用人毛乳头细胞和角质形成细胞构建带附属器的组织工程皮肤替代物的实验研究

发布时间：2018-07-05 11:35

本文选题：毛乳头细胞 + 角质形成细胞　；参考：《中国协和医科大学》2010年硕士论文

【摘要】： 实验研究(一) 目的：寻求一种洁净无需显微镜下操作的毛乳头分离方法。方法：头皮组织用中性蛋白酶消化后,将含有毛囊下段的皮下脂肪层剪下切碎后,用Ⅰ型胶原酶消化,最后通过多次离心的方法获得毛乳头。结果：新型的两步酶消化法无需显微镜下操作,就可以获得洁净的毛乳头。结论：新型的两步酶消化法是一种高效的毛乳头分离方法。实验研究(二) 目的：通过比较毛乳头细胞在含血清的角质形成细胞培养基(keratinocyte medium,KM)和普通培养基(normal medium, NM)中生物学特性差异来判断KM培养的毛乳头细胞是否可用于组织工程研究。方法：分别用角质形成细胞培养基和普通培养基培养毛乳头细胞,统计不同培养基中毛乳头贴壁率、细胞迁出时间、最大传代次数,镜下观察细胞形态差异,计数第3代细胞每皿聚集体个数,用MTT方法检.测细胞增殖,免疫荧光染色和特殊染色观察a-平滑肌肌动蛋白(a-smooth muscle actin, a-SMA)和ALP在两组细胞中的表达情况,并统计ALP蓝色阳性区域面积。结果：毛乳头在角质形成细胞培养基中贴壁率更高、细胞迁出更快,聚集生长能力更强,增殖迅速,可更多次持续传代,碱性磷酸酶的表达的持续代数更多。结论：用含血清的KM培养毛乳头细胞用于组织工程是可行的。实验研究(三) 目的：用人毛乳头细胞和角质形成细胞做为种子细胞构建带附属器的组织工程皮肤替代物。方法：将角质形成细胞和毛乳头细胞共同混入胶原蛋白中制成真皮替代物,在此真皮替代物表面种上角质形成细胞形成复合组织工程皮肤替代物。将此皮肤替代物在气-液界面培养3-5天后移植到裸鼠背部创面,观察创面愈合情况和收缩情况,分别于移植后4、6、8周取标本做组织学观察。结果：用毛乳头细胞和角质形成细胞构建的皮肤替代物可以促进创面的愈合、基底膜的形成。移植后6周替代物内可见毛囊样结构和皮脂腺样结构。结论：将人的毛乳头细胞和角质形成细胞共同种植在皮肤替代物的真皮层可以构建出带附属器的组织工程皮肤替代物。
[Abstract]:Objective: to explore a new method for the separation of dermal papilla without microscope. Methods: after the scalp tissue was digested with neutral protease, the subcutaneous fat layer containing the lower part of the hair follicle was cut off and digested with type I collagenase. Finally, the dermal papilla was obtained by multiple centrifugation. Results: the new two-step enzyme digestion method can obtain clean dermal papilla without operating under microscope. Conclusion: a new two-step enzyme digestion method is an effective method for the separation of dermal papilla. Objective: to compare the biological characteristics of dermal papilla cells in keratinocyte medium and (normal medium, NM medium. Whether cells can be used in tissue engineering. Methods: dermal papilla cells were cultured in keratinocyte culture medium and common culture medium respectively. The rate of dermal papilla adhesion, the time of cell migration, the maximum number of passage, and the morphological differences were observed under microscope. The number of aggregates per dish of the third passage cells was counted and detected by MTT method. The expression of a-smooth muscle actin (a-SMA) and a-smooth muscle in the two groups were observed by immunofluorescence staining and special staining. Results: the dermal papilla had higher adhesion rate, faster cell migration, stronger aggregation and growth ability, rapid proliferation, more continuous passage and more sustained algebra of alkaline phosphatase expression in keratinocyte culture medium. Conclusion: it is feasible to culture dermal papilla cells with km containing serum for tissue engineering. Objective: human dermal papilla cells and keratinocytes were used as seed cells to construct tissue engineered skin substitutes with appendages. Methods: keratinocytes and dermal papilla cells were mixed into collagen to make dermis substitutes. The keratinocytes were planted on the surface of the dermal substitutes to form composite tissue engineered skin substitutes. The skin substitute was cultured at the gas-liquid interface for 3-5 days and then transplanted to the back wound of nude mice. The wound healing and contraction were observed. The specimens were taken for histological observation at 48 weeks after transplantation. Results: dermal substitutes constructed from dermal papilla cells and keratinocytes could promote wound healing and basement membrane formation. Hair follicle like structure and sebaceous gland like structure were found in the substitute 6 weeks after transplantation. Conclusion: human dermal papilla cells and keratinocytes can be implanted in the dermis of skin substitutes to construct tissue engineered skin substitutes with appendages.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R751

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