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R E S U M E N En pacientes con ortodoncia aparecen eventos patológicos no deseados como agrandamiento gingival inducido por tratamiento de ortodoncia (AGTO) o hipertrofia gingival. El objetivo del estudio es identificar la distribución inmunohistoquímica de citoqueratina CK-14, CK-19 y Ki-67 en epitelio gingival de pacientes con AGTO. Se seleccionaron I3 pacientes divididos en: grupo control (n=6), conformado por individuos periodontalmente sanos no portadores de aparatología ortodóntica y grupo test (n=7), integrado por pacientes con AGTO. Los marcadores CK-14, CK-19 y Ki-67 fueron identificados mediante inmunohistoquímica con anticuerpos monoclonales y observados en un microscopio óptico Leica DM 500. En los pacientes del grupo test el tejido epitelial se mostró hipertrófico con pérdida en la continuidad de la membrana basal. La CK-14 y CK-19 fue positiva en el epitelio de todos los sujetos evaluados, con una expresión positiva de alta intensidad en células de la lámina basal del grupo test. El promedio de células positivas para Ki-67 en el grupo test fue de 56%. En conclusión, la CK-14, CK-19 y Ki-67 son marcadores con elevada inmunoreactividad en tejido gingival de pacientes con AGTO portadores de ortodoncia.
During orthodontic treatment, unwanted pathological events such as gingival overgrowth induced by orthodontic treatment or gingival hypertrophy may appear The objective of this study is to identify immunohistochemical distribution of cytokeratin CK-14, CK-19 and Ki-67 in the gingival epithelium of patients with gingival overgrowth induced by orthodontic treatment. Thirteen patients were selected divided into: control group (n = 6), conformed of periodontally healthy individuals without orthodontic appliances and the test group (n = 7), conformed of patients with gingival overgrowth induced by orthodontic treatment. The biomarkers CK-14, CK-19 and Ki-67 were identified by immunohistochemistry with monoclonal antibodies and observed in a Leica DM 500 optical microscope. Hypertrophic epithelial tissue with loss of continuity of the basement membrane was found in the test group patients. CK-14 and CK-19 were positive in the epithelial tissue of all the subjects evaluated, with a high intensity positive expression in the cells of the basal lamina of the test group. The average number of cells positive for Ki-67 in test group was 56%. In conclusion, CK-14, CK-19 and Ki-67 are biomarkers with high immunoreactivity in the gingival tissue of patients with gingival overgrowth induced by orthodontic treatment.
Durante o tratamento ortodôntico, eventos patológicos indesejados como o crescimento gengival induzido pelo tratamento ortodôntico (CGTO) ou hipertrofia gengival podem aparecer: O objetivo deste estudo é identificar a distribuição imuno-histoquímica das citoqueratinas CK -14, CK-19 e Ki-67 no epitélio gengival de pacientes com CGTO. Foram selecionados 13 pacientes divididos em: grupo controle (n=6), conformado por indivíduos periodontalmente saudáveis sem aparelhos ortodônticos e o grupo teste (n=7), conformado por pacientes com CGTO. Os biomarcadores CK-14, CK-19 e Ki-67 foram identificados por imuno-histoquímica com anticorpos monoclonais e observados em microscópio óptico Leica DM 500. Tecido epitelial hipertrófico com perda de continuidade da membrana basal foi encontrado nos pacientes do grupo teste. CK-14 e CK-19 foram positivos no tecido epitelial de todos os sujeitos avaliados, com expressão positiva de alta intensidade nas células da lâmina basal do grupo teste. O número médio de células positivas para Ki-67 no grupo teste foi de 56%. Em conclusão, CK-14, CK-19 e Ki-67 são biomarcadores com alta imunorreatividade no tecido gengival de pacientes com CGTO.
