Preparation of tissue-engineered skin grafts with hTERT cells carrying human papillomavirus type 6 genome in vitro：a preliminary study / 中华皮肤科杂志

Objective To establish a model for preparation of tissue-engineered skin grafts with hTERT cells carrying human papillomavirus type 6 (HPV 6) genome in vitro, so as to lay a foundation for studying HPV life cycle. Methods The full-length linear HPV6 genome and plasmid pEGFP-▲EGFP were electrophoretically cotransferred into hTERT cells. After selection using G418 resistance, Southern blotting was performed to determine the viral load of HPV6 in transfected cells. 3T3 J2 trophoblastic cells, type I rat-tail collagen and hTERT cells containing the full-length HPV6 genes (HPV6.hTERT cells)were mixed and cocultured on metal meshes to form skin graft-like structures. Hematoxylin and eosin (HE)staining was performed to observe the structure of formed skin grafts, an immunohistochemical assay to measure the expression of HPV6 L1 protein, and electron microscopy to observe virus particles in the skin grafts. Results The linear HPV6 gene was successfully transferred into hTERT cells, and Southern blotting showed the presence of HPV6 DNA in the transferred hTERT cells. The HPV6.hTERT cells, which were cocultured with 3T3 J2 trophoblastic cells and type I rat-tail collagen, proliferated and differentiated over time, and gradually formed skin grafts giving the appearance of verrucous hyperplasia. HE staining showed that the cocultured HPV6.hTERT cells could form typical stratified structure of skin after 7 days of cultivation, and histopathologic features of HPV infection, including obvious papillomatous hyperplasia, presence of vesicular cells, hyperkeratosis and parakeratosis, could be observed after 21 days. The immunohistochemical assay showed the expression of HPV6 L1 protein in the upper portion of skin grafts, and electron microscopy revealed the presence of HPV6 virus particles in skin grafts. Conclusions The established model for preparation of tissue-engineered skin grafts using HPV 6 genome-carrying cells provides a basis for biological studies of HPV, but its application is limited to some degree.

The Development of a Wound Healing Model Using Nerve Growth Factor(NGF) Expression in Raft Culture

BACKGROUND: The wound healing process is impaired or delayed in aged patients. The development of a new wound healing model is needed. Nerve growth factor (NGF) plays a special role in wound healing because NGF is expressed only in proliferating tissues such as wounds. OBJECTIVE: The aim of our study was to develop a wound healing model using a 3-dimensional culture system, raft culture, by comparing the level of NGF expression according to the wound stage after an artificial wound was made to the raft samples. We tried to specifically localize the site of NGF expression both in mRNA and protein level. METHODS: Raft culture using normal human keratinocytes was done and a 2 mm slit wound was made in the center of the raft samples. Raft samples of no wound, 4 d, 7 d, and 9 d after wounding were prepared. In situ RT-PCR and immunohistochemistry were performed to detect and localize NGF expression after making wounds and the addition of substance P (SP). RESULTS: We failed to localize NGF mRNA expression in raft samples by in situ RT-PCR. Immunohistochemistry showed NGF staining throughout the epidermis although a little more dense staining was found in the basal layer. NGF(+) cells tended to increase until 7 d after wounding, but there were no significant differences according to the wounding days. There was `a tendency that the SP(+) group showed more NGF(+) cells than the SP(-) group, but there were no statistical differences. CONCLUSION: We think that our in vitro raft wound model using NGF expression could be used, at least in part, as an objective indicator for wound healing. In our raft model lacking nerve, NGF may not be suitable for representing wound healing process because this model can not reflect the interaction between the skin and the nervous system. Expression of growth factors or cytokines other than NGF need to be applied to our raft culture system.

Effects of IFN - gamma on Spheroid and Raft Culture of Squamous Cell Carcinoma of the Head and Neck / 대한암학회지

PURPOSE: To establish new in vitro model systems that better reflect in vivo condition, multicellular tumor spheroids(MTS) and raft culture were developed using cell lines of squamous cell carcinoma(SCCHN) of the head and neck. In these 3-dimensional systems, the expression of cell surface molecules which are important for modulation of physiology of tumor cells were studied with or without the treatment of interferon(IFN)-gamma. MATERIALS AND METHODS: Four SCCHN cell lines were used for MTS and raft culture. The effects of interferon-gamma on SCCHN cells were examined by immunohistochemistry. RESULTS: All cell lines formed MTS, but only Tu-138 showed a good stratification at the air-liquid interface in the raft culture system. Immunohistochemical studies of MTS using monoclonal antibodies revealed a strong staining for MHC class I, no staining for MHC-DR, a weak patch expression of ICAM-1 and a central strong staining for integrin a 6. Staining patterns were similar for the raft cultures except integrin a 6(intense full-thickness positivity). In both systems, IFN-gamma enhanced the expression of MHC-DR and ICAM-1. No significant change was found in the expression of MHC class I and integrin a 6. CONCLUSIONS: MTS and raft culture system were established successfully from the SCCHN cell lines. IFN-gamma can modulate the surface molecules of tumor cells in the 3-dimensional culture systems.

Expression of Skin Sulfhydryl Oxidase in Normal Skin , Cultured Skin and Several Abnormally Keratinizing Dermatoses / 대한피부과학회지

BACKGROUND: Skin sulfhydryl oxidase(SSO) is the enzyme catalyzing the covalent crosslinking of the protein molecules via disulfide bond formation, which is probably responsible for the formation of tight and stable structures in the skin tissues, However, the lack of its gene cloning and antibody preparation limited the study for the role of the enzyme in the tissues. OBJECTIVE: It is required to study the role of the enzyme in the normal skin tissues and several dermatosis with abnormal keratinization and its regulation by several materials and drugs in the culture models. METHODS: We performed immunohistochemical analysis to determine the localization and expression of the enzyme in the normal skin and mucosa and hair tissues and in the skin explant cultures and artificial skins with irritant- and differentiation modifieradded conditions. RESULTS: In the skin, SSO was most strongly expressed in the granular layers and mostweakly in the basal layers. The similar but weaker expression pattern was observed in the mucosa with stronger expression in the upper flattened layers compared to the lower layers. In the hair, it was expressed in the inner and outer root sheaths at the isthmus portion in weaker stainable patterns. The strong expression of SSO was not observed beneath the Monro microabscess or parakeratosis in the involved aren of psoriasis. In actinic keratosis and Bowen disease, it was expressed more strongly in the dyskeratotic cells or parakeratotic areas. And in the horn pearls of the squamous cell carcinoma, the strong expression of SSO was observed. In the skin ezplant culture, the expression of SSO is induced by treatment with sodium lauryl sulfate or retinoic acid with more extended stained areas compared to the control. In contrast, its expression is not basically modified on the cellular level but inhibited dynamically by a decrease of the granular layers with retinoic acid in the artificial skin(raft culture). CONCLUSION: And the inducibility of the enzyme by the irritating agents in the skin organ explant culture suggests the role of the enzyme as one of skin defense system. The decrease of granular layers by retinoic acid in the artificial skin(raft culture) reflects the fact that its expression can be inhibited indirectly during the keratinization process. Therefore, it can be summarized that SSO is the enzyme involved in the keratinizing process of skin tissues as well as the protective function for the skin tissues.