Comparison of primary keloid fibroblast cultivation methods and the characteristics of fibroblasts cultured from keloids, keloid-surrounding tissues, and normal skin tissues / Comparación de los métodos de cultivo de fibroblastos queloides primarios y las características de los fibroblastos cultivados a partir de queloides, tejidos circundantes queloides y tejidos cutáneos normales

SUMMARY: The establishment of primary keloid fibroblast culture has always been a fundamental measure for studying mechanisms of keloid disease. The quality of the primary cell culture can directly affect the results of further experiments. This study was performed to investigate the optimal growth conditions, including the optimal storage time and collagenase treatment time, for in vitro cell culture models and the suitable methods for epidermis-dermis separation in different tissues. Keloid tissues, keloid-surrounding tissues, and normal skin tissues were collected from patients, for primary fibroblast culture. Two methods, tissue explant and collagenase digestion, were deployed and compared. Expression levels of the keloid-related genes α -SMA, Col1, and Col3 were assessed in cells cultured using both methods, to verify the qualities of the primary cells. A comparative analysis was conducted between the two methods and among the three different tissues used. Bacterial and lipid contamination was immediately minimized after the samples were processed. Different methods of epidermis removal and different durations of collagenase digestion were required in different tissues to generate optimal results. Real-time PCR results showed that the mRNA expression levels of keloid-related genes in cultured fibroblasts correlated to their in vivo expression profile, as previously reported in other studies. The results of this study have revealed several key points in the culture of primary keloid fibroblasts and demonstrated the correlation in gene expression between in vivo keloid fibroblasts and in vitro primary keloid fibroblasts.

RESUMEN: La identificación de un cultivo de fibroblastos queloides primarios, siempre ha sido una medida fundamental para estudiar los mecanismos de la enfermedad queloide. La calidad del cultivo de células primarias puede afectar directamente los resultados de otros experimentos. Este estudio se realizó para investigar las condiciones óptimas de crecimiento, incluido el tiempo óptimo de almacenamiento y el tiempo de tratamiento con colagenasa, para modelos de cultivo celular in vitro y los métodos adecuados para la separación epidermis-dermis en diferentes tejidos. Se recogieron de los pacientes tejidos queloides, tejidos circundantes queloides y tejidos cutáneos normales, para cultivo primario de fibroblastos. Se implementaron y compararon dos métodos, explante de tejido y digestión con colagenasa. Los niveles de expresión de los genes relacionados con queloides α -SMA, Col1 y Col3 se evaluaron en células cultivadas usando ambos métodos, para verificar las cualidades de las células primarias. Se realizó un análisis comparativo entre los dos métodos y entre los tres tejidos diferentes utilizados. La contaminación de bacterias y lípidos se minimizó inmediatamente después de que se procesaron las muestras. Se requirieron varios métodos de eliminación de la epidermis y diferentes tiempos de digestión con colagenasa en los tejidos para generar resultados óptimos. Los resultados de la PCR en tiempo real mostraron que los niveles de expresión de ARNm de genes relacionados con queloides en fibroblastos cultivados se correlacionaban con su perfil de expresión in vivo, como se informó en estudios anteriores. Los resultados de este studio indicaron varios puntos clave en el cultivo de fibroblastos queloides primarios y han demostrado la correlación en la expresión génica entre fibroblastos queloides in vivo y fibroblastos queloides primarios in vitro.

Expression of nerve growth factor p75 receptor and sortilin in the skin fibroblasts and scar fibroblasts / 中国组织工程研究

BACKGROUND: Previous studies have found that nerve growth factors play an important role in the process of wound healing, but there is less research for the low-affinity nerve growth factor receptor p75 and sortilin in fibroblasts, and no reports on whether there are differences in expression of p75 and sortilin in the scar fibroblasts and normal skin fibroblasts. OBJECTIVE: To study the expression of low-affility nerve growth factor receptor p75 and sortilin in the normal human skin fibroblasts and the human keloid fibroblasts. METHODS: The keloid fibroblasts and normal hunman skin fibroblasts were cultured in vitro, and the immortalized epithelial cells HaCaT were used as the positive control. The real-time PCR was used to detect the mRNA expression of the p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts, and western blot and immunocytochemical staining were used to detect the protein expression of p75 and sortilin. RESULTS AND CONCLUSION: The real-time PCR and western blot results showed that in the protein and mRNA levels, p75 and sortilin showed positive expression in the keloid fibroblasts and normal human skin fibroblasts, and there was no significant difference in the expression of p75 between keloid fibroblasts and normal human skin fibroblasts, and the expressions of p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts were significantly lower than those in HaCaT. There was no significant difference of p75 expression between keloid fibroblasts and normal human skin fibroblasts, and the expression of sortilin in the keloid fibroblasts was significantly lower than that in the normal human skin fibroblasts (P < 0.05). Immunocytochemical staining result showed that the expression of p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts were distributed in the membrane and cytoplasm. Precursor nerve growth factor combined with high-affinity p75 receptor could promote the apoptosis of the cells with the help of sortilin, and the expression of sortilin in the keloid fibroblasts was significantly lower than that in the normal human skin fibroblasts, which may associated with the high proliferation of the keloid fibroblasts. The results provide a new target for the prevention and treatment of pathological scars.

