【作者】 曹玉萍；

【导师】 刘维达； 马鹏程；

【作者基本信息】 北京协和医学院 ， 皮肤病性病学， 2011， 博士

【摘要】 在欧洲,组织工程皮肤作为一种重要的实验动物替代工具,已经被应用于包括皮肤腐蚀性、刺激性、光毒性及基因毒性等在内的多项安全性检测,以及皮肤念珠菌病、黑素瘤等多种皮肤相关疾病模型的构建。而在我国,关于组织工程皮肤作为动物替代方法进行化学物质安全性检测以及利用组织工程皮肤建立皮肤疾病模型的报道十分有限。本课题就构建化学物质致敏性检测的体外模型和组织工程皮肤真菌感染模型两方面展开研究,主要包括以下三部分内容：第一部分组织工程皮肤的构建目的构建组织工程真皮、组织工程表皮及组织工程皮肤(全层)。方法1.利用原代培养的成纤维细胞与含有10%胎牛血清和10%DMEM的牛胶原溶液混合孵育,液下培养3d构建组织工程真皮；2.以成纤维细胞的动态培养为饲养层,分别气液界面培养角质形成细胞和HaCaT细胞,或气液界面培养角质形成细胞和黑素细胞,培养10d构建组织工程表皮；3.液下培养含成纤维细胞的胶原凝胶3d后,在其表面分别接种角质形成细胞和HaCaT细胞,或者角质形成细胞和黑素细胞,气液界面培养10d构建组织工程皮肤(全层)；所有培养物用常规石蜡包埋切片方法制备组织切片,通过苏木精-伊红染色观察培养物的组织结构；不含色素的组织工程表皮角蛋白免疫组化染色,含色素的表皮和皮肤S100免疫组化染色。结果1.成功建立组织工程真皮,其外观形态为淡红色半透明胶状,具有一定韧性和弹性,HE染色显示胶原呈束状分布,结构致密,成纤维细胞呈梭形平行于上表面；2.成功建立不含或者含色素的组织工程表皮,苏木精-伊红染色观察,表皮细胞形成多达十余层结构的表皮,HaCaT构建者较角质形成细胞构建者表皮层数更多,但表皮内存在空泡；K1/K10角蛋白免疫组化染色中,随着细胞层数的增加,角质形成细胞构建者K1/K10角蛋白染色阳性逐渐增强,而HaCaT构建者为阴性,K5/K14角蛋白免疫组化染色中,两者均为阳性；含色素的组织工程表皮S100染色可见随着培养时间的延长,黑素细胞由最初分布于基底层及上方发展到表皮全层均有分布；3.不含色素的组织工程皮肤HE染色显示,角质形成细胞和HaCaT细胞生长分化均形成具有十余层细胞结构的表皮,但后者表皮结构不如前者有序,细胞形态变化不如前者规律,且HaCaT构建的表皮层较角质形成细胞构建者更易从真皮表面脱落；含有色素的角质形成细胞和黑素细胞共同构建组织工程皮肤在宏观形态上,随着培养时间的延长,培养物表面的颜色逐渐加深,色素颗粒增多；S100染色显示随着培养时间的延长,黑素细胞的分布从基底层逐渐发展到角质层。结论本研究成功建立了组织工程真皮、表皮和皮肤(全层),为今后皮肤相关化学物质的安全性评价,皮肤疾病相关机制的研究及多种药物的药效学研究提供了有利工具。第二部分致敏性体外模型的构建目的构建角质形成细胞/THP-1细胞共培养模型和含色素的组织工程表皮与THP-1细胞的三维分室共培养模型用于化学物质的致敏性检测。方法1.将THP-1细胞与角质形成细胞进行分室共培养,浓度为0.1×,0.5x和1×最小ICso的待检测物质作用24h后,流式细胞仪检测THP-1细胞表面的CD86和CD54表达水平；2.