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Objective:To investigate protective effect of Pinus massoniana needle extract (PMNE) against oxidative stress in human dermal papilla cells (HDPC) , and to explore its mechanisms. Methods:As research objects, some cultured HDPC were treated with H 2O 2 at different concentrations of 0 (control group) , 0.1, 0.2, 0.4, 0.8 and 1.0 mmol/L, in order to establish the optimal condition for in vitro oxidative stress in HDPC; some other HDPC were transfected with nuclear factor-erythroid 2-related factor 2 (Nrf2) specific small interfering RNAs (siRNA1, siRNA2, siRNA3) or a Nrf2-overexpressing plasmid (pCMV6-XL5-Nrf2) , the HDPC transfected with a scrambled-siRNA and an empty plasmid pCMV6-XL5 served as the control siRNA group and control plasmid group respectively, and HDPC subjected to conventional culture served as the blank group; after the above treatment, real-time fluorescence-based quantitative PCR and Western blot analysis were performed to determine Nrf2 mRNA and protein expression, respectively; cell viability and apoptosis were detected in the above transfected cells after the treatment with H 2O 2 at an optimal concentration. In the subsequent experiment, some HDPC were divided into several groups: control group subjected to conventional culture, dihydrotestosterone group treated with 0.03 μg/ml dihydrotestosterone, proanthocyanidin group treated with 0.03 μg/ml dihydrotestosterone and 6.00 μg/ml proanthocyanidin B2, PMNE groups treated with 0.03 μg/ml dihydrotestosterone and PMNE at different concentrations of 1, 5, 25 and 100 μg/ml; after the above treatment, cell viability and apoptosis were detected, relative fluorescence intensity of intracellular reactive oxygen species (ROS) , malondialdehyde (MDA) content, mRNA and protein expression of Nrf2, quinone oxidoreductase 1 (NQO1) , heme oxygenase 1 (HO-1) , Kelch-like ECH-related protein 1 (Keap1) , transforming growth factor (TGF) -β1, Sma- and Mad-related protein 2/3 (Smad2/3) , phosphorylated Smad2/3 (p-Smad2/3) were determined in HDPC. One-way analysis of variance was used for comparisons among multiple groups, and least significant difference- t test for multiple comparisons. Results:The viability of HDPC ranged from 75% to 85% after the treatment with 0.4 mmol/L H 2O 2, which was selected as the optimal condition for in vitro oxidative stress in HDPC. Compared with the blank group and control siRNA group, the Nrf2-siRNA1, Nrf2-siRNA2, Nrf2-siRNA3 groups showed significantly decreased Nrf2 mRNA and protein expression (all P < 0.05) , but significantly increased apoptosis rate (Nrf2-siRNA1, Nrf2-siRNA2, Nrf2-siRNA3 groups, blank group and control group: 12.50% ± 0.05%, 26.07% ± 0.05%, 58.44% ± 1.03%, 10.38% ± 0.64%, 13.05% ± 0.12%, respectively; all P < 0.05) . Nrf2 protein expression was the lowest in the Nrf2-siRNA2 group, so Nrf2-siRNA2 was selected as the optimal interfering fragment for subsequent experiments. Compared with the blank group and control plasmid group, the Nrf2 overexpression group showed significantly increased Nrf2 mRNA and protein expression (both P < 0.05) , but a significantly decreased apoptosis rate (all P < 0.05) . After the treatment with 0.4 mmol/L H 2O 2, the Nrf2 overexpression group showed a significantly decreased apoptosis rate, but significantly increased cell viability compared with the empty vector group ( t = 3.66, 40.40, respectively, both P < 0.001) ; the Nrf2-siRNA2 group showed a significantly increased apoptosis rate, but significantly decreased cell viability compared with the control group ( t = 13.13, 67.37, respectively, both P < 0.001) . In the PMNE treatment experiment, the proanthocyanidin group and PMNE groups showed significantly increased cell viability, but significantly decreased apoptosis rates compared with the dihydrotestosterone group (all P < 0.01) ; proanthocyanidin and PMNE at different concentrations could significantly inhibit dihydrotestosterone-induced overexpression of ROS and MDA in HDPC (all P < 0.01) ; the protein expression of Nrf2, NQO1 and HO-1 was significantly higher in the proanthocyanidin group, 5-, 25- and 100-μg/ml PMNE groups than in the dihydrotestosterone group (all P < 0.05) , while the protein expression of Keap1 and TGF-β1, and the Smad2/3 phosphorylation level were significantly lower in the proanthocyanidin group, 25- and 100-μg/ml PMNE groups than in the dihydrotestosterone group (all P < 0.05) . Conclusion:Nrf2 plays an important role in protecting against oxidative damage in HDPC, and PMNE may exert marked protective effect on HDPC by activating the Nrf2-antioxidant responsive element signaling pathway.