Physiological Characteristics of Skin of Normal Mongolian Subjects Determined by Non-invasive Bioengineering Methods / 대한피부과학회지

BACKGROUND: Physiological parameters of the skin measured by non-invasive methods have been considerably developed. It is known that there are some differences in physiologic parameters between different races. OBJECTIVE: The purpose of this study is to understand the differences between the races. METHODS: A total of 757 Mongolian participated in this study. All subjects had no major history of skin diseases requiring medical treatment. Several instruments were used such as Sebumeter, Corneometer, Mexameter for measuring sebum excretion rate, values of capacitance, melanin index and erythema index respectively. These were measured on various areas of the skin such as cheek, forehead, palm, outer arm, inner arm, back and buttock. RESULTS: The sebum excretion rates showed higher in males than in females on the cheek and forehead in Mongolian. However, they showed higher in the females on the palm. There were good correlation between the skin sebum level and the capacitance in males and females. The melanin index and erythema index showed higher in males than in females at all sites. When we compared this with the data published in other countries, many data were similar to Korean rather than a western country's. However, the capacitance data were lower than those of Korean. CONCLUSION: Although, this study has some limit for direct comparison between each race, our results can be used as basic data for the comparison between Mongolian and other races in the future.

The Influence of Physiologic Lipid Containing Moisturizer on the Normal Skin Barrier / 대한피부과학회지

BACKGROUND: Moisturizers are commonly used for the management of many skin problems and sometimes they are used to maintain healthy skin. Previous studies on the short-term treatment with moisturizers have shown that moisturizers can weaken or strengthen the skin barrier function. OBJECTIVE: We evaluated the effect of long-term treatment with a physiologic lipid-containing moisturizer on the barrier function of normal skin. METHODS: 39 healthy volunteers applied the moisturizer on one forearm 3 times daily for 8 weeks and then they were followed-up for the next 2 weeks (the regression period). No moisturizer was used on the other forearm as a control. The TEWL, skin capacitance and skin lipid content were evaluated weekly during the 8 weeks treatment period and then daily during 2 weeks of the regression period. RESULTS: During the treatment period, the skin capacitance and lipid content values of the treated side were significantly higher than that of the control side, and the TEWL values of the treated side were significantly lower than that of the control side. During the regression period, all the values of the treated side steadily became closer to those of the control side. These results suggest that long term treatment with moisturizer does not deteriorate the skin barrier function. CONCLUSION: Long-term treatment with moisturizer containing physiologic lipid not only enhances the normal skin barrier, it does not suppress the endogenous lipid synthesis of the skin.

The Influence of Physiologic Lipid Containing Moisturizer on the Normal Skin Barrier / 대한피부과학회지

BACKGROUND: Moisturizers are commonly used for the management of many skin problems and sometimes they are used to maintain healthy skin. Previous studies on the short-term treatment with moisturizers have shown that moisturizers can weaken or strengthen the skin barrier function. OBJECTIVE: We evaluated the effect of long-term treatment with a physiologic lipid-containing moisturizer on the barrier function of normal skin. METHODS: 39 healthy volunteers applied the moisturizer on one forearm 3 times daily for 8 weeks and then they were followed-up for the next 2 weeks (the regression period). No moisturizer was used on the other forearm as a control. The TEWL, skin capacitance and skin lipid content were evaluated weekly during the 8 weeks treatment period and then daily during 2 weeks of the regression period. RESULTS: During the treatment period, the skin capacitance and lipid content values of the treated side were significantly higher than that of the control side, and the TEWL values of the treated side were significantly lower than that of the control side. During the regression period, all the values of the treated side steadily became closer to those of the control side. These results suggest that long term treatment with moisturizer does not deteriorate the skin barrier function. CONCLUSION: Long-term treatment with moisturizer containing physiologic lipid not only enhances the normal skin barrier, it does not suppress the endogenous lipid synthesis of the skin.

Eczema Herpeticum in Normal Skin of Two Siblings / 대한피부과학회지

Eczema herpeticum is a herpes simplex virus infection with disseminated skin involvement, which superimposes on a pre-existing dermatosis. Two siblings with simultaneous primary herpes simplex virus (HSV) infection on the normal skin of their backs, followed by their mother's herpes labialis. Eczema herpeticum was diagnosed on the basis of history, clinical feature, Tzanck test, histologic examination and a serologic test. Their skin lesions were healed by treatment with oral acyclovir and mupirocin ointment.