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Objective To investigate the wound-healing process in a rat model of skin full-thickness incisions and to detect related possible mechanism.Methods Twenty-four female rats were selected and the dorsal skin of rats was used as the experimental area.A cutaneous excision (6 mm diameter) was made on the back of each animal,close to the cervical area.The dorsal skin of every rat was allocated to three groups which were treated with physiological saline,human recombinant epidermal growth factor (rhEGF),and CDPs,respectively.After making a rat model of skin incisions,we observed the wound healing process,took photos of the wounds under a digital microscope,and use sulfuric graph paper to record the size of every wound.At the 3rd,6th,9th,12th day after modeling,6 rats were killed,and mRNA expression of K10,K14,and EGF was detected in the skin tissues using a RT-PCR technique.Results At the 6th and 12th day after modeling,there were significant differences between the experimental group and the blank control group (P＜0.05).The gene expression of EGF,K-14 in the third day and that of EGF,K-10 and K-14 in the 6th and 12th day were upregulated compared with control group,and there were significant differences between them (P ＜ 0.05).Conclusions CDPs have a beneficial effect on the acceleration of skin wound healing,possibly due to increasing keratinocyte proliferation and up-regulating the expression of K10,K14 and EGF genes.
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Objective@#To evaluate the effect of atmospheric fine particulate matter (PM2.5) on the expression of skin barrier-associated proteins and proinflammatory cytokines in human keratinocytes.@*Methods@#Atmospheric PM2.5 samples were given by Professor Yufeng Zhou in Children′s Hospital of Fudan University. Human primary keratinocytes were isolated from circumcised foreskins of 5 males, and subjected to culture. These human primary keratinocytes were divided into several groups to be stimulated with PM2.5 at different concentrations of 0 (control group) , 10, 50, 100, 200 mg/L for 24 hours, and cell counting kit (CCK) -8 assay was performed to determine the survival rates of keratinocytes. Fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of filaggrin, keratin-14 and claudin-1 in these keratinocytes respectively, and enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of interleukin (IL) -1α, thymic stromal lymphopoietin (TSLP) and IL-33 in the culture supernatant of these keratinocytes. Statistical analysis was carried out with SPSS13.0 software by using one-way analysis of variance, least significant difference (LSD) -t test and Pearson correlation analysis.@*Results@#After 24-hour treatment with different concentrations of PM2.5, there was no significant difference in the survival rate of keratinocytes between the 10-mg/L PM2.5 group and control group (P > 0.05) , while the survival rates of keratinocytes were significantly lower in the 50-, 100-, 200-mg/L PM2.5 groups than in the control group (all P < 0.05) . After the treatment with 50 mg/L PM2.5, the cell survival rate gradually decreased and then remained stable along with the increase of PM2.5 treatment duration, and the cell survival rate at 24 hours was 72.37% ± 3.12%. After 24-hour treatment with PM2.5, the mRNA expression of filaggrin and keratin-14 was significantly higher in both the 10- and 50-mg/L PM2.5 groups (filaggrin: 1.27 ± 0.15, 1.32 ± 0.09 respectively; keratin-14: 1.15 ± 0.13, 1.08 ± 0.16 respectively) than in the control group (all P < 0.05) , but lower in both the 100- and 200-mg/L PM2.5 groups (filaggrin: 0.84 ± 0.11, 0.42 ± 0.12 respectively; keratin-14: 0.67 ± 0.09, 0.74 ± 0.11 respectively) than in the control group (all P < 0.05) . The 10-, 50-, 100- and 200-mg/L PM2.5 groups showed significantly decreased mRNA expression of claudin-1 compared with the control group (all P < 0.05) . Western blot analysis showed that filaggrin expression was significantly higher in the 50- and 100-mg/L PM2.5 groups than in the control group (both P < 0.05) , while no significant difference was observed between the 10-, 200-mg/L PM2.5 groups and the control group (both P > 0.05) . The 10-, 50-, 100- and 200-mg/L PM2.5 groups showed significantly increased keratin-14 expression compared with the control group (all P < 0.05) . The claudin-1 expression did not differ between the 10-mg/L PM2.5 group and control group (P = 0.87) , but significantly higher in the 50-, 100- and 200-mg/L PM2.5 groups than in the control group (all P < 0.05) . The stimulation with PM2.5 at 10, 50, 100 and 200 mg/L could induce an increase in the supernatant levels of TSLP, IL-1α and IL-33 (all P < 0.01) . Pearson correlation analysis showed that the supernatant levels of the proinflammatory cytokines TSLP, IL-1α and IL-33 were positively correlated with the concentration of PM2.5 (r = 0.57, 0.67, 0.91 respectively, all P < 0.05) .@*Conclusion@#The exposure to PM2.5 can induce decreased survival rate of keratinocytes, aberrant expression of filaggrin, keratin-14 and claudin-1, and elevated secretion of the proinflammatory cytokines TSLP, IL-1α and IL-33, which may lead to impaired skin barrier function.
