Quantification of In Vivo Epidermal Keratinocyte Architecture Associated with the Signs of Skin Aging and the Skin Benefit Evaluation by Application of Galactomyces Ferment Filtrate (Pitera)-Containing Skin Care Product

Background: Aged skin exhibits visual alterations such as wrinkles, rough texture, pore dilation, and dull skin tone, as well as physiological aging, namely, decreased hydration and increased transepidermal water loss (TEWL). Recent advances in coherence tomography have also revealed that skin aging affects in vivo epidermal keratinocyte architecture. However, the interconnectivity between spatial architectural aging and visual/physiological aging parameters remains largely unknown. Purpose: To elucidate whether the tomographic keratinocyte architectural aging is correlated with visual and physiological skin aging parameters and to quantitatively evaluate the improvements of the architectural, visual, and physiological aging parameters by the daily treatment of the skin care formula containing Galactomyces Ferment Filtrate (GFF, 8X PiteraTM). Method: We measured the in vivo keratinocyte cellular architecture with two-photon stereoscopic tomography obtaining by-layer epidermal section images in 78 Asian females of various ages. Visual aging parameters were analyzed using a portable image capture system. Hydration and TEWL were also assessed. The anti-aging effects of GFF-containing skin moisturizer (SK-II LXP CreamTM) were also examined in two studies after twice-daily application for 2 (N = 35) and 4 (N = 32) weeks. Results: As for the keratinocyte cellular architecture, skin aging was significantly associated with decreased cell density and increased cell uniformity. These architectural aging parameters were significantly correlated with visual and physiological aging parameters, namely, rough texture, wrinkles, pore dilation, dull skin tone, dehydration, and increased TEWL. The strong interconnectivity allowed us to develop formulae to estimate the keratinocyte architecture from visual aging parameters. Moreover, twice-daily application of SK-II significantly improved the keratinocyte architecture associated with multiple skin aging visual and physiological parameters. Conclusion: Skin aging is a process involving mutual interconnections among epidermal keratinocyte cellular architecture, visual, and physiological parameters. The GFF-containing moisturizer SK-II effectively improves spatial architecture of keratinocytes in epidermis and these evaluated skin aging parameters in a new trajectory over the course of treatment. .

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.

c-Myc、CDK12在胃癌组织中的表达及临床意义

目的 探讨胃癌组织中c-Myc、细胞周期蛋白依赖性激酶12(CDK12)表达及其与患者临床特征及预后的关系。方法 收集80例胃癌患者的胃癌组织和癌旁组织标本,采用免疫组化法检测标本中的c-Myc、CDK12表达水平。比较胃癌组织和癌旁组织中c-Myc、CDK12阳性表达情况;分析胃癌组织中c-Myc、CDK12阳性表达与患者临床病理特征的关系;分析c-Myc与CDK12阳性表达的相关性,胃癌组织中c-Myc、CDK12阳性表达与胃癌患者预后的关系。结果 胃癌组织中c-Myc、CDK12阳性表达率(77.5%、87.5%)均明显高于癌旁组织(13.8%、15.0%)(P<0.05)。不同年龄、性别、肿瘤最大直径患者胃癌组织中c-Myc、CDK12阳性表达率比较差异无统计学意义(P>0.05);中低分化、Ⅲ~Ⅳ期、侵犯浆膜、有淋巴结转移患者胃癌组织中c-Myc和CDK12阳性表达率分别为88.0%、87.0%、88.9%、90.0%和94.0%、92.6%、97.2%、100.0%,均明显高于高分化、Ⅰ~Ⅱ期、未侵犯浆膜、无淋巴结转移患者胃癌组织的60.0%、57.7%、68.2%、70.0%和76.7%、76.9%、79.5%、80.0%(P<0.05)。相关分析发现,c-Myc、CDK12在胃癌组织中的表达呈正相关性(r=0.487,P=0.016<0.05)。胃癌组织中c-Myc、CDK12阳性患者的3年生存率分别为19.4%、21.4%,均明显低于c-Myc、CDK12阴性患者的55.6%、70.0%(P<0.05)。结论 c-Myc、CDK12在胃癌组织中异常高表达,两者呈正相关性,并与肿瘤的TNM分期、分化程度、肿瘤侵袭深度及淋巴结转移有关,对患者的预后有明显影响,通过检测两者水平可能评估胃癌患者的预后。

