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背景:浸没式体外培养角质形成细胞的技术已得较普遍的应用,但角质形成细胞的体外器官样培养技术尚存在不足。目的:以铺有鼠尾胶原的TransWell培养板套皿,提供的气-液交界面体外培养角质形成细胞,以改进角质形成细胞的体外器官样培养技术。设计、时间及地点:体外人工皮肤器官实验,于2008-07/2009-02在河北医科大学第一医院实验室完成。材料:角质形成细胞由解放军第二军医大学王教授馈赠。方法:将浸没式培养的角质形成细胞接种到铺有鼠尾胶原的Transwell培养板套皿当中,Transwell培养板套皿每板有6个孔,每孔当中有一插件,插件悬挂于孔的边缘上,插件底面为一孔径为0.4μm的高分子膜,距离培养板孔的底面有1.5mm的高度,使细胞处于气-液交界面上并培养4周,然后将不同培养时段的培养物进行苏木精-伊红染色、免疫组织化学、流式细胞仪检测,进而观察细胞分层和分化的状况。主要观察指标:观察细胞的生长状态及分层和分化状态。结果:培养2周的角质形成细胞大部分为单层结构,细胞彼此衔接成铺路石状结构,多数细胞为三角形或多角形结构,细胞核清晰可见,细胞比较饱满。部分区域细胞出现双层结构,表现为单层的角质形成细胞上面铺有连接成条索状的细胞。3周的角质形成细胞大部分为双层结构,异层及同层细胞之间彼此衔接,呈索形结构,细胞核仍然清晰可见,细胞不如2周时饱满。4周的角质形成细胞部分区域为三层结构,且细胞较前变得萎缩,表现为细胞浆减少,细胞核变小。角质形成细胞进行气-液交界面培养至4周,细胞会分为3层,表层的细胞会表达终末分化的标志物角蛋白10。结论:在Transwell培养板套皿膜上,角质形成细胞会出现分层和分化,形成体外的皮肤样器官。
BACKGROUND: Submerged in vitro culture of keratinocytes has been more commonly used techniques, but keratinocytes in vitro organ-like culture techniques are still inadequate. OBJECTIVE: To culture keratinocytes cultured in vitro on a transwell plate coated with rat tail collagen and provide a gas-liquid interface to improve the ex vivo organ-like culture of keratinocytes. DESIGN, TIME AND SETTING: In vitro artificial skin organ experiment was performed at the Laboratory of the First Hospital of Hebei Medical University from July 2008 to February 2009. Materials: Keratinocytes were donated by Professor Wang of the Second Military Medical University of PLA. Methods: Immersed keratinocytes were inoculated into rat collagen-coated Transwell plates containing 6 wells per plate. Each well contained an insert that hung on the edge of the well , The bottom of the insert is a polymer membrane with a diameter of 0.4 μm, 1.5 mm height from the bottom of the hole of the culture plate, so that the cells are at the gas-liquid interface and cultured for 4 weeks, and then the cultures in different culture periods are subjected to the superoxide dismutase Ameloblanil-Eosin staining, immunohistochemistry, flow cytometry, and then observe the status of cell delamination and differentiation. MAIN OUTCOME MEASURES: Observation of cell growth status and stratification and differentiation status. Results: Most of the keratinocytes cultured for 2 weeks were monolayer. The cells coalesced into paving stones, most of the cells were triangular or polygonal. The nuclei were clearly visible and the cells were full. Some regions of the cells appear double-layered structure, the performance of a single layer of keratinocytes connected with bar-like cells connected to the top. The majority of keratinocytes in 3 weeks had a double-layer structure. The different layers and the same layers of cells were connected with each other, showing a cable-shaped structure. The nuclei were still clearly visible, and the cells were not as full as those at 2 weeks. Four weeks of keratinocyte partial area of three-tier structure, and the cells became atrophy, showing the cytoplasm decreased, smaller nuclei. Keratinocytes were cultured at gas-liquid interface for 4 weeks. The cells were divided into three layers, and the surface cells expressed keratin 10, the terminal differentiation marker. CONCLUSIONS: Keratinocytes delaminate and differentiate into keratinocytes on a Transwell plate and form an in vitro dermal-like organ.