人阴道黏膜干细胞的筛选与培养

Isolation and culture of human vaginal mucosa stem cells

目的:探讨人阴道黏膜干细胞(vaginal mucosa stem cells,VMSCs)的分离、筛选与适合的体外培养方法。方法:用胰酶、胶原酶混合消化法获得阴道黏膜细胞。以丝裂酶素C处理的小鼠成纤维细胞作滋养层细胞。将20min内黏附于Ⅳ型胶原的阴道黏膜细胞定为VMSCs。对筛选的VMSCs与原代人阴道黏膜细胞分别用碘化丙啶(PI)一步荧光染色法,用流式细胞仪检测其细胞周期。分为4组体外培养原代VMSCs,第1组用滋养层细胞+上皮细胞完全培养液;第2组滋养层细胞+DMEM/F12(3:1);第3组无滋养层细胞+上皮细胞完全培养液;第4组无滋养层细胞+DMEM/F12(3:1)。培养至12天,比较各组的CK19、CK10阳性细胞的比例、克隆形成率(clony forming efficiency,CFE)与凋亡前传代的次数。结果:筛选的VMSCs与原代人阴道黏膜细胞的G_0/G_1细胞比例、CK19、CK10细胞阳性比例均有统计学差异(P＜0．05)。用滋养层细胞+上皮细胞完全培养液培养VMSCs12天的CFE与CK19阳性细胞比例最高,CK10阳性细胞比例最低,体外培养VMSCs能稳定传代15代以上。结论:Ⅳ型胶原快速黏附法可有效地筛选VMSCs。滋养层细胞+上皮细胞完全培养液为体外培养原代VMSCs的较佳方法。

Objective：To explore the suitable method of isolation, identification and culture of human vaginal mucosa stem cells （VMSCs）. 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 20minutes. The cell cycles of the VMSCs and primary human vaginal mucosa cells were analyzed by flow cytometry. Culture VMSCs in vitro with 4 different media, including feeder layer cells ＋ epidermal total culture media, feeder layer cells ＋ DMEM/F12 （ 3 ： 1 ）, epidermal total culture media and DMEM/F12 （ 3 ： 1 ）. On the 12th day,clony forming efficiency （CFE）, CK19 and CK10 positive rates were examined. Then VMSCs were continuously cultured and passaged till they were apoptosis. Results：The ration of G0 - G1 ,CK19 and CK10 posotive rates between the VMSCs and primary human vaginal mucosa cells were statistically different. On the 12th day ,the VMSCs cultured with feeder layer and epidermal total culture media showed the highest CFE and CK19 positive rate and lowest CK10 postive rate. They could be passaged over 15 times in vitro. Conclusion ：It is an effective way to select VMSCs by adhesion to type Ⅳ collagen for 20minutes. Based on our research, culturing VMSCs in viro with feeder layer and epidermal total culture media is the best way to keep VMSCs＇stem cell characteristics.