本文選題：表皮干細胞 + IV型膠原　； 參考：《第三軍醫(yī)大學》2009年碩士論文

【摘要】： 隨著組織工程皮膚在皮膚及系統(tǒng)性疾病臨床應用(燒傷治療和基因療法)上的不斷發(fā)展,種子細胞的選擇成為一個關(guān)鍵因素。皮膚組織工程的研究顯示表皮干細胞較其他多能干細胞有更大的優(yōu)勢,因此如何快速分選表皮干細胞也就成為一個熱門的課題,本實驗通過不同時間內(nèi)角質(zhì)形成細胞對IV型膠原的粘附特性不同,對其進行分時分選,并對分選后的細胞進行生物學特性的研究,為表皮干細胞的分選提供實驗依據(jù)。 將分離后人包皮的角質(zhì)形成細胞接種到預先鋪有I和IV型膠原的六孔板中,在370C、CO2孵箱中分別孵育10、20、30、60min后,吸去未粘附細胞,離心法測粘附細胞在上述四個時段粘附在IV型膠原上的粘附力及粘附在I、IV型膠原20min粘附細胞的粘附力。細胞計數(shù)器測粘附數(shù)及細胞大小,并用β1整合素、外皮蛋白、CK10、CK19對貼壁10min的細胞進行鑒定,流式細胞儀測β1整合素在四時段的陽性表達量,相差顯微鏡進行細胞形態(tài)學觀察,并對四時段進行克隆形成率的測定。結(jié)果顯示:形態(tài)上貼壁細胞小而圓,反光能力強,核漿比大。隨著時間的延長,貼壁細胞數(shù)逐漸增多,20min內(nèi)粘附細胞數(shù)與10min有統(tǒng)計學意義(P0.05),而與30min、及60min無統(tǒng)計學意義,在30min粘附的細胞最小,克隆形成率20min與30、60min有統(tǒng)計學意義(P0.05),而與10min無統(tǒng)計學意義,粘附力10min與20、30、60min有統(tǒng)計學意義,20、30分鐘之間無統(tǒng)計學意義,I型膠原與IV膠原無差別。整合素β1間接免疫熒光、免疫組化顯示貼壁細胞10min后均為陽性,CK19免疫組化顯示部分陽性,外皮蛋白陰性表達,CK10偶有陽性,流式細胞儀測四時段整合素β1陽性率表達量均為100%。結(jié)論:利用IV型膠原分選后的角質(zhì)形成細胞具有干細胞的特征,以粘貼20min最為理想,可用于表皮干細胞的生物學特性的研究。
[Abstract]:With the development of tissue engineering skin in clinical application of skin and systemic diseases (burn therapy and gene therapy), the selection of seed cells has become a key factor. Skin tissue engineering studies have shown that epidermal stem cells have greater advantages than other pluripotent stem cells, so how to quickly separate epidermal stem cells has become a hot topic. In this experiment, the adhesion of keratinocytes to type IV collagen was different in different time, and the biological characteristics of the cells were studied in order to provide experimental basis for the sorting of epidermal stem cells. The keratinocytes isolated from human prepuce were inoculated into a six-hole plate precoated with type I and IV collagen. After incubating in the incubator of 370C- CO _ 2 for 30 minutes, the unadherent cells were sucked out. The adhesion force of adhesion cells to type IV collagen and adhesion to type IV collagen 20min were measured by centrifugation. The adhesion number and cell size were measured by cell counter, and the adherent 10min cells were identified by 尾 1 integrin and CK10 CK19. The positive expression of 尾 1 integrin in four periods was measured by flow cytometry, and cell morphology was observed by phase contrast microscope. The clone formation rate was determined in four periods. The results showed that the adherent cells were small and round in shape, with strong reflectivity and high nuclear / cytoplasmic ratio. With the extension of time, the number of adherent cells increased gradually within 20 minutes and the number of adherent cells was significantly higher than that of 10min (P 0.05), but had no statistical significance with 30 min and 60min, and the number of adherent cells in 30min was the smallest. The clone formation rate of 20min was significantly different from that of 30min (P 0.05), but not to 10min. There was no significant difference between type I collagen and IV collagen between 10min and 2030 min. The expression of integrin 尾 1 was indirect immunofluorescence. The positive expression of integrin 尾 1 was partly positive in adherent cells after 10min. The positive rate of integrin 尾 1 expression was 100% in four periods by flow cytometry. Conclusion: the keratinocytes separated by type IV collagen have the characteristics of stem cells, and the adhesion of 20min to keratinocytes is ideal for the study of the biological characteristics of epidermal stem cells.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

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