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目的建立从人足月胎盘中大量分离纯化滋养细胞的实验方法。方法收取20例正常足月妊娠胎盘,剪取50 g绒毛组织,2.5 g.L-1胰酶联合300 000 U.L-1DNaseⅠ反复消化组织,简化的密度梯度离心法(100 mL.L-1、300 mL.L-1、500 mL.L-1、700 mL.L-1Percoll细胞分离液)联合反复贴壁法分离纯化滋养细胞,锥虫蓝染色计算细胞活力,细胞免疫荧光标记、激光扫描共聚焦显微镜(LSCM)、流式细胞技术(FCM)检测滋养细胞内细胞角蛋白7(CK-7)的表达,计算滋养细胞纯度,比较2种方法测得的滋养细胞纯度是否有差异;FCM计算细胞数目。结果经胰酶、DNaseⅠ联合消化胎盘组织,简化的密度梯度离心法联合反复贴壁法,每50 g胎盘组织可获得细胞数为(0.95±0.25)×108、纯度约85%、活力为(88.86±2.83)%的滋养细胞。LSCM和FCM测得的胎盘滋养细胞的纯度比较差异无统计学意义(P>0.05)。结论简化的密度梯度离心法联合反复贴壁法,可获得大量纯化的足月胎盘滋养细胞。
Objective To establish a method for the isolation and purification of trophoblasts from human full term placenta. Methods Twenty normal placenta of term pregnancy were collected and 50 g of villi tissue were cut and digested with 2.5 g L-1 trypsin and 300 000 UL-1 DNase I repeatedly. The density gradient centrifugation (100 mL.L-1,300 mL) was performed. L-1,500mL.L-1,700mL.L-1Percoll cell separation fluid) combined with repeated adherent method separation and purification of trophoblast cells, trypan blue staining cell viability, immunofluorescence labeling, laser scanning confocal microscopy ( LSCM) and flow cytometry (FCM) were used to detect the expression of cytokeratin 7 (CK-7) in trophoblast cells. The purity of trophoblasts was calculated. The purity of trophoblasts was compared by FCM and FCM. Results The number of cells per 50 g of placenta was (0.95 ± 0.25) × 108, the purity was about 85% and the viability was (88.86%) with trypsin and DNase Ⅰ combined with digestion of placenta and simplified density gradient centrifugation. ± 2.83)% of trophoblast cells. There was no significant difference in the purity of placental trophoblast cells between LSCM and FCM (P> 0.05). Conclusion The simplified density gradient centrifugation combined with repeated adherent method can provide a large amount of purified term placental trophoblast cells.