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Objective:To investigate the potential anti-aging mechanism of 9-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-associated β-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 pRb 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.
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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-associated β-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 400 μM 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 anti-senescence and anti-wrinkle activities in vitro.
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At present, acellular matrix is an effective replacement material for the treatment of skin damage, but there are few systematic evaluation studies on its performance. The experimental group of this study used two decellularization methods to prepare the matrix: one was the acellular matrix which sterilized with peracetic acid first (0.2% PAA/4% ethanol solution) and then treated with hypertonic saline (group A), the other was 0.05% trypsin/EDTA decellularization after γ irradiation (group B); and the control group was soaked in PBS (Group C). Then physical properties and chemical composition of the three groups were detected. Hematoxylin eosin (HE) staining showed that the acellular effect of group B was good. The porosity of group A and B were both above 84.9%. In group A, the compressive modulus of elasticity was (9.94 ± 3.81) MPa, and the compressive modulus of elasticity was (12.59 ± 5.50) MPa in group B. There was no significant difference between group A or B and group C. The total content of collagen in acellular matrix of group A and B was significantly lower than that of group C (1. 662 ± 0.229) mg/g, but there was no significant difference in the ratio of collagen Ⅰ/Ⅲ between group B and group C. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that there was no significant difference in microstructure. Qualitative detection of fibronectin and elastin in each group was basically consistent with that in group C. Therefore, acellular matrix of group B had better performance as scaffold material. The experimental results show that the acellular matrix prepared by γ-ray sterilization and decellularization of 0.05% Trypsin enzyme/EDTA could be used for the construction of tissue-engineered skin. It could also provide reference for the preparation and mounting of heterogeneous dermal acellular matrix. It was also could be used for electrostatic spinning or three-dimensional printed tissue engineered skin scaffold which could provide physical and chemical parameters for it.
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Acellular Dermis , Cells, Cultured , Extracellular Matrix , Porosity , Tissue Engineering , Tissue ScaffoldsABSTRACT
In postmenopausal women, oral or topical administration of estradiol increases skin thickness and collagen synthesis,such as collagen type 1 alpha 1 (COL1A1) and collagen type 3 alpha 1 (COL3A1). Due to undesirable side effectsof estradiol, such as risks of breast and endometrium pathology, topical phytoestrogens are alternative treatments foraging-related skin changes. Phytoestrogen is a nonsteroidal substance derived from plants, like fenugreek (Trigonellafoenum-graceum L.), which has an estrogen like composition that appears to mimic estradiol. The mechanism ofaction remains unknown, especially in fibroblast-associated COL1A1 and COL3A1 production. In vitro experimentswere conducted using postmenopausal women's fibroblasts with estrogen receptor (ER) antagonists. Cell isolationused explant and enzymatic techniques with ELISA kit (MyBioSource, California) for COL1A1 and COL3A1. Pairedstudent t-tests compared results between control (no treatment), fenugreek extract 2 µg/ml alone, fenugreek extract 2µg/ml with receptor antagonists for ERα, ERβ, and both receptors. Greater suppresion of COL1A1 and COL3A1 wereshown by both antagonists ERα / ERβ group and antagonist ERβ group compared to antagonist ERα group. Theseresults indicate that the fenugreek increases secretion of COL1A1 and COL3A1 through ERα, ERβ, and is mainlymediated by ERβ in post menopausal women’s fibroblasts.
