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1.
Reactions of keratinocytes to in vitro millimeter wave exposure.   总被引:2,自引:0,他引:2  
The effects of millimeter waves (MW) on human keratinocytes were studied in vitro using the HaCaT keratinocyte cell line. MW-induced modulation of keratinocyte function was studied in proliferation, adhesion, chemotaxis, and interleukin-1beta (IL-1beta) production assays. Spontaneous proliferation, adhesion to tissue culture plate, random migration, and IL-8- and RANTES induced chemotaxis were not affected by exposure of cells to millimeter waves under the following conditions: frequency, 61.22 GHz; SAR, 770 W/kg; duration of exposure, 15-30 min. However, MW irradiation resulted in a modest but statistically significant increase in the intracellular level of IL-1beta. These data suggest that exposure of human skin (with keratinocytes being the major component of epidermis) to MW can cause activation of basal keratinocytes resulting in an elevated level of IL-1beta production.  相似文献   

2.
Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co‐cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm2), an increase in melanin synthesis whereas in melanocyte mono‐cultures, UVB doses up to 50 mJ/cm2 had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono‐ and co‐cultures. Removing certain keratinocyte growth factors from the co‐culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte–melanocyte co‐cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB‐induced pigmentation, (ii) UVA‐induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV‐induced pigmentation in vitro.  相似文献   

3.
3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') produces acute hyperthermia which increases the severity of the selective serotoninergic neurotoxicity produced by the drug in rats. Heat shock protein 70 (Hsp70) is a major inducible cellular protein expressed in stress conditions and which is thought to exert protective functions. MDMA (12.5 mg/kg, i.p.), given to rats housed at 22 degrees C, produced an immediate hyperthermia and increased Hsp70 in frontal cortex between 3 h and 7 days after administration. MDMA, given to rats housed at low ambient temperature (4 degrees C) produced transient hypothermia followed by mild hyperthermia but no increase in Hsp70 expression, while rats treated at elevated room temperature (30 degrees C) showed enhanced hyperthermia and similar expression of Hsp70 to that seen in rats housed at 22 degrees C. Fluoxetine-induced inhibition of 5-HT release and hydroxyl radical formation did not modify MDMA-induced Hsp70 expression 3 h later. Four- or 8-day heat shock (elevation of basal rectal temperature by 1.5 degrees C for 1 h) or geldanamycin pre-treatment induced Hsp70 expression and protected against MDMA-induced serotoninergic neurotoxicity without affecting drug-induced hyperthermia. Thus, MDMA-induced Hsp70 expression depends on the drug-induced hyperthermic response and not on 5-HT release or hydroxyl radical formation and pre-induction of Hsp70 protects against the long-term serotoninergic damage produced by MDMA.  相似文献   

4.
Heat shock protein (Hsp) 25 is a member of the small Hsp family. High levels of Hsp25 can be detected in skin. During adult epidermis differentiation, the concentration of Hsp25 increases as the distance of keratinocytes from the basal layer increases, in parallel with the extent of keratinization. We previously showed that Hsp25, mouse keratin (MK) 5, and MK14 participated in the formation of characteristic ring-shaped aggregates during the differentiation of the PAM212 keratinocyte cell line. We suggested that Hsp25 was involved in the disorganization of the MK5-MK14 keratin network before the establishment of the MK1-MK10 keratin network at the beginning of epidermis stratification. In this study, we have investigated the distribution of Hsp25 and keratins throughout skin development. We demonstrate that the distribution of Hsp25 and MK5 in the epidermis at the beginning of stratification and before keratinization is similar to that observed in PAM212 keratinocytes. These results indicate that there is a strong correlation between the mechanism we described ex vivo and the events taking place in vivo. Moreover, we show that Hsp25 is produced in different cell types in the epidermis and in the hair follicle at different stages of their development. Thus, our results suggest that Hsp25 is involved in more than one process during skin development.  相似文献   

