人羊膜上皮细胞和间质细胞的分离培养  

The primary culture and isolation of human amnion epithelial and mesenchymal

作　　者：刘芳[1] 漆洪波[1] 

机构地区：[1]重庆医科大学附属第一医院妇产科,重庆400016

出　　处：《激光杂志》2008年第2期86-87,共2页Laser Journal

基　　金：国家自然科学基金项目(30772336)

摘　　要：目的:探讨人羊膜上皮细胞和羊膜间质细胞的分离、培养及鉴定。方法:取足月妊娠剖宫产术中的羊膜组织剪成碎片,胰蛋白酶消化15min,共四次,取消化液1000rpm离心5min收集细胞。剩余羊膜组织加入1.0g/L的胶原酶和0.1g/L的DNA酶,39℃消化120min,消化液1000rpm离心5min收集细胞。用含10%胎牛血清的DMEM/F12培养液培养、传代。倒置显微镜下观察其细胞形态及细胞生长情况。通过免疫细胞化学的方法对细胞进行细胞鉴定。结果:通过不同的酶消化分离法可以将羊膜上皮细胞和间质细胞进行细胞分离培养,羊膜细胞体外一段时间内表现出良好的细胞增殖能力和传代能力。胰蛋白酶消化的细胞角蛋白染色呈阳性,波形蛋白染色呈阴性。胶原酶消化的细胞波形蛋白色阳性,角蛋白染色阴性。结论:羊膜上皮细胞和间质细胞能够在体外分离、扩增。这为羊膜细胞进行细胞治疗和组织工程技术奠定了实验基础。Objective：To investigate the isolation, culture and identification of human amniatic epithelial cells and mesenchymal cells. Methods： Amnion tissues were collected immediately after elective cesarean section from term placentas. The harvested pieces of tissue were mechanically minced and treated 4 times with 0.25% trypsin for 15 minutes. The cells obtained during trypsin treatments were collected by centrifugation at 1000 rpm for 5 minutes. The remaining tissue were incubated with 1.0g/L collagenase and 0.1g/L DNase at 37℃ for 120 minutes. The cells were collected by centrifugation at 1000 rpm for 5 minutes. Both types were cultured in a complete medium consisting of DMEM/FI2 and 10% fetal bovine serum for primary culture and passage. The cultured cells were investigated morphologically under inverted microscope and were identified by immunohistochemistry. Results：Human anniotic cells can be successfully isolated by trypsin and colagenase respectively, and the cells can proliferate and passaged in vitro, for a certain period. Cells dispersed with trypsin stained positive for cytokeratin and negative for Vimentin. Cell dispersed with collagenase stained negative for cytokeratin and positive for Vimentin. Conclusion： Human amniotic epithelial and mesenchymal cells can be isolated and proliferated in vitro. These findings suggest that amnion cells may be a practical cell source for cell therapy and tissue engineering.

关 键 词：羊膜上皮细胞 羊膜间质细胞 细胞培养 细胞鉴定 

分 类 号：TN248.1[电子电信—物理电子学]