兔胎盘源性间充质干祖细胞的分离方法比较  

Comparison with methods in separating placenta-derived mesenchymal stem cells from rabbits' placenta

作　　者：姚旺祥[1] 裴国献[1] 刘勇[1] 梅良斌[1] 马安[2] 

机构地区：[1]南方医科大学南方医院创伤骨科,广东广州510515 [2]南华大学医学院免疫学教研室,湖南衡阳421001

出　　处：《生物医学工程与临床》2007年第2期85-87,F0003,共4页Biomedical Engineering and Clinical Medicine

基　　金：国家高新技术发展项目(2003AA205010)

摘　　要：目的探讨几种酶消化方法对兔胎盘源性间充质干细胞(rPDMSCs)的分离效果。方法采用孕兔剖宫产取其胎盘,用Ⅱ型和Ⅳ型胶原酶、分散酶、DNaseⅠ组合成3组混合消化液:Ⅳ型胶原酶、DNaseⅠ与分散酶组(A组);Ⅳ型胶原酶与DNaseⅠ组(B组);Ⅱ型胶原酶与DNaseⅠ组(C组),分别对兔胎盘进行消化。分离间充质干细胞(MSCs)后经原代及传代培养,倒置显微镜逐日观察、HE及碱性磷酸酶染色、苔盼蓝拒染法计数胎盘源性细胞数目及倍增时间,流式细胞仪检测细胞表面标志。结果分步分离法对细胞的活力影响较小。C组分离的MSCs较其他两组细胞的数量多、活力好,细胞倍增时间约28～56h,组间差异有统计学意义(P<0.05);C组细胞形态规则,不表达碱性磷酸酶,诱导培养后可形成钙结节,流式细胞仪示CD105阳性率为91.9%,CD29阳性率89.1%,CD44阳性率93.6%,CD45阳性率3.2%。结论Ⅱ型胶原酶与DNaseⅠ混合消化液、分步分离法可以较好地从兔胎盘中提取MSCs。Objective To investigate the results of several digestive methods in separating placenta-derived mesenchymal stem cells （PDMSCs） from rabbits＇ placenta. Methods Placentas were adoped from the pregnant rabbits model by uterine-incision delivery and were processed by three combinations of digestive juice mixed with collagenase Ⅱ ,collagenase Ⅳ ,dispase and DNase Ⅰ ：group A （collagenase Ⅳ ＋ dispase ＋ DNase Ⅰ ） ;group B （collsgenase Ⅳ ＋ DNase Ⅰ ）;group C （collagenase Ⅱ ＋DNase Ⅰ ）. The isolated adherent cells were cultured and observed with inverted microscope day by day. The HE and alkali-phosphatase staining were used to observe the primary cell cultured. The viability and the doubling generation time were determined by staining with trypan blue. Results The step-by -step separation had less effect on cell vigoration. The adherent cells obtained from group C were more vigorous compared with group B and group A, and the statistical difference between each group was remarkable （P 〈 0.05）. The cells from group C did not express alkali-phosphatase ,the morphous of those cells were regulated ,but the alizarin Bordeaux staining was positive after inducing. The doubling generation time of the rabbit placenta-derived mesenchymal cells was 28 ～ 56 h. Conclusion The enzymatic digestion mixed with collagenase Ⅱ and DNase Ⅰ and step-by-step separation methods could effectively extract mesenchymal stem cells（MSCs） from the rabbits＇ placenta.

关 键 词：兔 胎盘 间充质干细胞 分离方法 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]