大鼠骨髓Thy-1.1^+干细胞的分选与表型研究

Isolation and Phenotype of Thy-1.1^+ Stem Cells from Rat Bone Marrow

目的采用免疫磁珠分选系统(MACS)分离大鼠骨髓Thy-1.1+干细胞群,研究Thy-1.1+干细胞与肝脏分化潜能相关的表型特征。方法收集大鼠胫骨、股骨骨髓细胞,Percoll密度梯度离心分离单个核细胞,Thy-1.1单抗标记,间接免疫磁珠系统分离纯化Thy-1.1+干细胞群,流式细胞仪检测其纯度,锥虫蓝拒染法检测细胞活力,计算回收率。同时流式检测分选前细胞及分选后Thy-1.1+干细胞的表型。结果经MACS分选后Thy-1.1+细胞比例由56.65%升至94.20%;分选后的细胞活力为99.62%,与分选前的99.79%无明显差异;MACS分选Thy-1.1+细胞的回收率为64.65%。Thy-1.1+细胞不表达CD34和c-kit;高表达β2微球蛋白(β2M)和CD45;部分表达Flt-3。分选前后CD34+和c-kit+比例无显著差异;β2M+的比例由93.38%下降至86.39%(P<0.05);CD45+和Flt-3+的比例则由67.18%、14.36%升高至分选后的81.12%和58.72%(均P<0.01)。结论MACS方法能有效分选大鼠骨髓Thy-1.1+干细胞群,所得细胞纯度高,细胞活力保持好。大鼠Thy-1.1抗原与CD34、c-kit分子无明显相关性;而与CD45、Flt-3有一定的正相关性;与β2M呈一定的负相关性。

Objective To isolate Thy-1.1^＋ stem cells from rat bone marrow by magnetic activated cell sorting （MACS） and investigate their phenotypes which are associated with the hepatic differentiation capability. Methods Rat bone marrow cells were collected from rat tibias and femurs, and mononuclear cells were isolated by Percoll density gradient centrifugation. After incubation with Thy-1.1 monoclonal antibody, the Thy-1.1^＋ stem cells were purified by indirect magnetic activated cells sorting. The purity and viability were evaluated by flow cytometry and by typanblue staining respectively. And the recovery rate was calculated. Simultaneously, the phenotypes of the cells before and after MACS were detected by flow cytometry. Results The purity of Thy-1.1^＋＂ cells was elevated from 56. 65% to 94.20% by MACS, and the viability was 99. 62%, which had no significant difference in comparison with that before MACS （99.79%）. The recovery rate was 64. 65%. Thy-1.1^＋ cells were negative for CD34 and c-kit, but positive for β2M and CD45, and partially expressed Flt-3. There was no significant difference in the proportion of CD34^＋ and c-kit^＋ before and after MACS, while the percentage of β2M^＋ was decreased from 93. 38% to 86.39%, and CD45^＋ and Flt-3^＋ increased from 67.18^, 14.36% to 81.12% and 58.72% respectively. Conclusion High purified Thy-1.1^＋ cells can be obtained from rat bone marrow using MACS, and the viability of the cells was not impaired. There was no obvious relationship between the Thy-1.1 antigen and the CD34. c-kit molecule in rats, but Thy-1.1 was positively correlated with CD45 and Fit-3, and negatively with β2M to some extent.