本文選題：毛乳頭細(xì)胞 + 角質(zhì)形成細(xì)胞��； 參考：《中國(guó)協(xié)和醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 實(shí)驗(yàn)研究(一) 目的：尋求一種潔凈無需顯微鏡下操作的毛乳頭分離方法。方法：頭皮組織用中性蛋白酶消化后,將含有毛囊下段的皮下脂肪層剪下切碎后,用Ⅰ型膠原酶消化,最后通過多次離心的方法獲得毛乳頭。結(jié)果：新型的兩步酶消化法無需顯微鏡下操作,就可以獲得潔凈的毛乳頭。結(jié)論：新型的兩步酶消化法是一種高效的毛乳頭分離方法。 實(shí)驗(yàn)研究(二) 目的：通過比較毛乳頭細(xì)胞在含血清的角質(zhì)形成細(xì)胞培養(yǎng)基(keratinocyte medium,KM)和普通培養(yǎng)基(normal medium, NM)中生物學(xué)特性差異來判斷KM培養(yǎng)的毛乳頭細(xì)胞是否可用于組織工程研究。方法：分別用角質(zhì)形成細(xì)胞培養(yǎng)基和普通培養(yǎng)基培養(yǎng)毛乳頭細(xì)胞,統(tǒng)計(jì)不同培養(yǎng)基中毛乳頭貼壁率、細(xì)胞遷出時(shí)間、最大傳代次數(shù),鏡下觀察細(xì)胞形態(tài)差異,計(jì)數(shù)第3代細(xì)胞每皿聚集體個(gè)數(shù),用MTT方法檢.測(cè)細(xì)胞增殖,免疫熒光染色和特殊染色觀察a-平滑肌肌動(dòng)蛋白(a-smooth muscle actin, a-SMA)和ALP在兩組細(xì)胞中的表達(dá)情況,并統(tǒng)計(jì)ALP藍(lán)色陽(yáng)性區(qū)域面積。結(jié)果：毛乳頭在角質(zhì)形成細(xì)胞培養(yǎng)基中貼壁率更高、細(xì)胞遷出更快,聚集生長(zhǎng)能力更強(qiáng),增殖迅速,可更多次持續(xù)傳代,堿性磷酸酶的表達(dá)的持續(xù)代數(shù)更多。結(jié)論：用含血清的KM培養(yǎng)毛乳頭細(xì)胞用于組織工程是可行的。 實(shí)驗(yàn)研究(三) 目的：用人毛乳頭細(xì)胞和角質(zhì)形成細(xì)胞做為種子細(xì)胞構(gòu)建帶附屬器的組織工程皮膚替代物。方法：將角質(zhì)形成細(xì)胞和毛乳頭細(xì)胞共同混入膠原蛋白中制成真皮替代物,在此真皮替代物表面種上角質(zhì)形成細(xì)胞形成復(fù)合組織工程皮膚替代物。將此皮膚替代物在氣-液界面培養(yǎng)3-5天后移植到裸鼠背部創(chuàng)面,觀察創(chuàng)面愈合情況和收縮情況,分別于移植后4、6、8周取標(biāo)本做組織學(xué)觀察。結(jié)果：用毛乳頭細(xì)胞和角質(zhì)形成細(xì)胞構(gòu)建的皮膚替代物可以促進(jìn)創(chuàng)面的愈合、基底膜的形成。移植后6周替代物內(nèi)可見毛囊樣結(jié)構(gòu)和皮脂腺樣結(jié)構(gòu)。結(jié)論：將人的毛乳頭細(xì)胞和角質(zhì)形成細(xì)胞共同種植在皮膚替代物的真皮層可以構(gòu)建出帶附屬器的組織工程皮膚替代物。
[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.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R751

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相關(guān)期刊論文 前8條

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