【作者】 张莉萍；

【导师】 韩春茂；

【作者基本信息】 浙江大学 ， 外科学， 2009， 博士

【摘要】 皮肤移植是治疗大面积烧伤或创伤后皮肤缺损的主要手段。其中自体皮移植是临床最为有效的方法，但受到自体皮源不足的限制。随着组织工程的兴起，人们意识到开发出能有效促进创面愈合与皮肤组织再生的新型皮肤再生产品具有重要意义。组织工程三大要素是种子细胞，支架材料及合适的微环境。种子细胞一直是组织工程研究的关键要素。理想的种子细胞应该具有以下的特点：来源方便；在体外增殖迅速可以短期扩增到所需的数量；长期体外传代不改变生物学特性等。骨髓间充质干细胞是成体干细胞的一种，具有自我更新能力和多向分化潜能。在特定的条件下可以成骨细胞，成软骨细胞及成脂肪细胞分化。骨髓间充质干细胞的获取不受伦理学的限制，且取材方便，免疫排斥反应小，增殖能力强，是一种理想的种子细胞。骨髓间充质干细胞能否向表皮细胞分化?能否作为构建组织皮肤工程的种子细胞?我们的试验基于这一思路展开。我们之前的研究结果表明骨髓间充质干细胞有很大可能分化为表皮细胞。于是我们从重复体外诱导分化，到动物创面愈合中再上皮化的表皮细胞中有无人源的骨髓间充质干细胞，再到体外用不对称支架构建看骨髓间充质干细胞能否形成表皮层或协同表皮细胞形成表皮。我们进行了大量的试验，但总的结果显示骨髓间充质干细胞尽管能促进创面的愈合，但分化为表皮细胞的确有难度。最后我们又从大面积烧伤病人着手，探讨在应激反应中，机体能否动员人的骨髓间充质干细胞到血液中而参与烧伤创面的修复，及今后血源性间充质干细胞有无可能作为表皮的种子细胞?结果显示可能性存在，但分离纯化的方法上还需努力。本实验的主要结果及推论如下：1．采用Percoll密度梯度离心结合贴壁筛选分离并培养扩增成人骨髓间充质干细胞，所得骨髓间充质干细胞呈典型的成纤维细胞形态，漩涡状生长；具有骨髓间充质干细胞的表面标志物。2．人骨髓间充质干细胞在特殊诱导液的作用下具有向表皮细胞分化的可能性。3．人骨髓间充质干细胞和体外定向诱导分化后的骨髓间充质干细胞对裸鼠的皮肤创面都有促进愈合作用，但在特殊诱导液作用下的骨髓间充质干细胞并没有表现出表皮细胞的功能；并没有出现在上皮化的裸鼠皮肤细胞中。4．我们采用纤维蛋白凝胶能够重复不对称支架的构建，并证实该支架对骨髓间充质干细胞没有毒性作用。但骨髓间充质干细胞作为种子细胞用于不对称支架进行构建组织工程化皮肤难度大5．利用Percoll分离液进行密度梯度离心法从大面积烧伤病人的外周血中没有分离培养出间充质干细胞，但是可能性存在。更多还原

【Abstract】 Skin transplantation is the main means of treating skin defect caused by large-area burn or wound. In addition, deemed as the most effective in clinic treatment, autogenous skin transplantation has always been restricted for lack of autogenous skin source. Along with the development of tissue engineering, people has begun to realize the significance of developing new-type products to accelerate the concrescence of wound area and promote skin regeneration. Tissue engineering has three main factors, i.e. seeding cells, scaffold and extracelluar matrix(proper microenvironment), among which, the seeding cells has been viewed as the most crucial. An ideal seeding cell ought to have the following features: firstly, it has a convenient source; secondly, it has enough proliferation capacity to increase as many cell population as needed; thridly, it passages in vitro in the long term without changing the biological properties, and etc. This research finds out that human bone marrow-derived mesenchymal stem cells (BM-MSCs) are ideal as seeding cells. As one kind of adult stem cells, BM-MSCs has the self-renewal and differentiation potential. In certain conditions, they can differentiate into different embryonic layer cells as osteoblast, adipocyte, chondrocyte etc. Moreover, with low antigenicitiy and strong proliferation capacity, they are convenient to obtain. As well, the acquisition of BM-MSCs is not restricted from ethnics perspective.But there is seldom report of using MSCs in tissue engineered skin. Our experiments aim at finding out whether MSCs can differentiate into epidermal cells and whether they can be used as seeding cells in tissue engineering. Our previous research shows that BM-MSCs are likely to differentiate into epidermal cells. The current experiments range from repeating differentiation induction in vitro, to observing whether BM-MSCs can form epidermal layer or form epidermis together with epidermal cells with asymmetric scaffold in vitro. A large number of experiments are made, which show that although BM-MSCs succeed in advancing the concrescence of wound area, they have difficulty in differentiating into epidermal cells. In the end, the study proceeds with large-area burned patients to discuss whether BM-MSCs can be mobilized to blood to participate in the repair of burn wound and whether blood-bom MSCs can be used as the seeding cells of epidermis. The result shows the possibility exists while great efforts should be made in the method of separation and purification. The main findings and conclusions of the study are as follows:1. Both primary and passage human BM-MSCs, separated and cultivated with the method of Percoll gradient, showed typically fibroblast-like morphology and swirl growth with MSC’s surface markers.2. There is possibility that BM-MSC differentiate into epidermal cells in vitro in inducing culture medium.3. Both BM-MSCs and BM-MSCs after directional inducing differentiation in vitro could promote the concrescence of skin wound area of naked mice. However, in special inducing culture medium, BM-MSCs neither performed the function of epidermal cells, nor emerged in the epidermal cells of the naked mice.4. Fibrin glue was useful to repeat the construction of symmetric porous scaffold and proofed the scaffold innocuous to MSCs. However, it is difficult to use BM-MSCs as seeding cells in the symmetric porous scaffold for the construction of tissue engineered skin.5. No MSCs were separated and cultured from peripheral blood of large-area burned patients in the way of density gradient centrifugation by use of Percoll separation medium. However, the possibility may exist.更多还原