCs and the dose-and time-response were investigated.Three chemically-synthetic si RNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs,and the effects on STAT3 expression were detected,then the most effective si RNA was selected for the subsequent experiments.The negative controls of siR NA（si RNA-NC） labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles were transfected into KCs,then the optimal parameters of ultrasonic irradiation were determined.The most effective si RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and Sono Vue microbubbles was transfected into KCs,and the dose-and time-response of RNA interference was determined.The effect of RNA interference by the most effective si RNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles（LUS group） was compared with that only carried by Lipofectamine 3000（L group）.The results showed that si RNA-3 achieved the highest silencing efficacy.0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation.The si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles could effectively knock down the STAT3 expression at m RNA and protein levels in dose-and time-dependent manners determined at 100 nmol/L with maximum downregulation on m RNA at 48 h,and on protein at 72 h after transfection.The LUS group achieved the highest silencing efficacy.It was concluded that si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoV ue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs,and the optimized transfection condition and the sequence of si RNA-3 could serve for further research on gene therapy of psoriasis.

Growth dynamics of individual clones of normal human keratinocytes: observations and theoretical considerations

The life histories of 429 individual epidermal keratinocyte clones picked at random were studied. Individual basal keratinocytes were derived from asynchronous rapidly proliferating subconfluent cultures propagated in either a low calcium (0.1mM) or a high calcium (2mM) serum-free medium. Single-celled clones were isolated by seeding trypsin-EDTA dissociated cells into a Petri dish containing cloning chips. Chips with only one cell per chip were transferred into dishes containing either low calcium or high calcium growth factor replete serum-free medium. Clone formation was monitored microscopically and the number of cells in each colony tallied at least twice daily for further analysis. A total of 369 clones were established from seven different neonatal foreskin cell strains (A-F), and 60 clones were derived from one adult human skin cell strain (G). During a five-day culture interval, among 32 clones of strain A, 83% divided at least once, 50% divided once in 24 hours, 86% divided at least three times within three days, and more than 50% divided at least four to five times in five days. Of 231 clones amongst the other five cell strains (B-F), an average of 63% (±12 S,E) divided more than three times in an eight day period, the remainder divided either once, twice or not at all. Of the 106 clones of strain G, reared in high calcium serum-free medium, 67% divided more than three times in a six-day period, and 55% divided five or more times in 6 days. Clones derived from adult skin strain H had a lower clone forming potential with 70% dividing at least once in seven days, and only 30% dividing three or more times. By contrast, the average generation time (AvGT) for second and third passage keratinocytes derived from neonatal foreskin cultures was 24 hrs. Detailed dendrograms were constructed for many of the proliferating clones. The majority of clones expressed a synblastic division pattern with every cell dividing at least once per day. A fraction of clones either exceeded this circadian division rate or displayed a biphasic division pattern with all cells initially dividing once a day and then abruptly slowing to once every other day or to an intermediate rate. A minority of clones was committed to a few terminal divisions. The division patterns of the non-synblastic clones fit an alternating bifurcated branching mode of clonal expansion expressed by the Fibonacci sequence for numbers of accumulated cells per clone per day. These results were analyzed in terms of deterministic, probabilistic and a limit cycle oscillator models of cell division timing.

Up-regulation of tight junction-related proteins and increase of human epidermal keratinocytes barrier function by Saccharomycosis ferment filtrate

Saccharomycopsis ferment filtrate (SFF), mainly used in skin care products, has been reported to inhibit inflammatory nitric oxide production and prevent epidermal damage. However, the effects of SFF on epidermal keratinocytes have not yet been explored. We investigated the effects of SFF on skin barrier function using human primary epidermal keratinocytes. Cell viability was determined by MTT assay. The mRNA and protein expression levels of tight junction proteins (claudin-1, -3, -4, occludin, ZO-1) were analyzed by RT-PCR and Western blotting, respectively. The effect of SFF on the barrier formation of epidermal keratinocytes was measured by transepithelial electrical resistance (TER). Rescue of cell death from H2O2 treatment was evaluated by annexin V staining. SFF, at concentrations that did not cause significant change of cell viability, induced dose-dependent cell-cell adhesion and formation of an organized monolayer structure. Pretreatment of keratinocytes with EGTA, a Ca2+ chelator, did not inhibit the cell-cell adhesion of keratinocytes by SFF, indicating a Ca2+-independent mechanism. The mRNA and protein levels of claudin-1 in keratinocytes were up-regulated by SFF treatment in a dose-dependent manner. The expressions of other tight junctions (TJs) including claudin-3 & 4, occludin and ZO-1 were also similarly increased in SFF-treated epidermal keratinocytes. The promoting effect of SFF on the barrier function of epidermal keratinocytes was further confirmed by the increased TER value in SFF-treated epidermal keratinocytes. Annexin V staining confirmed that SFF markedly decreased the number of dead cells resulted from H2O2 injury. Taken together, our results provided the first evidence that SFF enhanced keratinocytes barrier function by increasing the expression of TJs and TER.

