人脐带间充质干细胞不同分离方法的效果比较

Comparison of different methods for the isolation of human umbilical cord mesenchymal stem cells

目的对比分析分离人脐带间充质干细胞（MSCs）常用方法的效果，筛选比较高效的分离方法。方法采集足月剖宫产手术胎儿的脐带组织（n=15），充分洗涤后去除3条脐血管及脐带外膜，将其剪碎至1mm^3的组织块，洗涤离心确定组织块的总体积，平均分为4组，分别采用组织块法和单纯胶原酶Ⅱ消化法、胶原酶Ⅱ＋胰蛋白酶消化法、胶原酶Ⅱ＋透明质酸酶消化法，分离获得人脐带MSCs，行锥虫蓝染色计数细胞；倒置相差显微镜观察细胞形态；使用免疫荧光染色检测细胞表面标志物CDl05、CD90、CD73、CD31、CD44、CD45、人类白细胞抗原Ⅰ（HLA—Ⅰ）和人类白细胞抗原Ⅱ类分子（HLA—DR）的表达情况，进行细胞鉴定；噻唑蓝（MTY）法检测细胞的增殖曲线。结果组织块法和单纯胶原酶Ⅱ消化法、胶原酶Ⅱ＋胰蛋白酶消化法、胶原酶Ⅱ＋透明质酸酶消化法4种方法均可分离获得干细胞，但获得细胞的数量和增殖能力不同。4种方法获得的细胞数量分别为（5．44±0．21）×10^5、（4．03±0．24）×10^5、（4．91±0．33）×10^5和（5．94±0．40）×10^5，胶原酶Ⅱ＋透明质酸酶消化法显著多于其他3种方法，差异均有统计学意义（均P〈0．05）。组织块法种植后第9～11天观察到细胞在组织块周围生长，而其他3种方法种植后第2天即可见贴壁生长的细胞。4种方法细胞达到90％-95％融合的时间分别为（18．5±3．5）、（8．0±1．0）、（7．5±1．5）、（3．5±0．5）d，胶原酶Ⅱ＋透明质酸酶消化法显著短于其他3组，差异均有统计学意义（均P〈0．05）。组织块法原代细胞的形态多为多角形，不规则，细胞的体积较大，单纯胶原酶Ⅱ消化法和胶原酶Ⅱ＋胰蛋白酶消化法原代细胞的形态均较短小，而胶原酶Ⅱ＋透明质酸酶消化法细胞形态呈细长的梭形。4种方法得到的细胞均阳性表达CDl05、CD90和CD73，弱表达HLA-I，不表达CD31、CD45和HLA—DR。胶原酶Ⅱ＋透明质酸酶消化法细胞的增殖速率显著快于其他3种方法。结论胶原酶Ⅱ＋透明质酸酶消化法是一种高效的提取人脐带MSCs的方法。

Objective To explore the most appropriate method for the isolation of human umbilical cord mesenchyamal stem cells （MSCs） through a comparison of different methods. Methods Fifteen umbilical cord specimens from full-term healthy fetus with caesarean birth were completely rinsed with phosphate buffer saline （PBS） and sliced into 1 mm3 tissue blocks after removal of umbilical vessels and external membrane. These tissue blocks were averagely divided into 4 groups after washing and centrifuge. Then four methods for the isolation of human umbilical cord MSCs were compared： an explant culture and three enzymatic methods of collagenase 1] , collagenase II/trypsin and eollagenase II/hyaluronidase. The count of living cells was evaluated by trypan blue dye exclusion test. Cell morphology was observed under inverted microscope. The expressions of cell surface markers CD105, CD90, CD73, CD31, ,CD44, CD45, human leukocyte antigen- I （ HLA- I ） and human leukocyte antigen class II molecules （HLA-DR） were detected by immunofluoreseent staining. Cell proliferation was assayed by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyhetrazolium bromide （MTT）. Results The human umbilical cord MSCs were successfully isolated by four isolated methods. However the isolation method used profoundly altered the cell number and proliferation capacity of isolated cells. Isolated cells using four methods were counted at （5.44 ±0. 21 ） ×10^5 , （4. 03 ±0. 24） ×10^5, （4. 91 ±0. 33） ×10^5 and （5.94 ±0. 40） ×10^5 respectively. More cells were obtained with collagenase II/hyaluronidase than other three methods （ all P 〈 0.05 ）. Cells out of tissue blocks were observed at Day 9 - 11 and cells were observed at Day 2 with three types of enzyme digestion. The fusion time of cells were （18.5 ±3.5）, （8.0 ± 1.0）, （7.5 ± 1.5） and （3.5 ±0.5） days respectively. The fusion time of cells obtained with collagenase II/hyaluronidase was lower than other methods （ all P 〈 0.05 ）. Cell morphology ： polygonal, irregular and of large volume for explant culture; relatively short and small for collagenase II and collagenase II/trypsin methods ; thin spindle for collagenase II/hyaluronidase method. Immunofluorescent staining revealed that CD105, CD73, CD90 and CD44 were expressed in all groups while there was no expression of CD31, CD45 or HLA-DR. And the cells obtained with collagenase II/hyaluronidase method were in a higher cell proliferation rate and activity compared to other methods. Conclusion The collagenase I1/hyaluronidase method is optimal for the isolation of human umbilical cord MSCs than other methods.