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Objective@#To study the protective effect of adipose-derived stem cell conditioned medium against ultraviolet radiation B (UVB)-induced photoaging and replicative senescence in human dermal fibroblasts (HDFs).@*Methods@#HDFs were cultured and passaged in vitro, and treated with ADSC-CM after being irradiated once with UVB. The mRNA expression of CollagenⅠ(Col1), CollagenⅢ(Col3) and Elastin were detected by real-time PCR to define the anti-aging effects of ADSC-CM. The protein expressions of phosphorylated c-Jun N-terminal kinase (p-JNK) and p-p38 were detected by Western blot.@*Results@#①Both successive passage and UVB irradiation reduced the expression of Col1, Col3 and Elastin in HDFs.②ADSC-CM inhibited the reduction of Col1, Col3 and Elastin protein expressions induced by successive passage and UVB. ③ADSC-CM activated p38 and JNK pathway. Downregulation of p38 MAPK by SB203580 decreased the mRNA expression of Col1, Col3, and Elastin. Inhibiting JNK by SP600125 did not induce significant ECM changes.@*Conclusions@#Both intrinsic and extrinsic stimuli can decrease the expression of collagen and elastin, common markers of skin aging in HDFs, and ADSC-CM can attenuate the above decreasing and promoting the expression of ECM by p38 signaling pathway.
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AIM:To investigate the effect of sulodexide(SDX)on the apoptosis of human dermal microvascu-lar endothelial cells(HDMECs)exposed to hypoxia and its underlying mechanism.METHODS:The HDMECs were cul-tured and divided into normoxia control group cultured under normoxic condition;hypoxia control group cultured in a humid incubator maintained at 37 ℃with 5% CO2and 1% O2for 24 h;treatment groups treated with SDX at 0.25,0.5 and 1 LSU/mL for 24 h under hypoxic condition.The cell viability was measured by CCK-8 assay.The apoptotic rate of the HD-MECs was analyzed by flow cytometry.The activity of caspase-3 in HDMECs was examined by caspase-3 activity assay kit. The expression of pro-apoptotic factor P53,caspase-3,Bax and anti-apoptotic factor Bcl-2 at mRNA and protein levels was determined by real-time PCR and Western blot.RESULTS:SDX increased the viability of HDMECs exposed to hypoxia, but also decreased the apoptosis.Furthermore, SDX down-regulated the expression of pro-apoptotic factor P53, Bax and caspase-3 at mRNA and protein levels as well as the activity of caspase-3,while the expression of anti-apoptotic factor Bcl-2 was up-regulated.CONCLUSION:SDX significantly increases the viability and decreases the apoptosis of HDMECs ex-posed to hypoxia.Inhibition of the mitochondrial apoptosis pathway may be involved in the underlying mechanism.
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@#Introduction: Induced pluripotent stem cells (iPSC) that exhibit embryonic stem cell-like properties with unlimited self-renewal and multilineage differentiation properties, are a potential cell source in regenerative medicine and cell-based therapy. Although retroviral and lentiviral transduction methods to generate iPSC are well established, the risk of mutagenesis limits the use of these products for therapeutic applications. Materials and Methods: In this study, reprogramming of human dermal fibroblasts (NHDF) into iPSC was carried out using non-integrative Sendai virus for transduction. The iPSC clones were characterised based on the morphological changes, gene expression of pluripotency markers, and spontaneous and directed differentiation abilities into cells of different germ layers. Results: On day 18-25 post-transduction, colonies with embryonic stem cell-like morphology were obtained. The iPSC generated were free of Sendai genome and transgene after passage 10, as confirmed by RT-PCR. NHDF-derived iPSC expressed multiple pluripotency markers in qRT-PCR and immunofluorescence staining. When cultured in suspension for 8 days, iPSC successfully formed embryoid body-like spheres. NHDF-derived iPSC also demonstrated the ability to undergo directed differentiation into ectoderm and endoderm. Conclusion: NHDF were successfully reprogrammed into iPSC using non-integrating Sendai virus for transduction.
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Objective To study the effects of hypoxia on the cell migration,apoptosis and expression of related genes and proteins of human dermal microvascular endothelial cells(HDMEC). Methods To culture HDMEC in hypoxia condition by putting CoCl2in the cell incubator and all the subjects were divided to two groups which included normal group and hypoxia group.To find the appropriate CoCl2dose by CCK-8 experiment and the 200 μmol/L CoCl2was the best to establish hypoxia model.The migration of cells was measured by wound healing test.Apoptosis rate was detec-ted by flow cytometry. The gene expressions were detected by RT-PCR and the protein levels were detected by West-ern blot. Results The migration ability of cells was enhanced in hypoxia condition (P<0.05). The apoptosis rate was increased in hypoxia condition(P<0.05).The gene expressions of HIF-1α,HIF-1β,VEGF,iNOS were increased comparing with normal groups depending on time(P<0.05).The protein expressions of HIF-1α,VEGF,iNOS were in-creased comparing with normal groups depending on time(P<0.05). Conclusions Cell migration, apoptosis of HDMEC were influenced by hypoxia. It may be explained by increasing expression of related gene and proteins like HIF-1α,VEGF,iNOS in hypoxia condition.
