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Construction of in Vitro Sensitization Testing Model for Chemicals and Fungal Infected Tissue-engineered Skin

Author: CaoYuPing
Tutor: LiuWeiDa;MaPengCheng;CuiPanGen;ZhouWuQing
School: Beijing Union Medical College
Course: Dermatology and Venereology
Keywords: Tissue engineering skin THP-1 cell sensitization mycology Pharmacodynamics
CLC: R329
Type: PhD thesis
Year: 2011
Downloads: 38
Quote: 0
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Abstract


In Europe, the tissue-engineered skin as an important tool for the animal alternative testing has been used in many kinds of safety tests, such as skin corrosion, skin irritation, skin photo-toxicity and genetic toxicity, and also for the construction of several skin-related disease models, such as cutaneous candidiasis, melanoma and so on. However, in China, such reports are very limited. In this study, the in vitro sensitization testing model and fungal infected tissue-engineered skin model were constructed successfully, and reported here as following three parts:Part 1 Construction of tissue-engineered skinObjective To construct tissue-engineered dermis, tissue-engineered epidermis and tissue-engineered skin.Methods 1. Primary cultured fibroblasts were incubated in bovine collagen solution containing 10% fetal bovine serum and 10% DMEM for 3 days. 2. The keratinocytes, HaCaT cells and keratinocytes with melanocytes were seeded respectively on the surface of feeder layer, which was prepared by the dynamic culture of fibroblasts. Then, the air-liquid interface culture for 10 days was carried on. 3. After fibroblasts were cultured in collagen gel for 3 days, keratinocytes, HaCaT cells and keratinocytes with melanocytes were seeded respectively on the surface. Then air-liquid interface culture for 10 days was carried on.The paraffin embedded sections method was used to prepare the tissue slides and the hematoxylin-eosin staining was performed for the observation. Immunohistochemistry staining of keratins were used for non-pigmented tissue-engineered epidermis. Pigmented tissue-engineered epidermis and skin were stained by immunohistochemistry of S100. Results 1. Tissue-engineered dermis was successfully established. It was the translucent pink colloid-liked matter, with some flexibility and elasticity. It was shown by hematoxylin-eosin staining that collagen was in the fasciculated distribution with compact structure and spindle-shaped fibroblasts were parallel to the interface.2. Non-pigmented and pigmented tissue-engineered epidermis was successfully established. It was shown by hematoxylin-eosin staining that keratinocytes and HaCaT cell formed more than ten layers in the epidermis and it was thicker by HaCaT cells than keratinocytes. But there were some vacuoles in the epidermis of HaCaT cells. K1/K10 keratin immunohistochemistry was positive in tissue-engineered epidermis constructed by keratinocytes, but negative in that constructed by HaCaT cells. K5/K14 keratin immunohistochemistry was positive in both keratinocyte-derived tissue-engineered epidermis and HaCaT-derived tissue-engineered epidermis. S100 immunohistochemistry staining was used for tracking the distribution of melanocytes in pigemnted tissue engineering epidermis. It showed that melanocytes migrated from the basal layer to the horny layer with times.3. Hematoxylin-eosin staining of both keratinocyte-derived and HaCaT-derived non-pigmented tissue-engineered skin showed that more than ten layer epidermis formed. But the structure of the latter is less orderly and regular. The HaCaT-derived epidermis is more easily detached from the dermal sheet. The color of tissue-engineered skin with melanocytes gradually deepeded as the culture time prolonged. S100 staining showed the same characteristcs.Conclusion In Part 1, tissue-engineered dermis, epidermis and full thickness skin were successfully established, which provided a useful tool for the safety evaluation of chemicals, researches on pathogenesis of skin diseases and pharmacokinetics studies of the medicines.Part 2 Construction of sensitization testing in vitro model Objective To construct the co-culture model of keratinocytes/THP-1 cells and three-dimensional co-culture model of pigmented tissue-engineered epidermis/THP-1 cells to evaluate the sensitization potential of chemicals.Methods 1. THP-1 cells and karatinocytes were co-cultured in different compartments. After 24h treatment of chemicals at 0.1×0.5×and 1×the minimal IC50, the expression of CD86 and CD54 were evaluated by flow cytometry. 