将THP-1细胞培养于含色素的组织工程表皮下方的培养液内,用0.1×ICso的待检物质凝胶作用于组织工程皮肤表面,24h后,流式细胞仪检测THP-1细胞表面的CD86和CD54表达水平,用ELISA法测定培养液中IL-1p、IL-6和IL-8含量。结果1.致敏物质二硝基氯苯、苯二胺、甲醛、硫酸镍、丁香酚、异丁香酚可诱导THP-1表面的共刺激分子CD86和CD54不同程度的表达,而被非致敏物月桂基硫酸钠、乳酸、苯扎氯铵等理后,THP-1细胞表面共刺激分子CD86和CD54与阴性对照组比较未见统计学差异；2.致敏物二硝基氯苯、苯二胺、硫酸镍、丁香酚、苯佐卡因凝胶在0.1×IC50浓度时,可明显诱导THP-1细胞表面CD86和CD54的表达,非致敏物月桂基硫酸钠、苯扎氯铵凝胶无此效应,同时仅在强效致敏物作用时,培养液中IL-1β含量明显升高,在中弱效致敏物和非致敏物作用下未见明显变化,培养液中IL-6和IL-8在致敏物作用组和非致敏物作用组未见明显差异。结论1.角质形成细胞/THP-1细胞共培养模型用于检测可溶性化学物质的致敏性,其操作步骤简单,灵敏度高,是一种较好的致敏性检测方法,有较广泛的应用前景,其不足在于无法用于非可溶性化学物质及制剂的检测；2.含色素的组织工程表皮/THP-1细胞三维分室共培养模型的成功建立,解决了不溶性化学物质和外用制剂的致敏性检测的难题,它更符合变态反应致敏阶段的发生机制,有更强的可比性和可信度。第三部分组织工程皮肤在真菌感染性皮肤病中的应用目的构建白念珠菌、须癣毛癣菌、红色毛癣菌和糠秕马拉色菌感染的组织工程皮肤模型,为各种常见皮肤真菌病致病机制及病原菌毒力的研究及药效学的研究提供一种新型的工具；同时,初步探讨红色毛癣菌感染的组织工程皮肤模型在抗真菌药物药效学评价方面的应用。方法1.将浓度为约1×106/ml的白念珠菌、须癣毛癣菌、红色毛癣菌和糠秕马拉色菌菌悬液5μl接种于含色素的组织工程皮肤表面,于35℃,5%CO2孵箱内培养,分别于6h、12h、24h、48h和72h后,石蜡包埋切片,HE染色和PAS染色观察；2.将红色毛癣菌菌悬液加至含色素的组织工程皮肤表面,与此同时,向组织工程皮肤下方的培养液中分别加入0.001μg/ml,0.01μg/ml,0.1μg/ml和1μg/ml的特比萘芬溶液,48h后,石蜡包埋切片,HE染色和PAS染色观察。结果1.白念珠菌、须癣毛癣菌和红色毛癣菌组中可见菌丝及孢子随着时间的推移,渐渐侵入表皮层甚至真皮层,同时真菌对皮肤产生明显的破坏作用,而糠秕马拉色菌仅生长于表皮层上方或表皮浅层,对皮肤的破坏作用不明显；2.在红色毛癣菌感染模型中,随着特比萘芬浓度的升高,红色毛癣菌的菌丝侵入数量减少,对皮肤的破坏程度逐渐减轻。结论成功建立了白念珠菌感染的组织工程皮肤模型、须癣毛癣菌感染的组织工程皮肤模型、红色毛癣菌感染的组织工程皮肤模型和糠秕马拉色菌感染的组织工程皮肤模型,有利于上述常见皮肤真菌体外模型的发展；通过研究特比萘芬对红色毛癣菌感染的组织工程皮肤模型的干预作用,提示今后抗真菌药物的体外药效学评价可以菌丝侵入程度的变化及皮肤破坏程度的严重程度为指标,作为一种新型的体外模型,亲人性红色毛癣菌感染的组织工程皮肤模型对临床用抗真菌药物及抗真菌新药的药效学评价有极为重要的价值。更多还原

【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.更多还原