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Objective To study the effects of hypoxia on the cell migration,apoptosis and expression of related genes and proteins of human dermal microvascular endothelial cells(HDMEC). Methods To culture HDMEC in hypoxia condition by putting CoCl2in the cell incubator and all the subjects were divided to two groups which included normal group and hypoxia group.To find the appropriate CoCl2dose by CCK-8 experiment and the 200 μmol/L CoCl2was the best to establish hypoxia model.The migration of cells was measured by wound healing test.Apoptosis rate was detec-ted by flow cytometry. The gene expressions were detected by RT-PCR and the protein levels were detected by West-ern blot. Results The migration ability of cells was enhanced in hypoxia condition (P<0.05). The apoptosis rate was increased in hypoxia condition(P<0.05).The gene expressions of HIF-1α,HIF-1β,VEGF,iNOS were increased comparing with normal groups depending on time(P<0.05).The protein expressions of HIF-1α,VEGF,iNOS were in-creased comparing with normal groups depending on time(P<0.05). Conclusions Cell migration, apoptosis of HDMEC were influenced by hypoxia. It may be explained by increasing expression of related gene and proteins like HIF-1α,VEGF,iNOS in hypoxia condition.
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BACKGROUND: The human dermal papilla cells (hDPCs) play an important role in regulation of hair cycling and growth. OBJECTIVE: The aim of this study was to investigate the effect of different wavelengths of light-emitting diode (LED) irradiation on the proliferation of cultured hDPCs and on the growth of human hair follicles (HFs) in vitro. METHODS: We examined the effect of LED irradiation on Wnt/β-catenin signaling and mitogen-activated protein kinase (MAPK) pathways in hDPCs. Anagen HFs were cultured with LED irradiation and elongation of each hair shaft was measured. RESULTS: The most potent wavelength in promoting the hDPC proliferation is 660 nm and 830 nm promoted hDPC proliferation to a lesser extent than 660 nm. Various wavelengths significantly increased β-catenin, Axin2, Wnt3a, Wnt5a and Wnt10b mRNA expression. LED irradiation significantly increased β-catenin and cyclin D expression, and the phosphorylation of MAPK and extracellular signal-regulated kinase (ERK). HFs irradiated with 415 nm and 660 nm grew longer than control. CONCLUSION: Our result suggests that LED has a potential to stimulate hDPC proliferation via the activation of Wnt/β-catenin signaling and ERK pathway. To our best knowledge, this is the first report which investigated that the effect of various wavelengths of LED on hDPC proliferation and the underlying mechanisms.
Subject(s)
Humans , Cyclin D , Extracellular Signal-Regulated MAP Kinases , Hair Follicle , Hair , In Vitro Techniques , MAP Kinase Signaling System , Phosphorylation , Phosphotransferases , Protein Kinases , RNA, MessengerABSTRACT
Objective:To reveal the primary mechanism of changing permeability in DENV-2 infected pHDMECs. Methods:pHDMECs was incubated by DENV-2 on the concentration of 103 TCID50 ,and the penetrability of the cell was detected by Transwell at 4,8,12,24,48 h,respectively. Then,the partial sequence of DENV-2 NS1 was analyzed by Real time-PCR,and NS1 protein was detected by immunofluorescence and flow cytometer (FCM). The apoptosis rate of pHDMECs was assayed by FCM. Finally,IL-6 and IL-8 secreted by pHDMECs were analyzed by Real time-PCR and double antibody sandwich ELISA. Results:The relative expression of NS1 gene was elevated but NS1 protein was not detected;the permeability of DENV-2 infected pHDMECs had dramatically increased both at 24,48 h,but the apoptosis rate has little changed even been influenced by DENV-2 at 72 h. However,the relative expression of IL-6/IL-8 mRNA was boosted at 8,24 h[(2. 49±0. 50) and (6. 82±1. 69) fold,respectively,P<0. 05]. In protein level,compared with control(869. 6±50. 70)pg/ml,IL-6 secreted by DENV-2 infected pHDMECs could reach by(1 248. 8±86. 9)pg/ml(P<0. 05),and IL-8 was(1 331. 0±86. 3)pg/ml(P<0. 05) while the control was (967. 6±156. 6)pg/ml. Conclusion:Indeed,pHDMECs can be infected by DENV-2;the increasing permeability of DENV-2 infected pHDMECs may not be caused by the pHDMECs′ apoptosis but the enhancing of pro-inflammatory cytokine IL-6 /IL-8.