Differential Anticancer Effect of an Apple Extract (Applephenon^&reg), Polyphenols and Isoflavones on Normal Human Keratinocytes and Epidermoid Cancer Cells

Applephenon&reg, a purified extract prepared from green apples, was examined for its cytotoxicity and inhibitory effects on the proliferation of cultures of normal human keratinocytes and several epidermoid cancer cell lines. Our HPLC studies demonstrated a high content of phenolic compounds (>65%), including catechin, epicatechin, caffeic acid and phloretin as well as polyphenols such as proanthocyanidins. Applephenon&reg demonstrated a greater cytotoxic effect against HeLa, A431 cancer cell lines and HaCaT, an immortalized keratinocyte cell line than serum-free cultures of proliferating normal human keratinocytes (NHK). Proliferation of NHK was inhibited at concentrations above 0.0013% while concentrations above 0.005% were cytotoxic. By contrast, Applephenon&reg solutions above 0.00025% killed each of the cancer cell lines. Treated cells displayed increased intercellular separation and evidence of keratinizing stratification. We also tested the effect of epicatechin, and two isoflavonoids, genistein and daidzein, on cancer cell lines. Hela cells were more sensitive to epicatechin and genistein inhibition of cell growth and cytotoxicity than were NHK. Daidzein at these concentrations had little effect on cancer cells. These results indicate that Applephenon&reg and some of its phenolic components have selective anticancer activity.

Induction of Phase II Enzymes Glutathione-S-Transferase and NADPH: Quinone Oxydoreductase 1 with Novel Sulforaphane Derivatives in Human Keratinocytes: Evaluation of the Intracellular GSH Level

Phase II enzymes including NADPH: Quinone Oxydoreductase 1 (NQO1) and Glutathione-S-Transferase (GST) represents a major and natural cellular protection system against deleterious environmental factors which cause skin damages. Sulforaphane is one of the most popular isothiocyanates found in cruciferous vegetables and known for its cytoprotective effects by inducing Phase II enzymes. Five novel sulforaphane derivatives were synthetized and tested for their activity on NQO1 and GST induction as well as for their effect on total GSH intracellular level using colorimetric assays on human keratinocytes cell line (HaCat). As sulforaphane and the synthetized components showed variable toxicity after their evaluation by means of in vitro cytotoxicity (MTT test), cells were treated at a concentration of 5 μM during 48 hours. The results showed that the addition products of sulforaphane decreased cytotoxity but none of those derivatives had a better effect than referenced sulforaphane on Phase II enzymes. It seems that the isothiacyanate function remains important for the sulforaphane activity.

Retinoid and Ethanol-Sensitive Benzo(<i>α</i>)Pyrene Induction of Cytochrome P450 in Human Keratinocytes

Polycyclic aromatic hydrocarbons (PAHs) induce cytochrome P-450 monoxygenase enzymes that catalyze the formation of DNA adducts. We investigated the effects benzo(α)pyrene (B[α]P) alone or in combination with ethanol on normal human keratinocyte (NHK) growth, induction of cytochrome P-4501A1 (CYP1A1), and modulation of these treatments by retinoic acid (RA) in a serum-free culture medium. Growth-arrested confluent NHK serum-free cultures were treated with B[α]P alone or in combination with ethanol and RA. The effects on CYP1A1 enzyme activity were investigated. B[α]P treatment alone was not toxic to post-confluent cells;sub-toxic ethanol stimulated cell growth regardless B[α]P treatment. No CYP1A1 activity was detected in control or ethanol-treated NHK cell cultures. B[α]P alone induced CYP1A1 activity, and B[α]P plus ethanol treatment further enhanced B[α]P-induced CYP1A1 activity. Pretreatment with all-trans-RA (t-RA) abolished ethanol enhancement of CYP1A1 activity. There is a synergistic action of ethanol in combination with PAH on induction of P-450 cytochrome enzymes. By contrast, RA reverses ethanol enhancement implying a role for retinoid therapy in counteracting the risk posed by combined alcohol and PAH exposure on epidermal cell carcinogenesis.