5.
FAS/CD95/Apo-1 is a ubiquitously expressed cell-surface receptor involved in the initiation of programmed cell death. Its function in epidermal keratinocytes has been incompletely defined. Available evidence from in vitro studies points to important roles of Fas in the pathogenesis of contact dermatitis and in keratinocyte apoptosis induced by ultraviolet light. To define functions of Fas in the epidermis in vivo, we have generated mice with epidermis-specific deletion of the fas gene and tested its requirement for 2,4-dinitrofluorobenzene-induced contact dermatitis and for ultraviolet light B (UVB)-induced keratinocyte apoptosis. We report here our unexpected finding that keratinocyte apoptosis induced by both a contact allergen and UVB irradiation was significantly enhanced in Fas-negative epidermis. Expression of Fas by epidermal keratinocytes was neither necessary for the normal development of contact hypersensitivity of the skin, nor required for keratinocyte apoptosis following UVB irradiation. Our study results thus show that in the epidermis in vivo Fas exerts antiapoptotic effects that outweigh its proapoptotic role in contact hypersensitivity responses of the skin and in the tissue response of the epidermis to UVB irradiation.  相似文献   

6.
Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co-cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm2), an increase in melanin synthesis whereas in melanocyte mono-cultures, UVB doses up to 50 mJ/cm2 had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono- and co-cultures. Removing certain keratinocyte growth factors from the co-culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte-melanocyte co-cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB-induced pigmentation, (ii) UVA-induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV-induced pigmentation in vitro.  相似文献   

7.
IL-4 enhances keratinocyte expression of CXCR3 agonistic chemokines   总被引:6,自引:0,他引:6  
IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC) belong to the non-glutamate-leucine-arginine motif CXC chemokine family and act solely through the CXCR3 receptor for potent attraction of T lymphocytes. In this study, we evaluated the capacity of the T cell-derived cytokines IL-4, IL-10, and IL-17 to modulate IP-10, Mig, and I-TAC in cultured human keratinocytes and CXCR3 expression in T cells from allergic contact dermatitis (ACD). IL-4, but not IL-10 or IL-17, significantly up-regulated IFN-gamma- or TNF-alpha-induced IP-10, Mig, and I-TAC mRNA accumulation in keratinocytes and increased the levels of IP-10 and Mig in keratinocyte supernatants. Immunohistochemistry of skin affected by ACD revealed that >70% of infiltrating cells were reactive for CXCR3 and that CXCR3 staining colocalized in CD4+ and CD8+ T cells. Nickel-specific CD4+ and CD8+ T cell lines established from ACD skin produced IFN-gamma and IL-4 and expressed moderate to high levels of CXCR3. Finally, CXCR3 agonistic chemokines released by stimulated keratinocytes triggered calcium mobilization in skin-derived nickel-specific CD4+ T cells and promoted their migration, with supernatant from keratinocyte cultures stimulated with IFN-gamma and IL-4 attracting more efficaciously than supernatant from keratinocytes activated with IFN-gamma alone. In conclusion, IL-4 exerts a proinflammatory function on keratinocytes by potentiating IFN-gamma and TNF-alpha induction of IP-10, Mig, and I-TAC, which in turn may determine a prominent recruitment of CXCR3+ T lymphocytes at inflammatory reaction sites.  相似文献   

8.
In rat portal veins (RPV) isolated from septic rats, we previously showed that the contractile response to angiotensin II (AT(II)) was significantly decreased and that the vascular failure was correlated with the severity of the disease. We hypothesized that hyperthermia might be one of the factors responsible for the vascular failure. Moreover, hyperthermia should concomitantly increase heat shock proteins (Hsps) expression. We then compared the vascular contractility and the heat shock protein 70 (Hsp70) expression in RPV incubated at 37 degrees C and 39.5 degrees C and sought for a relationship between both events. In our experimental model, hyperthermia increased the Hsp70 expression and decreased the contractile response to AT(II). Incorporation of the Hsp70 antisense oligonucleotide in RPV blocked the increase in Hsp70 expression but had no consequence on the contractile response to AT(II). In conclusion, hyperthermia increases Hsp70 expression but does not mediate the decreased response to AT(II). Hsp70 overexpression has no effect on the actin-myosin interaction in vascular smooth muscle.  相似文献   

9.
10.
Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70–80% confluence, they begin to float up into the overlying medium and are called “epithelial pop-up keratinocytes (ePUKs)” allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.  相似文献   