胃癌模型大鼠胃黏膜组织Cyclin D1、c-Myc、CKIT的表达意义及其与胃癌病变程度的相关性

目的探讨胃癌模型大鼠胃黏膜组织细胞周期蛋白D1(cyclinD1)、c-Myc、CKIT的表达意义及其与胃癌病变程度的相关性。方法选择48只健康成年雌性大鼠,按照随机数字表法将其分为对照组、研究1组、研究2组、研究3组,每组各12只。研究1组、研究2组、研究3组均制备成胃癌模型。对照组给予等量0.9%氯化钠溶液,第24周处死取材;研究1组、研究2组、研究3组制备成胃癌大鼠后分别于第8、16、24周取材。分析各组大鼠一般情况及胃黏膜组织病理切片,采用蛋白质印迹法检测胃黏膜组织Cyclin D1、c-Myc、CKIT蛋白的表达,采用逆转录聚合酶链式反应(RT-PCR)法测定胃黏膜组织Cyclin D1、c-Myc、CKIT mRNA的表达,采用Spearman分析法测定胃黏膜组织Cyclin D1、c-Myc、CKIT表达与胃癌病变程度相关性。结果对照组大鼠胃黏膜完整正常,外膜层、肌层、黏膜下层、黏膜层等结构清晰,且无炎症细胞浸润;研究1组大鼠胃黏膜组织与对照组大鼠接近,不存在炎症细胞,且黏膜腺体结构基本正常;研究2组大鼠胃黏膜组织存在破损,细胞核变大,基底部部分腺体细胞形态异常,存在轻度异型性,为早期胃癌;研究3组大鼠胃黏膜组织增加破损,核质比变大,细胞形态不规则,部分腺体存在扩张,黏膜下层及肌层存在炎症细胞浸润,为胃癌进展期。研究1组、研究2组、研究3组胃黏膜组织Cyclin D1、c-Myc、CKIT蛋白均呈显著高于对照组,差异均有统计学意义(P<0.05),胃黏膜组织Cyclin D1、c-Myc、CKIT蛋白在研究1组、研究2组、研究3组中逐渐升高趋势。研究1组、研究2组、研究3组胃黏膜组织Cyclin D1、c-Myc、CKIT mRNA均呈显著高于对照组,差异均有统计学意义(P<0.05),胃黏膜组织Cyclin D1、c-Myc、CKIT mRNA在研究1组、研究2组、研究3组中逐渐升高趋势。采用Spearman相关性结果分析显示,胃黏膜组织Cyclin D1、c-Myc、CKIT表达与胃癌病变程度呈正相关(r=0.382、0.781、0.993,均P<0.001)。结论胃癌模型大鼠胃黏膜组织Cyclin D1、c-Myc、CKIT呈高表达,其与胃癌病变程度密切相关。

Cultured keratinocyte grafting on various biologic matrices

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Keratinocyte growth factor-2 and autologous serum potentiate the regenerative effect of mesenchymal stem cells in cornea damage in rats

AIM:To investigate the healing process after severe corneal epithelial damage in rats treated with mesenchymal stem cells(MSCs)cultured with or without keratinocyte growth factor(KGF-2)and autologous serum(AS)on amniotic membrane(AM).Many patients are blind and devastated by severe ocular surface diseases due to limbal stem cell deficiency.Bone marrow-derived MSCs are potential sources for cellbased tissue engineering to repair or replace the corneal tissue,having the potential to differentiate to epithelial cells.METHODS:The study included 5 groups each including 10 female'Sprague Dawley'rats in addition to20 male rats used as bone marrow donors.Group I rats received AM+MSCs,Group II rats AM+MSCs cultured with KGF-2,Group III rats AM+MSCs cultured with KGF-2+AS,Group IV rats only AM and Group V rats,none.AS was derived from blood drawn from male rats and bone marrow was obtained from the femur and tibia bones of the same animals.Therapeutic effect was evaluated with clinical,histopathological and immunohistochemical assessment.MSC engraftment was demonstrated via detection of donor genotype(Y+)in the recipient tissue(X)with polymerase chain reaction.RESULTS:Corneal healing was significantly better in Groups I-III rats treated with MSC transplantation compared to Group IV and Group V rats with supportive treatment only.The best results were obtained in Group III rats with 90%transparency,70%lack of neovascularization,and 100%epithelium damage limited to less than 1/4 of cornea.CONCLUSION:We suggest that culture of MSCs with KGF-2 and AS on AM is effective in corneal repair in case of irreversible damage to limbal stem cells.

