Objective: To compare the efficacy of directional erythroid differentiation in different serum free culture systems and to screen the optimal culture systems for inducing the differentiation of umbilical cord blood hematopoietic stem and progenior cells (HSPC) to erythroid cells.
Methods: The CD34+ cells from umbilical blood munonuclear cells were sorted by using the magnetic beads, and were inoculated into 3 different of culture systems (system 1, 2 and 3 respectively), to induce erythrold differentiation by 3 stage culture. The living cells were counted in different differentiation stages and were observed by Wright-Giemsa staining; the expression of CD71 and CD235a on cell surface was detected by flow cytometry, the erythroid differentiation pteency was detected via colony-forming test.
Results: The ability of system 2 to promote the HSPC proliferation was the strongest, the efficacy of system 3 to promote the erythroid differentiation of HSPC was the most optimal; the proliferation ability of cells cultured in system 2 for 2-15 days all was higher than that of cells cutured in system 1 and 3 (P＜0.05). The flow cytometry detection showed that the expression of CD71 and CD235a on surface of cells cultured in system 3 was the highest, the CD235a percentage on day 15 of differentiation in system 3 was (92.33±3.89)%, that in system 2 was (84.67±3.12)%, while that in system 1 was (72.17±6.83)% (P＜0.05). Cell morplologic detection showed that throid differentiation was accelerated on day 12, the percentage of orthochromatic erythrocytes in system 3 was (67.67±2.08)% which was 10.69 and 25.34 times higher than that in system 2 and 1 respectively (P＜0.05). The colony-forming test showed the ratio of BFU-E in system 3 increased gradually on day 3-9 (r=0.99, P＜0.05), which was significanlly higher than that in system 2 and 1 on day 9 (90.35±5.52% vs 77.06±2.26% and 74.50±3.95%).
Conclusion: Culture system 3 is the most effective serum-free erythroid differentiation system, and the culture system 2 is the most powerful HSPC proliferation system. This study results provide a technical basis for further efficiently increasing and inducing the erythroid proliferation and differentiation of HSPC, and also provide culture system in vitro for the clinical application and basic research.
题目: 无血清培养体系对脐带血CD34+细胞定向红系分化影响的比较.
目的: 通过3种无血清红系定向诱导分化培养体系比较，优化培养体系诱导脐带血干/祖细胞（HSPC）体外红系定向分化，以满足基础研究与临床应用.
方法: 应用磁珠分选脐带血单个核细胞中的CD34+细胞；分别接种到3种培养体系（1、2、3）中并采用3阶段培养法诱导CD34+细胞红系定向分化，在分化不同阶段进行细胞计数，瑞氏吉姆萨染色，应用流式细胞术检测细胞表面CD71、CD235a的表达，集落形成能力检测鉴定红系分化的进程.
结果: 体系2促HSPC增殖能力最强，体系3促红系分化效果最佳。体系2培养的细胞增殖能力均明显高于体系1、2（P＜0.05）；FACS分析显示，红系表面分子CD71、CD235a在体系3培养的细胞表面表达最高，分化d 15 CD235a+百分率高达（92.23±3.89）%，体系2为（84.67±3.12）%，体系1为（72.17±6.83）%（P＜0.05）；细胞形态学染色显示，体系3培养的细胞在分化d 12的晚幼红细胞比例为（67.67±2.08）%，是体系2的10.69倍、体系1的25.34倍（P＜0.05）；造血集落形成实验发现在体系3中d 3-9 形成的BFU-E比例逐渐升高（r= 0.99, P＜0.05），体系3中BFU-E形成比率明显高于体系1、2（P＜0.05）.
结论: 通过比较3种培养体系，筛选出体系3是促进CD34+细胞体外红系分化最有效的体系，体系2是促增殖最有效的体系。本研究为进一步提高HSPC体外红系增殖与诱导效率奠定了技术基础，也为研究红系分化调控机制提供了体外培养体系.