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Objective@#To construct and identify a mouse model with conditional knockout (cKO) of p75 neurotrophin receptor (p75NTR-cKO) gene in epidermis cells by Cre-loxP system.@*Methods@#Five p75NTRflox/flox transgenic C57BL/6J mice (aged 6-8 weeks, male and female unlimited, the age and sex of mice used for reproduction were the same below) and five keratin 14 promotor-driven (KRT14-) Cre+ /- transgenic C57BL/6J mice were bred and hybridized via Cre-loxP system. Five p75NTRflox/+ ·KRT14-Cre+ /- mice selected from the first generation of mice were mated with five p75NTRflox/flox mice to obtain the second generation hybrids. After the second generation mice were born 20-25 days, the parts of the mice tail were cut off to identify the genotype by polymerase chain reaction method. Four p75NTR gene complete cKO mice (6 weeks old) and 4 wild-type mice (6 weeks old) were selected and sacrificed respectively. The abdominal skin tissue and brain tissue were excised to observe the expression of p75NTR in the two tissue of two types of mice by immunohistochemical staining. The abdominal skin tissue of two types of mice was obtained to observe the histomorphological changes by hematoxylin and eosin staining.@*Results@#(1) Twenty second generation mice were bred. The genotype of 4 mice was p75NTRflox/flox·KRT14-Cre+ /-(p75NTR-/-), i. e. p75NTR gene complete cKO mice; the genotype of 5 mice was p75NTRflox/+ ·KRT14-Cre+ /-, i. e. p75NTR gene partial cKO mice; the genotype of 5 mice was p75NTRflox/flox·KRT14-Cre-/-, and that of 6 mice was p75NTRflox/+ ·KRT14-Cre-/-, all of which were wild-type mice. (2) The expression of p75NTR was negative in skin epidermis tissue of p75NTR gene complete cKO mice, while numerous p75NTR positive expression was observed in skin epidermis tissue of wild-type mice. Abundant p75NTR positive expression was observed in brain tissue of both wild-type mice and p75NTR gene complete cKO mice. (3) There was no abnormal growth of skin epidermis tissue in both wild-type mice and p75NTR gene complete cKO mice, with intact hair follicle structure.@*Conclusions@#Applying Cre-loxP system can successfully construct a p75NTR-cKO mice model in epidermis cells without obvious changes in skin histomorphology.
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Objective To evaluate the effect of atmospheric fine particulate matter(PM2.5)on the expression of skin barrier-associated proteins and proinflammatory cytokines in human keratinocytes. Methods Atmospheric PM2.5 samples were given by Professor Yufeng Zhou in Children' s Hospital of Fudan University. Human primary keratinocytes were isolated from circumcised foreskins of 5 males, and subjected to culture. These human primary keratinocytes were divided into several groups to be stimulated with PM2.5 at different concentrations of 0(control group), 10, 50, 100, 200 mg/L for 24 hours, and cell counting kit(CCK)-8 assay was performed to determine the survival rates of keratinocytes. Fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of filaggrin, keratin-14 and claudin-1 in these keratinocytes respectively, and enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of interleukin (IL)-1α, thymic stromal lymphopoietin(TSLP)and IL-33 in the culture supernatant of these keratinocytes. Statistical analysis was carried out with SPSS13.0 software by using one-way analysis of variance, least significant difference (LSD)- t test and Pearson correlation analysis. Results After 24 - hour treatment with different concentrations of PM2.5, there was no significant difference in the survival rate of keratinocytes between the 10-mg/L PM2.5 group and control group (P > 0.05), while the survival rates of keratinocytes were significantly lower in the 50-, 100-, 200-mg/L PM2.5 groups than in the control group(all P<0.05). After the treatment with 50 mg/L PM2.5, the cell survival rate gradually decreased and then remained stable along with the increase of PM2.5 treatment duration, and the cell survival rate at 24 hours was 72.37％ ± 3.12％. After 24-hour treatment with