免疫磁珠分选骨髓衍生肝干细胞亚群c-Kit^+lin^-

Purification of c-Kit^+lin^- cells from murine bone marrow by magnetic activated cell sorting

目的:利用免疫磁性细胞分选系统分离纯化骨髓衍生肝干细胞亚群c-Kit+lin-。方法:实验于2006-07/08在南方医科大学实验动物中心完成。6～8周龄的SPF级纯系BALB/C雄性小鼠10只,体质量18～20g。收集小鼠股骨骨髓细胞,利用免疫磁性细胞分选系统,通过两步法分选纯化c-Kit+lin-:将获取的lin-细胞悬液8℃条件下1500r/min离心10min,弃上清,按80μL/107加入Buffer重悬细胞。按20μL/107加生物素抗体磁珠,混匀,4℃冰箱孵育15min,按1mL/107加入Buffer洗细胞1次,8℃条件下1500r/min离心10min,弃上清,按500μL/108加入Buffer重悬细胞。Buffer500μL润MS柱,悬液过柱后,Buffer500μL/次洗柱3次,柱子脱离磁场,加1mLBuffer,用配套柱塞推出柱中的c-Kit+lin-细胞,收集到c-kit+lin-细胞,细胞计数。取2.0×106个细胞分成10等份,流式细胞仪分析c-Kit+lin-细胞纯度,计算回收率,评估纯化效率,苔盼兰染色检测纯化前后的细胞活力。计算活细胞的百分率。细胞纯度和细胞回收率的计算:细胞纯度=分离产物中的阳性细胞数/分离细胞的总细胞数×100%,细胞回收率=分离产物中的阳性细胞数/起始标本阳性细胞总数×100%。结果:10只小鼠均进入结果分析。利用免疫磁性细胞分选系统分选出的骨髓衍生肝干细胞亚群c-Kit+lin-细胞纯度和回收率分别为(77.98±2.34)%,75.40%,纯化前后细胞活力不受影响。结论:免疫磁性细胞分选系统能有效分选骨髓衍生肝干细胞亚群c-Kit+lin-,纯度和回收率高,且不影响细胞活力。

AIM： To isolate and purify c-Kit^＋lin^- cells from murine bone marrow by magnetic activated cell sorting （MACS）. METHODS： This experiment was conducted at the Animal Experiment Center of Southern Medical University from July to August 2006. Totally 10 Balb/c male mice of SPF level with 6-8 weeks old and 18-20 g were randomly selected. Murine bone marrow cells collected were isolated and purified by MACS： at the 8 ℃, the lin-cells were centrifugated at the speed of 1 500 r/min for 10 minutes, then supematant were abandoned. Buffer by 80 μL/10^7 was added to weight the cells, and then put the cells into refrigerator in 4 ℃ for 15 minutes after added anti-biotin magnetic bead by 20 μL/10^7, misce bene. Buffer by 1 mL/10^7 was added to wash the cells once. The cells were centrifugated at the speed of 1 500 r/min for 10 minutes at 8 ℃, then supematant were abandoned. Buffer by 500 μL/10^8 was added to weight the cells, and then moistened column by Buffer 500μL. The column was washed three times at Buffer 500 μL/times and the column was separated from magnet after cells passed. 1 mL Buffer was added, and c-Kit^＋lin^- cells were put out of column using plunger, and counted. 2.0×10^6 cells were obtained and separated equal 10 parts. Flow cytometry was applied to analyze the purity of c-Kit^＋lin^- cells, calculate recovery rate, and evaluate purification efficiency. Trypan blue staining was used to identify the cell activity before and after the purification. Percentage of living cells was calculated. Calculation of cell purification and cell recovery rate： cell purification was equal to number of positive cells in separated products/total number of separated cells ×100%. Cell recovery rate was equal to number of positive cells in separated products/total number of positive cells of initial samples x100%. RESULTS： A total of 10 mice were involved in the result analysis. The cell purification and recovery rate of c-Kit^＋lin^- cells were （77.98：P.2.34）%, 75.40%, respectively by MACS. The cell activity did not be affected before and after purification. CONCLUSION： Highly purified c-Kit^＋lin^- cells can be obtained from murine bone marrow by MACS which does not impair the activity of the purified cells.