Mouthwash with Active Oxygen (blue^®m) Induces Keratinocytes Proliferation

Chlorhexidine is widely used in dentistry to treat various gingival conditions. Its side effects are widely described. Histologically, the gingiva consists of a stratified squamous epithelium, with a predominance of keratinocytes, the latter being fundamental in healing processes. Thus, the objective of this in vitro study was to evaluate the effects of a new product with active oxygen (blue®m) on keratinocytes. Keratinocytes (HACAT) were incubated with different concentrations of blue®m (1, 10 and 100 μl/ml), and another well was used as a control, without the presence of mouthwash. After 24, 48 and 72 hours, cell proliferation was analyzed by CyQUANT®. It was possible to observe that lower concentrations (1 μl/ml) of blue®m increased cell proliferation in HACAT cell lines, while moderate and higher concentrations of mouthwash may present a cytotoxic effect. This is the first in vitro study with showing that human keratinocytes cell line demonstrated greater proliferation rate when exposed to lower concentrations of blue®m mouthwash.

Modulation of Sodium-Dependent Transporters Expression in Normal Human Keratinocytes by a Sodium Rich Isotonic Thermal Water

Background/Aim: In order to show that water can participate to the skin defense in front of different stress, we investigated the effect of an isotonic thermal water notably rich in Sodium (i.e. the Uriage thermal water) on 1) The taurine transporter (TauT) expression in human normal keratinocytes irradiated or not by UVB;and 2) the Sodium-dependent vitamin C transporter 1 (SVCT1) expression in human normal keratinocytes issued from two “young” and two “aged” subjects, irradiated or not by UVB. Methods and Results: Using sensible and specific TAUT and SVCT1 ELISA assays developed in house, we provide 1) the unambiguous demonstration that the Uriage thermal water is able to help the epidermis to maintain its taurine content under UVB irradiation;2) the first example of an altered SVCT1 expression in “aged” keratinocytes and of a significant positive effect of the Uriage thermal water on this altered SVCT1 production;and 3) arguments showing that Uriage thermal water is also able to participate to the regulation of the SVCT1 production in UVB-irradiated keratinocytes. Conclusion: Taking together, these results suggest that the Uriage thermal water could act to efficiently protect the skin from dehydration through its effect on TauT and SVCT1 expression, and furthermore, to allow a more efficient taurine and ascorbic acid supplying to the epidermis in order to protect him from other aggressions such as oxidant stress for example.

Transient Expression of Human Papillomavirus Type 16(HPV 16) mRNA in Normal Human Keratinocytes Transfected by pSV2-neo/16

Objective: To investigate the expression of HPV16 mRNA innormal human keratinocytes transfected with pSV2-neo/16. Methods: First human keratinocytes were cultured in theserum-free medium M154.Second, the plasmid pSV2-neo/16was transfected into the human keratinocytes using atransfecting reagent. Third, RT-PCR and Southern Blottingwere used to detect the expression of HPV16 mRNA and DNAin the transfected keratinocytes, respectively. Results: The expression of HPV 16 mRNA was successfullyamplified and an 110bp was detected by RT-PCR. A 7.9kbfragment was confirmed in the transfected keratinocytes bySouthern Blot analysis. Conclusion: HPV 16 mRNA and DNA were successfullydetected in the human keratinocytes.