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BACKGROUND: Dihydrotestosterone (DHT) induces androgenic alopecia by shortening the hair follicle growth phase, resulting in hair loss. We previously demonstrated how changes in the microRNA (miRNA) expression profile influenced DHT-mediated cell death, cell cycle arrest, cell viability, the generation of reactive oxygen species (ROS), and senescence. Protective effects against DHT have not, however, been elucidated at the genome level. OBJECTIVE: We showed that epigallocatechin gallate (EGCG), a major component of green tea, protects DHT-induced cell death by regulating the cellular miRNA expression profile. METHODS: We used a miRNA microarray to identify miRNA expression levels in human dermal papilla cells (DPCs). We investigated whether the miRNA expression influenced the protective effects of EGCG against DHT-induced cell death, growth arrest, intracellular ROS levels, and senescence. RESULTS: EGCG protected against the effects of DHT by altering the miRNA expression profile in human DPCs. In addition, EGCG attenuated DHT-mediated cell death and growth arrest and decreased intracellular ROS levels and senescence. A bioinformatics analysis elucidated the relationship between the altered miRNA expression and EGCG-mediated protective effects against DHT. CONCLUSION: Overall, our results suggest that EGCG ameliorates the negative effects of DHT by altering the miRNA expression profile in human DPCs.
Subject(s)
Humans , Aging , Alopecia , Cell Cycle Checkpoints , Cell Death , Cell Survival , Computational Biology , Dihydrotestosterone , Genome , Hair , Hair Follicle , MicroRNAs , Reactive Oxygen Species , TeaABSTRACT
BACKGROUND: Various health benefits have been attributed to Er-Miao-San (EMS), a traditional Chinese herbal formulation that contains equal amounts of cortex phellodendri (Phellodendron amurense Ruprecht) and rhizoma atractylodis (Atractylodes lancea D.C). However, its effect on the anti-inflammatory activity in human dermal fibroblasts (HDFs) and the mechanism underlying this effect are unknown. RESULTS: This study investigated the effects of EMS on TNF-α-induced MMP-1 expression in HDFs. Our data show that EMS inhibited TNF-α-induced MMP-1 expression in a concentration-dependent manner. Interestingly, EMS maintained IkB content without inhibiting the phosphorylation of MAPKs, which are well-established upstream kinases of NF-kB. Moreover, EMS reduced the level of nuclear p65 protein in HDFs. Luciferase assay revealed that EMS inhibits the transcriptional activity of NF-kBbystabilizing IkB. Our results show that EMS exerts its anti-inflammatory effect by inhibiting NF-kB-regulated genes such as IL-1ß and IL-8. Moreover, EMS effectively inhibited TNF-α-induced expression of MMP-1 via the NF-kBpathway. CONCLUSIONS: Taken together, our data suggest that EMS could potentially be used as an anti-inflammatory and anti-aging treatment.
Subject(s)
Humans , Aging/drug effects , Drugs, Chinese Herbal/pharmacology , Plant Extracts/pharmacology , Dermis/cytology , Matrix Metalloproteinase 1/biosynthesis , Fibroblasts/drug effects , Signal Transduction/drug effects , Cell Line , Cell Survival/drug effects , Enzyme Induction/drug effects , Gene Expression Regulation/drug effects , Interleukin-8/drug effects , Interleukin-8/metabolism , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/metabolism , Mitogen-Activated Protein Kinases/drug effects , Interleukin-1beta/drug effects , Interleukin-1beta/metabolism , Real-Time Polymerase Chain Reaction , Fibroblasts/enzymology , Anti-Inflammatory Agents/administration & dosageABSTRACT
Objective To investigate the expression of specific marker molecules in hair-inducing activity of long-term cultured human dermal papilla cells (HDPCs) in vitro.Methods After dissected and cultured the HDPCs in vitro,the cells of passages 1 to 8 were used for experiments.The growth appearances of HDPCs in different passages were observed under inverted microscope.To detect the expression of specific marker molecules of long-term cultured HDPCs,the alkaline phosphatase (ALP) activity of the HDPCs was examined,and the specific genes ALP and insulin-like growth factor-1 (IGF-1) expression levels of HDPCs were determined by real-time quantitative PCR.Results After long-term cultured in vitro,the ALP and IGF-1 expression levels of HDPCs gradually decreased in different passages,as well as the display of the aggregated and cartouche growth.The ALP and IGF-1 expression levels of HDPCs in passage 1 was the highest,they were almost about 6.8-fold and 3.5-fold higher than the HDPCs in passage 8.The ALP staining of the HDPCs in passage 1 and passage 2 were evident,but the cells' ALP staining gradually became much weaker than the cells in the previous passages after the long-term cultured in vitro.Conclusions The expression levels of specific marker molecules ALP and IGF-1 of the HDPCs decrease gradually after long-term cultured in vitro,and the higher passage HDPCs lost the special aggregated and cartouche growth appearance,and hence lead to the loss of hair-inducing activity of HDPCs.
