Ⅳ型胶原粘附分选后表皮干细胞基本生物学特性的研究

发布时间：2018-05-23 21:43

本文选题：表皮干细胞 + IV型胶原　；参考：《第三军医大学》2009年硕士论文

【摘要】： 随着组织工程皮肤在皮肤及系统性疾病临床应用(烧伤治疗和基因疗法)上的不断发展,种子细胞的选择成为一个关键因素。皮肤组织工程的研究显示表皮干细胞较其他多能干细胞有更大的优势,因此如何快速分选表皮干细胞也就成为一个热门的课题,本实验通过不同时间内角质形成细胞对IV型胶原的粘附特性不同,对其进行分时分选,并对分选后的细胞进行生物学特性的研究,为表皮干细胞的分选提供实验依据。将分离后人包皮的角质形成细胞接种到预先铺有I和IV型胶原的六孔板中,在370C、CO2孵箱中分别孵育10、20、30、60min后,吸去未粘附细胞,离心法测粘附细胞在上述四个时段粘附在IV型胶原上的粘附力及粘附在I、IV型胶原20min粘附细胞的粘附力。细胞计数器测粘附数及细胞大小,并用β1整合素、外皮蛋白、CK10、CK19对贴壁10min的细胞进行鉴定,流式细胞仪测β1整合素在四时段的阳性表达量,相差显微镜进行细胞形态学观察,并对四时段进行克隆形成率的测定。结果显示:形态上贴壁细胞小而圆,反光能力强,核浆比大。随着时间的延长,贴壁细胞数逐渐增多,20min内粘附细胞数与10min有统计学意义(P0.05),而与30min、及60min无统计学意义,在30min粘附的细胞最小,克隆形成率20min与30、60min有统计学意义(P0.05),而与10min无统计学意义,粘附力10min与20、30、60min有统计学意义,20、30分钟之间无统计学意义,I型胶原与IV胶原无差别。整合素β1间接免疫荧光、免疫组化显示贴壁细胞10min后均为阳性,CK19免疫组化显示部分阳性,外皮蛋白阴性表达,CK10偶有阳性,流式细胞仪测四时段整合素β1阳性率表达量均为100%。结论:利用IV型胶原分选后的角质形成细胞具有干细胞的特征,以粘贴20min最为理想,可用于表皮干细胞的生物学特性的研究。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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