2. The THP-1 cells were cultured in the medium below the pigmented tissue-engineered epidermis. After 24h treatment of the gels of chemicals with the concentration of 0.1×IC50, CD86 and CD54 expression was determined by flow cytometry. And the content of IL-1β, IL-6 and IL-8 in the culture medium was determined by ELISA.Results 1. Dinitrochlorobenzene, p-phenylenediamine, formaldehyde, nickel sulfate, eugenol, isoeugenol induced THP-1 expressing CD86 and CD54, rather than sodium lauryl sulfate, lactic acid and benzalkonium chloride in keratinocyte/THP-1 cell co-culture model; 2. The gel of dinitrochlorobenzene, p-phenylenediamine, nickel sulfate, eugenol, benzocaine at the concentration of 0.1×IC50, can significantly induce THP-1 expressing CD86 and CD54, while the gel of sodium lauryl sulfate, benzalkonium chloride had no such effect. Furthermore, IL-1βin the culture medium only increased by the strong allergens. The determination of IL-6 and IL-8 showed no significant difference between sensitizers and non-sensitizers.Conclusion 1. Keratinocytes/THP-1 cell co-culture model was useful for the detection of soluble chemical in sensitization testing, which had simple operation, high sensitivity. However, the disadvantage of this co-culture model was that insoluble chemicals can not be evaluated; 2. Pigmented tissue-engineered epidermis/THP-1 cell co-culture model were successfully established, which solved the sensitization evaluation of insoluble chemicals and topical preparations. It is more in line with the mechanism of sensitization stage so that it had stronger comparability and credibility.Part 3 The application of tissue-engineered skin in fungal infectionObjective To construct in vitro model of tissue-engineered skin infected by Candida albicans, Trichophyton mentagrophytes, Trichophyton rubrum and Malassezia furfur, which will provide a new tool for the studies about skin fungal infection and pharmacodynamics of anti-fungal medicines. Meantime, to evaluate the feasibility of the application of Trichophyton rubrum infected tissue-engineered skin in pharmacodynamic study of antifungal medicine.Methods 1. The suspension of Candida albicans, Trichophyton mentagrophytes, Trichophyton rubrum and Malassezia furfur at 1 cfu/ml were inoculated with pigmented tissue-engineered skin in the incubator at 35℃with 5% CO2. After 6h,12h,24h,48h and 72h, paraffin embedded sections, HE staining and PAS staining were performed. 2. The suspension of Trichophyton rubrum was added to the surface of pigmented tissue-engineered skin. At the same time, terbinafine was added to the culture medium of the tissue-engineered skin at 0.001μg/ml,0.01μg/ml, 0.1μg/ml, 1μg/ml,respectively.48h later, paraffin embedded sections, hematoxylin-eosin staining and periodic acid-schiff staining were performed.Results 1. It was shown by hematoxylin-eosin staining and periodic acid-schiff staining that the hyphae and spores were visible and gradually penetrated the epidermis and dermis in tissue-engineered skin infected by Candida albicans, Trichophyton mentagrophytes and Trichophyton rubrum. It was also found that skin damage was significant with time. It was shown by hematoxylin-eosin staining and periodic acid-schiff staining that blastospores of Malassezia furfur were only seen in the upper of epidermis. The damage of the skin was not significant. 2. In Trichophyton rubrum infected tissue-engineered skin, the number of invased hyphae reduced, and the damage of the skin gradually reduced as the concentration of terbinafine increased.Conclusion Candida albicans, Trichophyton mentagrophytes, Trichophyton rubrum and Malassezia furfur infected tissue-engineered skin were successfully established, respectively. It was significant for the development of in vitro models of Candida albicans, Trichophyton mentagrophytes, Trichophyton rubrum and Malassezia furfur. The results of Trichophyton rubrum infected tissue-engineered skin treated with terbinafine suggest that the depth and amount of invasive hyphae and the damage of the skin can be considered as indicators for pharmacodynamics studies of anti-fungal medicines.

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