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Oral administration of royal jelly (RJ) promotes wound healing in diabetic mice. Concerns have arisen regarding the efficacy of RJ on the wound healing process of normal skin cells. In this study, a wound was created by scratching normal human dermal fibroblasts, one of the major cells involved in the wound healing process. The area was promptly treated with RJ at varying concentrations of 0.1, 1.0, or 5 mg/ml for up to 48 hrs and migration was analyzed by evaluating closure of the wound margins. Furthermore, altered levels of lipids, which were recently reported to participate in the wound healing process, were analyzed by HPTLC and HPLC. Migration of fibroblasts peaked at 24 hrs after wounding. RJ treatment significantly accelerated the migration of fibroblasts in a dose-dependent manner at 8 hrs. Although RJ also accelerated the migration of fibroblasts at both 20 hrs and 24 hrs after wounding, the efficacy was less potent than at 8 hrs. Among various lipid classes within fibroblasts, the level of cholesterol was significantly decreased at 8 hrs following administration of both 0.1 ug/ml and 5 mg/ml RJ. Despite a dose-dependent increase in sphinganines, the levels of sphingosines, ceramides, and glucosylceramides were not altered with any concentration of RJ. We demonstrated that RJ enhances the migration of fibroblasts and alters the levels of various lipids involved in the wound healing process.
Subject(s)
Animals , Humans , Mice , Administration, Oral , Ceramides , Cholesterol , Chromatography, High Pressure Liquid , Fatty Acids , Fibroblasts , Glucosylceramides , Skin , Sphingosine , Wound HealingABSTRACT
Solar ultraviolet (UV) irradiation leads to distinct changes in the skin connective tissues by degradation of collagen, which is a major structural component in the extracellular matrix. UV irradiation induces the production of matrix metalloproteinases (MMP) capable of attacking native fibrillar collagen and responsible for inhibiting the construction of collagenous extracellular matrix. In this study, we attempted to investigate the protective actions of Rubus coreanus ethanol extract (RCE) on the MMP production and the consequent procollagen/collagen degradation in UV-B-irradiated human dermal fibroblasts. The analytical data showed that Rubus coreanus ethanol extract was mostly comprised of cyanidin 3-rutinoside. Pre-treatment of fibroblasts with this extract inhibited UV-B-induced production of MMP-1, MMP-8 and MMP-13 in dose-dependent manners. In addition, Western blot analysis and immunocytochemical staining assay revealed that RCE markedly augmented the cellular levels of procollagen/collagen declined in UV-B-exposed dermal fibroblasts. These results demonstrate that RCE blocks UV-B-induced increase of the collagen degradation by inhibiting MMP production. Thus, RCE may act as an agent inhibiting excessive dermal collagen degradation leading to the skin photoaging.
Subject(s)
Humans , Blotting, Western , Collagen , Connective Tissue , Ethanol , Extracellular Matrix , Fibrillar Collagens , Fibroblasts , Matrix Metalloproteinases , SkinABSTRACT
Employing electrophysiological and cell proliferation assay techniques, we studied the effects of Ca2+ -activated K+ channel modulators on the proliferation of human dermal fibroblasts, which is important in wound healing. Macroscopic voltage-dependent outward K+ currents were found at about -40 mV stepped from a holding potential of -70 mV. The amplitude of K+ current was increased by NS1619, a specific large-conductance Ca2+ -activated K+ (BK) channel activator, but decreased by iberiotoxin (IBTX), a specific BK channel inhibitor. To investigate the presence of an intermediate-conductance Ca2+ -activated K+ (IK) channels, we pretreated the fibroblasts with low dose of TEA to block BK currents, and added 1-EBIO (an IK activator). 1-EBIO recovered the currents inhibited by TEA. When various Ca2+ -activated K+ channel modulators were added into culture media for 1~3 days, NS1619 or 1-EBIO inhibited the cell proliferation. On the other hand, IBTX, clotrimazole or apamin, a small conductance Ca2+ -activated K+ channel (SK) inhibitor, increased it. These results suggest that BK, IK, and SK channels might be involved in the proliferation of human dermal fibroblasts, which is inversely related to the channel activation.
