【作者】 刘源；

【导师】 金岩； 诹访文彦；

【作者基本信息】 中国人民解放军第四军医大学 ， 口腔基础医学， 2003， 博士

【摘要】 组织工程皮肤产品的研制成功为治疗皮肤的急性和慢性损伤提供了一个极有前景的治疗方法。总的来说，组织工程皮肤可以针对各种病理情况(烧伤、疤痕、深度溃疡和先天畸形等)构建各种不同的生物产品。同时还可以在组织工程皮肤产品中整合各种针对性的治疗因子，从而大大降低全层皮肤缺损造成的发病率和死亡率。组织工程皮肤提供了足够的可供移植的皮肤来源，但是只能恢复部分解剖结构和部分生理功能。因此，组织工程皮肤的全部潜力还远远未被开发。在本实验中，我们准备构建两种组织工程皮肤，一个含有黑色素细胞，另一个含有血管样结构，具体实验如下： 1．探讨如何以简便快捷的方法获得大量生长状态良好的高纯度黑色素细胞：以包皮组织作为细胞来源，用消化法获得表皮细胞悬液，用本实验室设计的方法对黑色素细胞进行纯化，然后用两种不同培养 第四军医大学博士学位论文 基 MC、MCZ培养细胞，Dopa染色，免疫组化检测（S上）和透 射电镜鉴定细胞来源，通过MTT和流式细胞仪分析观察细胞的生长 状态。黑色素细胞中未见角肮细胞和成纤维细胞污染，MTT和流式 细胞仪检测结果显示用MCZ 培养的黑色素细胞处于旺盛的增殖状 态，明显好于 MC培养的黑色素细胞。结果表明本实验方法可以获 得大量处于良好生长状态的高纯度的黑色素细胞。2．运用组织工程方法构建含有黑色素细胞的组织工程皮肤：以包皮组 织作为细胞来源，用消化法获得角肮细胞、成纤维细胞和黑色素细 胞，用本实验室设计的组织工程皮肤培养系统构建组织工程皮肤。 用 Dopa染色、透射电镜和免疫组化检测（Sl）构建的皮肤中黑 色素细胞的分布和状态。用所构建的组织工程皮肤修复裸鼠的全层 皮肤缺损。结果表明所构建的组织工程皮肤结构完整，细胞状态良 好，Dopa染色、透射电镜和免疫组化检测（SJ）显示该皮肤中含 有大量黑色素细胞并处于良好状态。所构建的含黑色素细胞组织工 程皮肤可以成功修复裸鼠的皮肤缺损，并形成黑色的皮肤。3．探讨如何以简便快捷的方法获得大量生长状态良好的人血管内皮细 胞以作为构建含血管结构组织工程皮肤的种子细胞：以新生儿脐带 静脉作为细胞来源，用消化法获得血管内皮细胞，用本实验室设计 的方法对细胞进行纯化培养，倒置显微镜观察细胞形态，结合免疫 5 第四军医大学博士学位论文 组化检测（第皿因子）和透射电镜（WeibelPalade 小体）鉴定细胞 来源。结果表明本实验方法可以获得大量处于良好生长状态的高纯 度血管内皮细胞，可以用于含血管组织工程皮肤的构建。4．运用组织工程方法构建含有血管结构的组织工程皮肤；以新生儿脐 带静脉和包皮作为细胞来源，用消化法获得血管内皮细胞、皮肤的 角肮细胞和成纤维细胞，用本实验室设计的组织工程皮肤培养系统 构建组织工程皮肤，其中真皮层成纤维细胞和血管内皮细胞的比例 为1／1，在真皮表面种植角肌细胞以形成表皮。HE染色和免疫组化 检测（第皿因子）观察真皮层中血管结构的形成情况。用所构建的 组织工程皮肤修复裸鼠的全层皮肤缺损。结果表明所构建的组织工 程皮肤结构完整，细胞状态良好，真皮层中形成了微血管样结构。 所构建的含有血管结构的组织工程皮肤可以成功修复裸鼠的皮肤缺 损，移植成功率高。 本实验成功构建了含黑色素细胞和血管样结构的组织工程皮肤。含色素的组织工程皮肤的成功构建，为今后构建含不同比例的色素细胞，形成不同颜色的组织工程皮肤提供了技术支持，可以满足不同皮肤缺损修复的要求。含血管结构组织工程皮肤的成功构建，使得组织工程皮肤对于皮肤缺损创面的要求进一步降低，还可以构建厚度更大的皮肤以利于临床医生的操作，同时为构建更大体积的组织工程产品提供了积极的 6 第四军医大学博士学位论文技术参考。更多还原

【Abstract】 The ultimate objective for tissue engineering skin is restoration of the anatomy and physiology of uninjured skin after treatment and healing of the wound. At present, only an autologous full-thickness skin grafts, free flaps or pedicle flaps restore all of the structures and functions of uninjured skin but donor sites and treatment sites must be equal in size. Angiogenesis (the formation of new blood vessels) is essential for the growth of new tissue, tissue repair and wound healing. Tissue engineering, the construction of new tissue and organs for reparative purposes, relies on angiogenesis for the vascularisation of these new grafts. In tissue engineering, the emphasis to date has been on vascularisation of newly constructed tissue grafts by an extrinsic blood supply, and relatively littleattention has been given to the possibility of building these grafts around an intrinsic blood supply. In this study, two kinds of skin substitutes were reconstructed by employing tissue engineering method:1. To develop a rapid and reproducible method for the isolation of pure melanocytes in good conditions from foreskin: The melanocytes were isolated and purified by a method employed in our lab. Two different media MCI and MC2 were used to culture the cells. The biological conditions of the cells were examined by MTT detection and flowcytometry (FCM) analysis. The melanocytes obtained by this method were free of keratinocytes and fibroblasts contamination. The cells maintained in MC2 showed better proliferation ability than those cultured in MCI. The results showed that pure melanocytes culture in good conditions can be obtained rapidly by our method.2. To construct a tissue engineering skin containing melanocytes: The keratinocytes, dermal fibroblasts and melanocytes were isolated and purified from a single foreskin biopsy. Then the cells were used to construct a tissue engineering skin containing melanocytes. The location of melanocytes in the tissue engineering skin were detected byDopa staining, transmission electron microscopy (TEM) and S-100immunostaining. The reconstructed skin was used to repair the nude mice skin defects. Results demonstrated that the artificial skin containing melanocytes was successfully constructed in vitro and can be used to repair the full-thickness skin defects successfully.3. To develop a rapid and reproducible method for the isolation of pure human umbilical vein endothelial cells (HUVECs) in good conditions: The HUVECs were isolated and purified by a method employed in our lab. The HUVECs were identified by cell morphology, immunostaining and transmission electron microscopy. The HUVECs obtained by this method were free of fibroblasts contamination. And the cells showed very good proliferation ability. The pure HUVECs in good conditions can be attained rapidly and easily by our method.4. To construct a tissue engineering skin containing capillary-like network: The HUVECs were isolated and purified from a new-born umbilical cord. The keratinocytes and dermal fibroblasts were cultivated from a new-born foreskin biopsy. The skin equivalent containing capillary-like network was constructed by employing tissue engineering method. The reconstructed skin was used to repair the nude mice skin defects.Results showed that the artificial skin containing capillary-like networkwas successfully reconstructed in vitro and repaired the full-thicknessskin defects successfully.更多还原