PM2.5, the mRNA expression of filaggrin and keratin-14 was significantly higher in both the 10- and 50-mg/L PM2.5 groups (filaggrin: 1.27 ± 0.15, 1.32 ± 0.09 respectively;keratin-14:1.15 ± 0.13, 1.08 ± 0.16 respectively)than in the control group(all P<0.05), but lower in both the 100- and 200-mg/L PM2.5 groups (filaggrin: 0.84 ± 0.11, 0.42 ± 0.12 respectively;keratin-14:0.67 ± 0.09, 0.74 ± 0.11 respectively)than in the control group(all P<0.05). The 10-, 50-, 100-and 200-mg/L PM2.5 groups showed significantly decreased mRNA expression of claudin-1 compared with the control group(all P < 0.05). Western blot analysis showed that filaggrin expression was significantly higher in the 50- and 100-mg/L PM2.5 groups than in the control group (both P < 0.05), while no significant difference was observed between the 10-, 200-mg/L PM2.5 groups and the control group(both P > 0.05). The 10-, 50-, 100- and 200-mg/L PM2.5 groups showed significantly increased keratin-14 expression compared with the control group(all P<0.05). The claudin-1 expression did not differ between the 10-mg/L PM2.5 group and control group (P = 0.87), but significantly higher in the 50-, 100- and 200-mg/L PM2.5 groups than in the control group(all P<0.05). The stimulation with PM2.5 at 10, 50, 100 and 200 mg/L could induce an increase in the supernatant levels of TSLP, IL-1αand IL-33(all P<0.01). Pearson correlation analysis showed that the supernatant levels of the proinflammatory cytokines TSLP, IL-1αand IL-33 were positively correlated with the concentration of PM2.5(r=0.57, 0.67, 0.91 respectively, all P < 0.05). Conclusion The exposure to PM2.5 can induce decreased survival rate of keratinocytes, aberrant expression of filaggrin, keratin-14 and claudin-1, and elevated secretion of the proinflammatory cytokines TSLP, IL-1αand IL-33, which may lead to impaired skin barrier function.
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PURPOSE: Actinic keratosis (AK) is an incipient form of cutaneous squamous cell carcinoma (SCC). We determined if the pattern of expression of keratin-14 (K14) is a factor for tumor progression in AK and SCC. MATERIALS AND METHODS: Eighteen sections from the tissues of 16 patients were stained with anti-K14 antibody and p16(INK4a). Among the 16 patients, 4 were diagnosed with both SCC and AK at the same site, but AK developed first and SCC developed subsequently. Thus, SCC may have evolved from AK. The other 12 patients were only diagnosed with AK. RESULTS: In all of the AK and SCC tissues, basement membranes showed positive staining for K14. However, strong reactivities were shown in the spinous and granular layers and focuses of dermal invasion in the SCC tissues developed from AK. Two and 3 of the 12 AK cases had moderately positive reactions for K14 in the spinous and granular layers, respectively. Also, all SCC tissues except one had moderate-to-strong reactions in the basal, spinous, and granular layers for p16(INK4a). Two of the 12 AK cases had weak-to-moderate positive reactions in the basal, spinous, and horny layers for p16(INK4a). CONCLUSION: The results of our study advance our understanding of the pathogenesis of SCC developing from AK. The results also indicate a differential role in the control of K14 in normal epithelia, AK, and SCC. K14 expression in the spinous and granular layers may be a prognostic factor for tumor progression of AK.
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Humans , Actins , Basement Membrane , Carcinoma, Squamous Cell , Cyclin-Dependent Kinase Inhibitor p16 , Keratin-14 , Keratosis , Keratosis, ActinicABSTRACT
Objective To study the gene mutation in a pedigree with Dowling-Meara type epidermolysis bullosa simplex (DM-EBS). Methods Using the immuno-histochemistory, electron microscopy, PCR-HA and DNA sequencing. Results The cytolysis was observed throughout the lower basal layer, tonofilaments were decreased; using PCR-HA, the K14 gene mutation was detected. By DNA sequencing, we found gene mutation in this pedigree: K14 1A domain: N123R. Conclusion There is K14 1A domain gene mutation in this DM-EBS pedigree.