Keratinocyte growth factor gene therapy ameliorates ulcerative colitis in rats

AIM:To investigate the effect of keratinocyte growth factor(KGF) gene therapy in acetic acid-induced ulcerative colitis in rat model.METHODS:The colitis of Sprague-Dawley rats was induced by intrarectal infusion of 1 mL 5%(v/v) acetic acid.Twenty-four hours after exposed to acetic acid,rats were divided into three experimental groups:control group,attenuated Salmonella typhimurium Ty21a strain(SP) group and SP strain carrying human KGF gene(SPK) group,and they were separately administered orally with 10% NaHCO3,SP or SPK.Animals were sacrificed and colonic tissues were harvested respectively on day 3,5,7 and 10 after administration.Weights of rats,colonic weight/length ratio and stool score were evaluated.Histological changes of colonic tissues were examined by hematoxylin and eosin(HE) staining method.The expression of KGF,KGF receptor(KGFR) and TNF-α were measured either by enzyme-linked immunosorbent assay or Western blotting.Immunohistochemistry was used to detect the cellular localization of KGFR and Ki67.In addition,superoxide dismutase(SOD) activity and malondialdehyde(MDA) contents in the homogenate were measured.RESULTS:Body weight and colonic weight/length ratio were declined in SPK group compared with SP and control groups(body weight:272.78 ± 17.92 g vs 243.72 ± 14.02 g and 240.68 ± 12.63 g,P < 0.01;colonic weight/length ratio:115.76 ± 7.47 vs 150.32 ± 5.99 and 153.67 ± 5.50 mg/cm,P < 0.01).Moreover,pathological changes of damaged colon were improved in SPK group as well.After administration of SPK strain,KGF expression increased markedly from the 3rd d,and remained at a high level till the 10th d.Furthermore,KGFR expression and Ki67 expression elevated,whereas TNF-α expression was inhibited in SPK group.In the group administered with SPK,SOD activity increased significantly(d 5:26.18 ± 5.84 vs 18.12 ± 3.30 and 18.79 ± 4.74 U/mg,P < 0.01;d 7:35.48 ± 3.35 vs 22.57 ± 3.44 and 21.69 ± 3.94 U/mg,P < 0.01;d 10:46.10 ± 6.23 vs 25.35 ± 4.76 and 27.82 ± 6.42 U/mg,P < 0.01) and MDA contents decreased accordingly(d 7:7.40 ± 0.88 vs 9.81 ± 1.21 and 10.45 ± 1.40 nmol/mg,P < 0.01;d 10:4.36 ± 0.62 vs 8.41 ± 0.92 and 8.71 ± 1.27 nmol/mg,P < 0.01),compared with SP and control groups.CONCLUSION:KGF gene therapy mediated by attenuated Salmonella ameliorates ulcerative colitis induced by acetic acids,and it may be a safe and effective treatment for ulcerative colitis.

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.

Related gene expressions in anti-keratinocyte aging induced by Ganoderma lucidum polysaccharides

Objective:To examine the level of expression of anti skin aging gene of Ganoderma lucidum polysacchayides and clarify its mechanism with anti aging of this ancient Chinese medicine.Methods:HacaT cell of keratinocytes lines were cultured and treated with the polysaccharides.The total RNA was extracted with Trizol reagent and cDNA was synthesized by reverse thanscription.The obtained cDNAs were then fluorescently labeled with cy3 and cy5 respectively and hybridized with gene expressing pedigree cDNA chip.The images were scanned and analyzed with special software.The scan data were analyzed with software and checked by real time PCR.Results:Among total 18 346 human genes,the expression of 103 ones was up-regulated and 378 ones down-regulated.It was demonstrated evidently that Ganoderma lucidum polysaccharides affected the expression of genes of anti skin aging.Two ways are anastomotic.Conclusion:it is concluded by analysis of function of these up-regulation and down-regulation genes that Ganoderma lucidum polysaccharides may play an important role in boosting cell growth and against skin aging.It shows that the results of gene array reliable by real time PCR.