11.
Reconstructed pigmented epidermis was established by co‐seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air–liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3‐isobutyl‐1‐methyl‐xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.  相似文献   

12.
Reconstructed pigmented epidermis was established by co-seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air-liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3-isobutyl-1-methyl-xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.  相似文献   

13.
14.
In recent years, possible health hazards due to radiofrequency radiation (RFR) emitted by mobile phones have been investigated. Because several publications have suggested that RFR is stressful, we explored the potential biological effects of Global System for Mobile phone communication at 900 MHz (GSM-900) exposure on cultures of isolated human skin cells and human reconstructed epidermis (hRE) using human keratinocytes. As cell stress markers, we studied Hsc70, Hsp27 and Hsp70 heat shock protein (HSP) expression and epidermis thickness, as well as cell proliferation and apoptosis. Cells were exposed to GSM-900 under optimal culture conditions, for 48 h, using a specific absorption rate (SAR) of 2 W x kg(-1). This SAR level represents the recommended limit for local exposure to a mobile phone. The various biological parameters were analysed immediately after exposure. Apoptosis was not induced in isolated cells and there was no alteration in hRE thickness or proliferation. No change in HSP expression was observed in isolated keratinocytes. By contrast, a slight but significant increase in Hsp70 expression was observed in hREs after 3 and 5 weeks of culture. Moreover, fibroblasts showed a significant decrease in Hsc70, depending on the culture conditions. These results suggest that adaptive cell behaviour in response to RFR exposure, depending on the cell type and culture conditions, is unlikely to have deleterious effects at the skin level.  相似文献   

15.
Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)-induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m2 whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/m2 with a maximum at 40 J/m2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m2. These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation.  相似文献   

16.
Heat shock protein 70 (Hsp70), a protein induced in cells exposed to sublethal heat shock, is present in all living cells and has been highly conserved during evolution. The aim of the current study was to determine the role of heat shock proteins in the resistance of prostate carcinoma cell line spheroids to hyperthermia. In vitro, the expression of Hsp70 by the DU 145 cell line, when cultured as monolayer or multicellular spheroids, was studied using Western blotting and enzyme-linked immunosorbent assay methods. The level of Hsp70 in spheroid cultures for up to 26 days at 37 degrees C remained similar to monolayer cultures. However, in samples treated with hyperthermia at 43 degrees C for 120 min, the spheroid cultures expressed a higher level of Hsp70 as compared to monolayer culture. Under similar conditions of heat treatment, the spheroids showed more heat resistance than monolayer cultures as judged by the number of colonies that they formed in suspension cultures. The results suggest that cells cultured in multicellular spheroids showed more heat resistance as compared to monolayer cultures by producing higher levels of Hsp70.  相似文献   

17.
It has been demonstrated that hyperthermia protects keratinocytes from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if keratinocytes become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal keratinocytes (NHEK) and the carcinoma-derived cell line A431 were exposed to either 42 degrees C or to 15dPGJ2 for 4 hours at 24-hour intervals for 4 consecutive days. The intracellular level of the 72-kDa heat shock protein (Hsp72) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ2 treatment, and survival was determined 24 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ2 are potent inducers of Hsp72 expression and lead to increased resistance to UVB-induced cell death in human keratinocytes; (2) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp72 and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ2 is enhanced by repeated exposure without a further increase of Hsp72; (4) repeated heat shock and 15dPGJ2 up to a concentration of 1 microg/mL have no influence on cell growth over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing factors, stress tolerance can be maintained without additional toxicity in human keratinocytes. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.  相似文献   

18.
People exposed to sunlight can develop erythema, DNA damage, and photoimmunosupression. Extended exposure of normal epidermis to sunlight will induce dysmorphic keratinocytes with pyknotic nuclei scattered throughout the spinous layer. These 'sunburn cells' are apoptotic keratinocytes and are usually cleared within 48 hours after sunburn. Patients with lupus erythematosus, however, whether it be the discoid, subacute cutaneous, systemic, or tumid form, develop new cutaneous lesions and can experience systemic worsening of their disease. Are sunlight-induced keratinocyte apoptosis and the immune response to these cells abnormal in lupus patients?  相似文献   

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