细胞共培养皮肤类似物的制备及评价

发布时间：2018-02-27 18:04

本文关键词： 表皮生长因子皮肤类似物屏障功能气液界面培养相对湿度形态结构　出处：《北京中医药大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的：本研究拟为经皮给药系统研究提供新型皮肤模型。通过皮肤类似物的制备及评价,以初步了解其特性。在此基础上,以皮肤类似物的结构特征、分化标志物的表达、屏障功能、分子结构等为指标,优选皮肤类似物的气液界面培养时间、生长环境相对湿度及培养基中是否加入表皮生长因子,并与优化前屏障功能相比较,以期得到与人类皮肤相似、在经皮给药系统研究中能替代动物皮肤等皮肤模型的新型皮肤替代物。方法：(1)皮肤类似物的制备及评价设定培养环境为95%RH,不添加表皮生长因子,气液界面培养3周,制备皮肤类似物。通过肉眼观察、电镜观察、渗透性实验,初步了解皮肤类似物的特性。(2)皮肤类似物生长环境的优选：①设定不同的气液界面培养时间,收获皮肤类似物,通过肉眼观察、电镜观察、免疫组织化学法、傅里叶红外光谱法、渗透性实验筛选皮肤类似物的气液界面培养时间。②设定95%RH及75%RH两个环境相对湿度,分别制备皮肤类似物。以吲哚美辛为模型药,渗透性实验筛选培养环境相对湿度。③气液界面培养阶段培养基中添加或者不添加表皮生长因子,制备皮肤类似物。以吲哚美辛为模型药,渗透性实验研究气液界面培养阶段表皮生长因子对皮肤类似物屏障功能的影响。(3)皮肤类似物的评价①肉眼观察皮肤类似物的宏观形态结构特征,并与人类在体皮肤比较。②马松染色研究皮肤类似物中细胞及纤维蛋白的分布情况,并与人类皮肤比较。③扫描电镜观察皮肤类似物表面结构,并与人类皮肤比较。④透射电镜研究皮肤类似物角质层及皮肤各层间连接的变化,并与人类皮肤比较。⑤以吲哚美辛、氢化可的松、咖啡因为模型药,以大鼠、小鼠、裸鼠及Start-M膜为对照,考察皮肤类似物对不同油水分配系数模型药的渗透屏障特性。⑥优化前后,皮肤类似物屏障功能的比较。结果：(1)皮肤类似物具有与人类皮肤相似的表皮层和真皮层,并呈现出与人类皮肤相似的超微结构,但是表皮层不完整；对吲哚美辛的经皮渗透具有一定的屏障作用,其屏障作用与Start-M膜相似,但是与大鼠相比有较大差距。(2)皮肤类似物生长的最佳条件为75%RH,不添加表皮生长因子,气液界面培养3周。(3)皮肤类似物的形态结构、角蛋白10的表达及超微结构与人类皮肤非常相似,具有一定的机械强度。而丝聚合蛋白的表达被过度激活。皮肤类似物对不同油水分配系数模型药经皮渗透均有屏障作用,总体来说,对亲脂性化合物的屏障作用相对较高,接近小鼠,但与其他动物皮肤仍有一定差距；对亲水性化合物屏障作用相对较弱,与Start-M膜相似。结论：实验室制备的皮肤类似物在一定程度上与人类皮肤相似,通过培养环境的优化使其屏障作用有所提高,但是其在经皮给药系统研究中替代动物皮肤使用仍然需要通过努力提高其屏障功能。
[Abstract]:Objective: to provide a new skin model for the study of transdermal drug delivery system. Through the preparation and evaluation of the skin analogue, the characteristics of the skin analogue were preliminarily understood. On this basis, the structural characteristics of the skin analogues and the expression of differentiation markers were used. The barrier function, molecular structure and so on were used as indexes. The culture time of gas-liquid interface of skin analogue, the relative humidity of growth environment and whether the epidermal growth factor was added to the culture medium were selected, and compared with those before the optimization of barrier function. In order to obtain a new skin substitute, similar to human skin, which can replace animal skin and other skin models in the study of transdermal drug delivery system. Methods the preparation and evaluation of skin analogue was determined to be 95 RH and no epidermal growth factor was added. Skin analogue was prepared by gas-liquid interface culture for 3 weeks. Through naked eye observation, electron microscope observation and permeability experiment, the characteristics of skin analogues were preliminarily understood. The skin analogue was harvested. The relative humidity of 95RH and 75RH were set at the gas-liquid interface culture time of the skin analogue by naked eye observation, electron microscope observation, immunohistochemistry, Fourier transform infrared spectroscopy and permeability experiment. Indomethacin was used as model drug to screen the culture medium with or without epidermal growth factor (EGF) in the culture medium at the gas-liquid interface stage of relative humidity (RH. 3). Preparation of skin analogue. Using indomethacin as model drug, The effect of epidermal growth factor on the barrier function of skin analogue during gas-liquid interface culture. Evaluation of skin analogue: 1 the macroscopic morphological and structural characteristics of the skin analogue were observed with naked eyes. The distribution of cell and fibrin in the skin analogue was studied by using 2.2-#china_person0# staining compared with the human skin in vivo, and the surface structure of the skin analogue was observed by scanning electron microscope (SEM) compared with the human skin. Compared with human skin, transmission electron microscope (TEM) was used to study the changes of keratinocytes and interlaminar junctions of skin analogues, and to compare with human skin by indomethacin, hydrocortisone, coffee as model drug, rats and mice. In nude mice and Start-M membrane, the permeability barrier characteristics of skin analogue to different oil-water partition coefficient model drugs were investigated before and after optimization. Results the skin analogue has the similar epidermis and dermis to the human skin, and presents the ultrastructure similar to the human skin, but the epidermis is incomplete. The barrier effect of indomethacin was similar to that of Start-M membrane, but there was a big gap between rats and rats. The optimum condition for the growth of skin analogue was 75RH without adding epidermal growth factor (EGF). The morphology, expression and ultrastructure of keratin 10 were very similar to those of human skin. The expression of silk polymeric protein was over-activated. The skin analogue had a barrier effect on the transdermal permeation of different oil-water partition coefficient drugs, but the barrier effect on lipophilic compounds was relatively high. It is close to mice, but still has a certain gap with other animal skin, and has relatively weak barrier effect on hydrophilic compounds and is similar to Start-M membrane. Conclusion: the skin analogues prepared in laboratory are similar to human skin to some extent. The barrier function is improved by optimizing the culture environment, but it is still necessary to improve the barrier function by replacing the animal skin in the study of transdermal drug delivery system.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R96

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