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.

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.

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.

Activated protein C: A regulator of human skin epidermal keratinocyte function

Activated protein C(APC) is a physiological anticoagulant, derived from its precursor protein C(PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor(EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal keratinocytes. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-2 activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of keratinocyte monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal growth factor receptor or Tie2. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APC's function in skin epidermis/keratinocytes and its therapeutical potential in skin inflammatory conditions.

Issues within Keratinocyte Growth Factor （KGF） research

As the seventh member of Fibroblast Growth Factor （FGF） family, Keratinocyte Growth Factor （KGF or FGF-7） is observed tp mediate epithelial cell proliferation and differentiation in a variety of tissues. In this article, such following issues within KGF research were reviewed, as （1） KGF functioning pathways： experimental results demonstrated the paracrine pathway of KGF played main role in mesen- chymal-epithelial interactions whereas KGF itself was under the control of a feedback regulation, autocrine provided KGF alternative functioning way particularly in tumourogenesis; （2） KGF in apoptosis： a few of investigations recently illustrated KGF mediated cell survival was based on its mitogenic function via stimulating cell growth, moreover KGF could inhibit the ROS-induced apoptosis through Nrf-2 pathway; （3） KGF during tumourogenesis： high expression of KGF enhanced progression, motility and invasiveness of tumor cells and various cancers, in company with paracrine loop replaced by autocrine loop, meanwhile KGF clearly played the early signal in the progression of breast cancer; （4） Medical application and administration of KGF： KGF had been successfully used in several preclinical models of radiation and chemotherapy-induced mucositis, and developed into commercial medicine （i.e. Palifermin ）, however more effective delivery systems are still under trial.

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.

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.

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.

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.

华蟾素通过调控MYH9/USP7/c-MYC通路抑制急性髓系白血病细胞免疫逃逸

【目的】探讨华蟾素调控肌球蛋白重链9(MYH9)/泛素特异性蛋白酶7(USP7)/骨髓细胞瘤病毒癌基因(c-MYC)通路对急性髓系白血病(AML)细胞免疫逃逸的影响。【方法】(1)体内实验:建立裸鼠异种移植瘤模型,评估华蟾素对AML细胞在体内生长和免疫逃逸的影响。(2)体外实验:使用不同浓度的华蟾素处理人AML细胞株HL-60,细胞计数试剂盒8(CCK-8)法检测细胞活力,Transwell实验检测细胞侵袭能力。将HL-60细胞与活化的CD8^(+)T细胞共培养,流式细胞术检测CD8^(+)T细胞表面标志物CD25的表达,酶联免疫吸附分析(ELISA)检测共培养上清液中细胞因子[白细胞介素2(IL-2)和干扰素(IFN-γ)]的水平,CytoTox96非放射性细胞毒性分析评估CD8^(+)T细胞对HL-60细胞的毒性。Western Blot法检测MYH9、USP7和c-MYC的蛋白表达,免疫共沉淀(Co-IP)法检测MYH9、USP7和泛素化之间的相互作用。转染MYH9过表质粒,验证华蟾素在AML中的作用机制。【结果】华蟾素抑制裸鼠移植瘤生长,增强CD8^(+)T细胞抗肿瘤的能力。华蟾素浓度依赖性地抑制HL-60细胞活力、侵袭。华蟾素处理后上调CD8^(+)T细胞表面标志物CD25的表达,同时还上调IL-2和IFN-γ水平。华蟾素增强CD8^(+)T细胞对HL-60细胞的毒性。华蟾素抑制HL-60细胞MYH9、USP7和c-MYC的蛋白表达,MYH9通过募集USP7促进c-MYC去泛素化,华蟾素抑制MYH9介导的c-MYC去泛素化。【结论】华蟾素可通过抑制MYH9的表达进而减少去泛素化酶USP7对c-MYC的募集,促进c-MYC泛素化降解,从而抑制AML细胞免疫逃逸。

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.