Lipoxin A4 Inhibits Lipopolysaccharide-induced Production of Inflammatory Cytokines in Keratinocytes by Up-regulating SOCS2 and Down-regulating TRAF6

Liopxin A4（LXA4） is considered to be a crucial modulator in the inflammatory responses. In the present study, we aimed to study the effect of LXA4 on the inflammatory cytokines production induced by lipopolysaccharide（LPS） and the possible mechanism in normal human epidermal keratinocytes（NHEKs）. NHEKs were isolated and cultured. The expression of toll-like receptor 4（TLR4）, LXA4 receptor（ALXR） and aryl hydrocarbon receptor（Ah R） in NHEKs was detected by reverse transcription polymerase chain reaction（RT-PCR）. The m RNA and protein levels of tumor necrosis factor-alpha（TNF-α） and interleukin-1β（IL-1β） were determined in NHEKs stimulated by LPS（10 μg/m L） with or without preincubation with LXA4（100 nmol/L） for 30 min by real-time quantitative PCR（real-time q PCR） and enzyme-linked immunosorbent assay（ELISA）, respectively. The expression levels of tumor necrosis factor receptor-associated factor 6（TRAF6） and suppressors of cytokine signaling 2（SOCS2） m RNAs and proteins, and nuclear translocation of NF-k B-p65 were measured by real-time q PCR and Western blotting, respectively. The results showed that NHEKs expressed TLR4, ALXR and Ah R. LXA4 significantly inhibited the m RNA and protein expression levels of TNF-α, IL-1β and TRAF6 induced by LPS in NHEKs, and LXA4 obviously increased the expression of SOCS2 at m RNA and protein levels. The nuclear NF-k B-p65 protein expression induced by LPS was inhibited after preincubation with LXA4 in NHEKs. It was concluded that LXA4 inhibits the LPS-induced production of TNF-α and IL-1β in NHEKs by up-regulating SOCS2 and down-regulating TRAF6.

Enhancing effect of tazarotene on the HLA-DR expression of cultured human keratinocytes induced by interferon-gamma

Objective: To investigate the effect of tazarotene on the expression of HLA-DR induced by IFN-γ. Methods: (1) Keratinocytes from normal human skin were cultured in vitro;(2) Tazarotene, IFN-γ and the combination of the two compounds were incubated with the keratinocytes in medium, respectively. The expression of HLA-DR in keratinocytes was determined using immunocytochemistry techniques at 24h after incubation. Results: (1) There was rare expression of HLA-DR in normal human keratinocytes; (2) 10 -6mol/L tazarotene failed to induce the expression of HLA-DR in keratinocytes at 24h after incubation; (3) 500 U/ml IFN-γ obviously induced the HLA-DR expression in keratinocytes at 24h after treatment; (4) After 24h, 10 -7-10 -5 mol/L tazarotene had a significantly enhancing effect on the expression of HLA-DR induced by IFN-γ (P<0.005). Conclusion: Tazarotene up-regulates the expression of HLA-DR in keratinocytes cultured in vitro when combined with IFN-γ . Therefore, the reduction of HLA-DR positive keratinocytes in psoriatic lesions may be attributed to not direct interaction of tazarotene in combination with IFN-γ but other pathways.

KGF-transfected cells can stimulate growth and proliferation of human cultured keratinocytes in vitro

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The effect of keratinocyte growth factor-2(KGF-2) on the proliferation of human keratinocytes

Objective;In vitro studies have shown that KGF-2 has a proliferative effect on neonatal foreskin kerati-nocytes.Cells from adult donors have been shown to respond to KGF-1 to a lesser degree than neonatal keratino-cytes.The purpose of the study was to investigate the proliferative effect of KGF-2 on keratinocytes from an adultsubject.Methods;Standard medium was Keratinocyte Growth Medium without BPE,hydrocortisone and EGF.Ke-ratinocytes cultured from a 48-year-old subject were seeded at 2 10~4 in 32 mm ...

Biological properties of differently-aged human keratinocytes:population doubling time growth curve and cell cycle analysis

Objective To explore the biological properties of keratinocytes from differently-aged healthy human beings. Methods Keratinocytes from fetus,teenager and middle-aged groups were separated and cultured. The population doubling time (PDT) and cell growth curve in different cells were compared,and the cell cycles were analyzed by flow cytometry. Results ① In primary culture of keratinocytes,the adherence time in middle-aged group was longer than that in fetus and teenager groups. However,all cell morphology showed no obvious differences. In subculture of keratinocytes,with donator’s age increasing,time of cell adherence prolonged,passage number decreased and differences in cell morphology were obvious. ② The average PDT of keratinocytes was shorter in fetus group than in teenager and middle-aged groups. But difference in cell growth curve between different passages was not observed. ③ Keratinocytes showed G2/M period in fetus group but G0/G1 period in teenager and middle-aged groups mainly. Conclusion As age increases,the biological properties of keratinocytes change obviously.