利用体外构建的组织工程皮肤模型进行皮肤刺激性检测的相关研究

发布时间：2018-04-16 20:29

本文选题：上皮角质形成细胞 + 永生化上皮角质形成细胞HaCaT　；参考：《河北联合大学》2011年硕士论文

【摘要】：组织工程皮肤模型已应用于许多研究中,他们其中的主要目标之一是替代动物实验。目前大多数皮肤模型是用原代培养的细胞构建的,然而,细胞的来源有限,每代细胞之间有很大的差异,因此,皮肤模型的一致性和重复性很难保证。实验的主要目的是构建两种皮肤模型来进行皮肤刺激性测试。一种皮肤模型利用永生化上皮角质形成细胞HaCaT构建,另外一种皮肤模型利用原代培养的上皮角质形成细胞构建。我们根据欧洲替代实验确认中心规定的用于皮肤刺激性的物质中选取了20种化学品,在刺激物接触皮肤模型后,细胞活力通过MTT测得,然后收集条件培养基检测IL-1α。两种皮肤模型具有相似的结构。根据欧洲替代实验确认中心20种物质的检测结果显示两种模型都适用于进行皮肤刺激性检测,而利用永生化细胞构建的皮肤模型的敏感性和特异性均高于利用原代培养细胞构建的皮肤模型。结论:两种皮肤模型均适用于皮肤刺激性的检测,然而,在皮肤刺激性检测实验中含永生化细胞的皮肤模型具有更好的敏感性和特异性。因此,利用永生化细胞构建的皮肤模型更适合用于皮肤刺激性实验。实验分三部分: 第一部分:人原代培养上皮角质形成细胞和永生化上皮角质形成细胞HaCaT增殖能力的比较为了筛选适合充当组织工程皮肤的种子细胞,比较原代培养的人原代培养上皮角质形成细胞和永生化的上皮角质形成细胞HaCaT的增殖能力。比较两种细胞的细胞周期,细胞凋亡,生长曲线。结论:永生化上皮角质形成细胞HaCaT的增殖能力要高于原代培养的上皮角质形成细胞。第二部分:利用原代培养的上皮角质形成细胞和永生化的上皮角质形成细胞构建组织工程皮肤检测模型将生长状态良好的人成纤维细胞与小牛Ⅰ型胶原混合,37°C孵育0.5h凝固后,加入培养液连续培养10d,取上述真皮基质放入24孔培养孔内,在其表面以5Х106密度接种上皮角质形成细胞培养7d,再进行气液面培养7d,即获得检测用的组织工程皮肤模型。利用相同的方法构建含永生化上皮角质形成细胞的组织工程皮肤检测模型。比较两种模型的形态学,免疫组化检测,屏障功能检测。结论:两种模型都适合用于皮肤刺激性检测。第三部分:利用构建的两种组织工程皮肤模型进行皮肤刺激性检测依据ECVAM规定的用于皮肤刺激性检测的物质中选取20种物质作用于两种皮肤模型,检测标准依据EpiSkin检测标准,检测两种皮肤模型的敏感性和特异性及IL-1α。结论:含永生化细胞的组织工程皮肤模型检测的敏感性和特异性均高于利用原代培养细胞构建的皮肤模型。
[Abstract]:Tissue engineering skin model has been applied in many studies, one of the main goals of them are replacing animal experiments. At present most skin model with primary cultured cells, however, limited cell sources, there are great differences between each generation of cells, therefore, consistency and repeatability of skin model is very difficult guarantee. The main objective of the experiment was to build the skin irritation test of two kinds of skin models. An immortalized keratinocyte cell line HaCaT was constructed by means of the skin model to another model using the original skin keratinocytes. We constructed according to the European Center for alternative experimental verification provisions of the skin irritating substances selected 20 kinds of chemicals, in contact with skin irritants model, cell viability was measured by MTT, and then collected conditioned medium to detect IL-1 alpha two skin model. Has the similar structure. According to the detection results of European substitution experiments confirm the center of 20 substances showed that two kinds of models are suitable for skin irritation test and skin model containing HaCaT cells. The sensitivity and specificity were higher than the primary skin cells to construct the culture model. Conclusion: the two kinds of skin models the applicability of the stimulus to the skin detection, however, is better in the skin to stimulate the skin model containing immortalized cells of the experiment to detect the sensitivity and specificity. Therefore, by means of the skin model of establishing immortalized cells are more suitable for skin irritation.
The experiment is divided into three parts:
The first part: the comparison of primary cultured human keratinocyte cells and immortalized keratinocyte cell proliferation HaCaT
In order to select suitable to act as seed cells of tissue engineering skin. Comparison of primary cultured human primary cultured keratinocyte cells and immortalized keratinocyte cell line HaCaT proliferation. Cell cycle, comparison of two kinds of cell apoptosis, growth curve. Conclusion: immortalized keratinocyte cell line HaCaT proliferation ability than the primary cultured epithelial keratinocytes.
The second part: the use of primary keratinocytes and immortalized keratinocyte model detection of tissue engineering skin cells
The good growth state of human fibroblast and calf collagen mixed 37 ~ C after incubation of 0.5h solidified, cultured with 10d continuous medium, the dermal matrix into the 24 hole culture hole on the surface with 5 * 106 density inoculated epithelial keratinocytes cultured 7d, followed by gas liquid 7d culture, to obtain tissue engineering skin model for detection. To construct immortalized keratinocyte model for detection of tissue engineering skin cells by using the same method. The morphological comparison of two kinds of models, immunohistochemistry, barrier function detection. Conclusion: the two kinds of models are suitable for the skin irritation test.
The third part: using the constructed two tissue engineering skin models to detect skin irritation
According to the ECVAM regulations for skin irritation testing material in selected 20 kinds of substances in two kinds of skin model, according to the EpiSkin standard test standard detection, detection of two kinds of skin models the sensitivity and specificity of IL-1 and alpha. Conclusion: the model of tissue engineering skin containing detection of immortalized cells. The sensitivity and specificity were higher than that of the use of the original skin cell model constructing training.

【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R751

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