The Distribution of Malassezia Species on the Normal Human Skin According to Body Region / 대한의진균학회지

BACKGROUND: The seven species Malassezia yeasts are recognized in the genus Malassezia (M.). These species can be isolated from the normal skin of warm-blooded animals including humans and various dermatoses. In Korea, most surveys of Malassezia yeast distribution have been performed without classifying the genus Malassezia into the species. OBJECTIVE:The aim of this work was to study the qualitative distribution of the Malassezia species on the normal human skin to provide a basic data for a study of Malassezia species in patients with various dermatoses. METHODS: The Malassezia yeasts were cultured from clinically normal skin at four sites in 37 subjects. The isolated yeasts were identified into the species level using the cell types and physiological properties of the species. RESULTS: Six species of the genus Malassezia, M. furfur, M. sympodialis, M. globosa, M. Obtusa, M. restricta and M. slooffiae, were isolated. M. restricta was found to be predominant on the forehead and scalp. M. globosa was found to be predominant on the chest and back. The incidence of Malassezia yeasts was 78.4% on the scalp, 86.5% on the forehead, 100% on the chest and 97.3% on the back. CONCLUSION: The results showed a regional variation in the distribution of M, restricta and M, globosa. The information obtained from this study would be of great value in the evaluation of the role of Malassezia species in related diseases.

Expression of Metallothionein in the Neoplasms of Ectodermal Origin / 대한피부과학회지

BACKGROUND: Metallothioneins(MTs) are ubiquitous low molecular weight cytosolic proteins with high contents of cystein that xhibit a selective binding affinity for zinc, copper and other group II heavy metal ions. In additi~on to the sequestration function and detoxification action, MTs probably participate in the cellirlar protection against. ionizing radiation and alkylating agent cytotoxicity and in cellular pn:liferation. OBJECT: The objective of this study was to evaIuate the expression of MTs and to clarify t.heir role in carcinogenesis and/or biological behaviour in the neoplasms of ectoderrnal origin. METHODS: We performed immunohistochemical studies on the surgical specimens of rnalignant rnelanoma and squamous cell carcinoma and basal cell carcinoma. The results were compared with the normal skin and mu osa and hair t,issues and raf't-on culture tissues of skin and mucosal keratinocytes. RESULTS: 1) MTs were strongly expressed in epidermal basal cells of the normal skin and raft-on cultured artificial skin They were also rnore weakly expressed in epithelial basal cells of the normal mucosa and raft on cultured artificial mucosa and in matrix cells of the normal hair. 2) In rnalignant melanoma, MTs were meaningfully expressed, i.e., a strong expression was seen in the lesions showing a rapid vertical growth phase with fatal out,comes, but a weak expression in the cases whose precursor lesions were thought as dysplastic nevi, compared to the negative finding of norrnal melanocyt,es. The strong expression in a case of congenit.al rnelanocyt.ic nevus is considered as the activation of nevoid melanocytes. 3) MTs were weakly expressed in squarnous cell carcinoma with a diffuse aattern within the tumor mass. St,rong expression of MTs was particularly apparent in the poorl. differentiated squamous cell carcinoma and at the growing border of well differentiated squamous cell carcinoma. 4) In basal cell carcinoma, MTs were rather weakly expressed in solid cireumscribed types with a diffuse pattern and in cystic type with a patchy pattern. However they were not expressed in the solid infiltrative type. CONCLUSION: From these resuilts, we concluded that MTs were ubiquitously present in possible stem cells of various epithelial tissues except melanocytes irrespective of normal or artificial skin and/or mucosa including norrnal hair tissues. In the neoplasms of ectodermal origin, MTs might be involved in the invasive growth of tumor cells in the malignant melanoma and squamous cell carcinoma. It was implied that the role of MTs in basal cell carcinoma might be related to early events of hasal cell carcinogeresis and its down regulating tendency could be possible in its infil trative stage.

The study of incidence and distribution of pityrospoum on clinically normal skin / 대한피부과학회지

BACKGROUND: Previous studies have suggested that pityrosporurr yeasts, normal resident flora of skin, may be important in the pathogenesis of dandruff seborrheic dermatitis and confluent and reticulated papillomatosis. OBJECTIVE: We investigated the incidence and distribution of pityiosorum yeasts on clinically normal skin as a more reasonable method for providing the basic data or proving the relationship of pityrosporum and pathogenesi, of the diseases. MEHTODS: A total of 350 subjects were studied. Skin scrapings were stained with 20% Parker ink-KOH(P-KOH) solution. The numbers of pityrosporum spores and hyphe per high power field( x 400) were counted and designated according to a new grading method using bacterial index(BI) of lepra bacilli. RESULTS: The incidence rates of pityrosporum orbiculare(p. orbiculare) and pityrosporum ovale(p. ovale) were 60-70% and 20-50% of subjects respectively on seborrheic area., The incidence of p. orbicilare was highter than that of p. ovale on all examined sites except for the iterior scalp. And pityrosporal hyphae was present on anteriand posterior scalp and earwax in 24-25% of subjects respectively. The incidence of pityrosporum was higher in summer than other seasons The incidence rate of p. orbiculare and hyphae in the group aged below 9 (except neonates) and above 60, were lower than that of other age groups. The inciderice rates and distribution of grades of pityrosporum of neonates were lower than those of older subjects. CONCLUSION: The results of this study showed that the incidence and distribution of pityrosporum spores were similar to previoureports and hyphal forms were found 25% approximately on clinically normal skin of the scalp and carwax. The significance of the presenet of pityosporal hyphae and the relationship between normal skin and pityrosporal hyphae are to be firther elucidated.