人阴道黏膜干细胞的筛选与培养(英文)

Screening and culture of human vaginal mucosa stem cells

背景:目前国内外学者已经从人类或啮齿类等动物的皮肤、毛囊、角膜、口腔黏膜、肠道黏膜、牙齿断端、唾液腺等上皮组织中成功分离出干细胞,并应用于组织创伤修复的实验中,但人阴道黏膜干细胞的分离和应用的实验较少。目的:探讨人阴道黏膜干细胞的分离、筛选与适合的体外培养方法。设计:以细胞为对象的观察基础实验。单位:广州医学院第三附属医院(原广州市第二人民医院)妇产科。材料:实验于2004-10/2005-08在中山大学中山眼科中心实验室完成。实验室级别:卫生部眼科学重点实验室、广东省眼科视觉科学重点实验室、广东省教育厅眼科视觉科学重点实验室、广东省"五个一科教兴医工程"重点实验室。孕2周健康成年雌性昆明小鼠,由广州中医药大学实验动物中心提供。无细菌与病毒感染的患者的阴道黏膜标本5份,由广州医学院第三附属医院(原广州市第二人民医院)妇科提供。方法:采用胰酶、胶原酶混合消化法获得阴道黏膜细胞。将丝裂酶素C处理的小鼠成纤维细胞作滋养层细胞。采用Ⅳ型胶原快速黏附法筛选人阴道黏膜干细胞,并采用免疫细胞化学方法鉴定。将所筛选的人阴道黏膜干细胞与原代人阴道黏膜细胞分别用采用碘化丙啶一步荧光染色法、流式细胞仪分别检测其细胞周期。以4组方法体外培养原代人阴道黏膜干细胞:滋养层细胞+上皮细胞完全培养液、滋养层细胞+DMEM/F12(3∶1)、无滋养层细胞+上皮细胞完全培养液、无滋养层细胞+DMEM/F12(3∶1)。培养至12d,比较各组CK19、CK10阳性细胞比例、克隆形成率与凋亡前能传代的次数。主要观察指标:①所筛选的人阴道黏膜干细胞与原代人阴道黏膜细胞的G0/G1细胞比例、CK19、CK10阳性细胞的比例。②比较4种不同方法培养人阴道黏膜干细胞的CK19、CK10阳性的细胞比例、克隆形成率以及能传代的次数。结果:所筛选的人阴道黏膜干细胞与原代人阴道黏膜细胞的G0/G1细胞比例、CK19、CK10阳性的细胞比例均有统计学差异(P<0.05)。其中采用滋养层细胞+上皮细胞完全培养液培养人阴道黏膜干细胞12d,其克隆形成率与CK19阳性细胞比例最高,CK10阳性细胞比例最低,体外培养人阴道黏膜干细胞能稳定传15代以上。结论:①Ⅳ型胶原快速黏附法可有效筛选人阴道黏膜干细胞。②滋养层细胞+上皮细胞完全培养液为体外培养原代人阴道黏膜干细胞的较佳方法。

BACKGROUND： Nowadays stem cells have been isolated successfully from the epithelial tissue of human and rodents such as skin, hair follicle, cornea, oral mucous, intestinal mucosa dental epithelial cells, and salivary glands by the Chinese or foreign researchers. But according to the vaginal mucosa stem cells （VMSCs）, it is not known in the field of isolation and application. OBJECTIVE： To find out the suitable method of isolation, identification, and culture of human VMSCs. DESIGN： Observational basic study. SETTING： Department of Gynaecology and Obstetrics, the Third Affiliated Hospital of Guangzhou Medical College. MATERIALS： This study was performed in the Zhongshan Ophthalmology Center of Zhongshan Medical College from October 2004 to August 2005. Two-week gestation mice were provided by the Experimental Animal Center of Guangzhou University of Chinese Medicine. The sterile human vaginal mucosa was collected from five patients （25-38 years old） after the plastic operation of vagina from the 3^rd Affiliated Hospital of Guangzhou Medical College. METHODS： Vaginal mucosa cells could be harvested from human vagina mucosa treated with trypsin-collagenase digesting. The VMSCs were selected by adhesion to type Ⅳ collagen for 20 minutes. The cell cycles of the VMSCs and primary human vaginal mucosa cells were analyzed by flow cytometry. VMSCs were cultured in vitro with 4 different media, including feeder layer cells plus epidermal total culture media, feeder layer cells plus DMEM/F12 （3：1）, epidermal total culture media and DMEM/F12 （3：1）. At the 12^th day, their colony forming efficiency （CFE）, CK19 and CK10 positive rates were compared. Then VMSCs were continuously cultured and passaged till they were apoptosis. MAIN OUTCOME MEASURES： （1） Positive rate of CK19 and CK10 and G0/G1 proportion; （2） Positive rate of CK19 and CK10, the colony forming efficiency （CFE） and the times be passaged when primary VMSCs were cultured in different media after 12 days. RESULTS： The cells at the G0-G1 phase cell cycle, CK19 and CK10 positive rates between the VMSCs and primary human vaginal mucosa cells were statistical different （P 〈 0.05）. When cultured at 12^th day, the VMSCs cultured with feeder layer and epidermal total culture media had the highest CFE and CK19 positive rate and lowest CK10 positive rate. They could be passaged over 15 times in vitro. CONCLUSION： It is an effective way that select VMSCs by adhesion to type Ⅳ collagen for 20 minutes. In this research, culturing VMSCs in vitro with feeder layer and epidermal total culture media was the best way to keep VMSC characteristics.