Subject(s)
Humans , Apamin , Cell Proliferation , Clotrimazole , Culture Media , Fibroblasts , Hand , Tea , Wound HealingABSTRACT
It is not determined yet whether hyaluronic acid up- regulates or down-regulates wound healing. This study was designed to define the effect of hyaluronic acid on proliferation of human dermal fibroblasts in vitro and on skin wound closing in vivo. Fibroblasts were isolated from the dermis of adults and cultivated in the presence of either one of 6 concentrations of hyaluronic acid(0, 0.1, 0.2, 0.5, 1.0, 2.0 mg/ml). The fibroblasts were seeded at 2.0 x 10(4) cells/well in Dulbecco's Modified Eagle's Medium/Ham's F-12 nutrient including 10% fetal bovine serum with either one of 6 different concentrations of hyaluronic acid in 24-well plates. The cells were incubated for 6 days. All concentrations of hyaluronic acid stimulated the proliferation of fibroblasts. The best proliferation was seen at 0.2 mg/ml of hyaluronic acid concentration(p = 0.01). For in vivo study, 10 white rats were used. A 5 mm round punch was employed to excise skin and subcutaneous tissue at eight sites on the back. After creating 8 open wounds, 8 concentrations(0, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mg/ml) of hyaluronic acid were applied. The degrees of wound closing were compared the 6th day under light microscope. Low concentration of hyaluronic acid(0 - 2.0 mg/ml) stimulated the wound closing. However, high concentration of hyaluronic acid(5.0 -10.0 mg/ml) delayed the wound closing. The best wound closing was seen at 0.5 mg/ml of hyaluronic acid (p = 0.032). These results demonstrated that hyaluronic acid influenced human dermal fibroblast proliferation and the skin wound closing in rats, and its concentration was critically important factor.
Subject(s)
Adult , Animals , Humans , Rats , Dermis , Fibroblasts , Hyaluronic Acid , Skin , Subcutaneous Tissue , Wound Healing , Wounds and InjuriesABSTRACT
Granulocyte-Macrophage colony-stimulating factor(GM- CSF) is naturally generated protein that stimulates the survival, proliferation, and differentiation of myeloid progenitor cells. The determination of the molecular sequence of this protein by recombinant DNA technology enabled us to produce sufficient quantity for preclinical and clinical use. In the animal studies, rhGM-CSF to wounds has been reported to result in increased formation of granulation tissue, increased breaking strength, and reversal of wound contraction. A number of case reports have been published on the favorable effect of rhGM-CSF as a treatment for infected, nonhealing wounds, and ulcers. However, there are no clinical reports about the effect of GM-CSF on wound healing in normal patients. Therefore, in this report, we examined the effect of GM-CSF on the proliferation of human dermal fibroblasts which play a crucial role in wound healing process in vitro. To determine an optimal GM-CSF concentration for human dermal fibroblast proliferation, the cells were incubated with either one of 13 concentrations of GM-CSF(0 - 30mug/ml). The media used in this study was DMEM/F- 12(GIBCO, Grand Island, NY, USA). The fibroblasts were seeded at 1.5 x 104 cells/well in 500mul of medium including 10% fetal bovine serum and either one of 13 concentrations of GM-CSF in 24-well plates. The cells were incubated for 6 days at 5% CO2, 100% humidity at 37degrees C. On the 6th day of plating, fibroblast proliferation was determined by hematocytometer. Each concentration was tested 8 times.Low concentration of GM-CSF(below 5.0mug/ml) stimulated the proliferation of human dermal fibroblasts. How ever, high concentration of GM-CSF(over 10mug/ml) downregulated the proliferation of human dermal fibroblasts. The best fibroblast proliferation was seen at 1.0mug/ml of GM- CSF. These results demonstrated that GM-CSF influenced human dermal fibroblast proliferation and the GM-CSF concentration was critically important factor in vitro.