摘要
目的研究小鼠阴道黏膜干细胞的分离、富集和鉴定方法，为干细胞替代疗法或构建人工阴道黏膜治疗先天性无阴道提供依据。方法取7～8周龄小鼠的阴道黏膜上皮，用酶消化培养法原代培养阴道黏膜干细胞（VMSC）。用Ⅳ型胶原黏附法并连续传代分离和富集VMSC。显微镜下动态观察细胞生长特性；苏木精-伊红（HE）染色；透射电镜观察细胞超微结构；荧光显微镜下观察标记滞留细胞（LRC）。免疫组化sP法检测细胞表面标记物（β1整合素、细胞角蛋白19）进行鉴定。结果用酶消化培养法得到原代VMSC，用Ⅳ型胶原黏附法分离到较纯的VMSC，经连续传代，富集到大量VMSC。动态观察发现细胞呈克隆性生长的干细胞特性。HE染色细胞呈椭圆形或梭形、核质比例大等幼稚细胞特征。透射电镜发现细胞呈未成熟细胞特征。荧光显微镜下LRC的核呈亮黄色，标记滞留时间长。免疫组化SP法检测β1整合素和细胞角蛋白19呈阳性表达。结论用酶消化培养法可原代培养VMSC。用Ⅳ型胶原黏附法并连续传代可分离、富集VMSC。免疫组化SP法检测细胞表面标记物可鉴定VMSC。
Objective To evaluate the methods of the isolation, incubation and identification of mouse vaginal mucosa stem cells （VMSCs） in vitro. Methods The vaginal mucosa epithelial tissues were dissected from 7 - 8-week-old mice. VMSCs were primarily incubated by the enzyme digesting incubation method,isolated by type IV collagen sticking method, and enriched by successful passing for some generations. Cells growth properties were observed under the microscope and uhrastucture under the transmission electron microscope. Label-retaining cells （ LRCs ） were examined under the fluoroscope. Cell surface makers like integrin β1 and cytokeratin 19 were detected by immunohistochemistry for identification. Results The primary VMSCs were obtained by the enzyme digesting incubation method. The purer VMSCs were isolated by type IV collagen sticking method. VMSCs were enriched by successful passing for some generations. VMSCs grew in colony and presented the characteristic of stem cell by dynamic observation under the microscope. By HE staining, VMSCs showed oval or shuttled-shape and the larger ratio of nucleolus and cytolymph characteristics under the microscope. VMSCs possessed the characteristics of premature cell by observing cell uhrastructure under the transmission electron microscope. The nucleolus of LRCs presented light yellow color and labeling lasted for a long time under the fluoroscope. VMSCs positively expressed integrin β1 and cytokeratin 19 by immunohistochemically detecting with SP method. Condusions VMSCs can be primarily cultured by the enzyme digesting incubation method,isolated by type IV collagen sticking method,enriched by successful passing for some generations. VMSCs are identified by observing cells＇growth property under the microscope, cell characteristic after HE staining, LRCs under the fluoroscope, cell ultrastucture under the transmission electron microscope, and by immunohistochemically detection of some cell surface makers.
出处
《中华生物医学工程杂志》
CAS
2007年第3期171-175,共5页
Chinese Journal of Biomedical Engineering
基金
广东省医学科学技术研究基金（20040544）
关键词
阴道黏膜
干细胞
细胞培养
分离
富集
鉴定
Mouse
Vaginal mucosa stem cell
Cell culture
Isolation
Enrichment
Identification
