CAJ | 학술논문

目的探讨适用于临床的诱导脐血细胞向巨核细胞分化并扩增的方法.方法将脐血用羟乙基淀粉(HES)沉降红细胞,以Ficoll液密度梯度分离单个核细胞(MNC),并探索不同细胞因子组合以及不同培养基体外诱导其向巨核细胞分化及扩增的效果.结果 HES浓度为15 g/L沉降红细胞效果最好,可获得更多的有核细胞.分别采用116t(IL-11、IL-6、TPO)、st36(SCF、TPO、IL-3、IL-6)、pt36[血小板衍生生长因子(PDGF)、TPO、IL-3、IL-6]、pst36四种不同细胞因子组合的国产无血清培养基体外诱导培养脐血MNC7d,其中st36组(50 ng/ml SCF、50 ng/ml TPO、20 ng/ml IL-3、50 ng/mlIL-6)获得的CD41 +CD61+细胞数最高,为(6.79±1.97)×104.含st36的进口无血清培养基体外培养7d,细胞存活率为(82.85±0.64)％,优于含相同因子组合的国产无血清培养基[细胞存活率为(60.90±6.93)％],且获得CD41 +CD61+细胞数为(18.60-1.97)×104,高于国产无血清培养基.故以含st36的进口无血清培养基作为培养体系进行后续实验.倒置显微镜观察显示,脐血MNC随培养时间的延长,细胞数增多,细胞体积变大,培养7d细胞增殖明显.体外诱导培养10d的脐血MNC经Wright-Giemsa染色可见原巨核细胞、幼巨核细胞、颗粒型巨核细胞等不同阶段的巨核细胞;诱导培养14 d CD41+CD6+细胞率达到(54.27±6.31)％.巨核细胞集落形成单位(CFU-MK)实验表明,随着诱导时间延长,CFU-MK数呈增加趋势.体外诱导14 d CFU-MK数为1 236.0±32.9,高于未诱导的脐血MNC,差异有统计学意义(P＜0.01).收集体外诱导培养10d的细胞上清,加入凝血酶,可见聚集的血小板.结论采用HES浓度15 g/L沉降红细胞,含st36四种因子组合的进口无血清培养基诱导巨核祖细胞可获得最佳的细胞数和诱导效率,且诱导的细胞能够产生有功能的血小板.
목적 탐토괄용우림상적유도제혈세포향거핵세포분화병확증적방법.방법 장제혈용간을기정분(HES)침강홍세포,이Ficoll액밀도제도분리단개핵세포(MNC),병탐색불동세포인자조합이급불동배양기체외유도기향거핵세포분화급확증적효과.결과 HES농도위15 g/L침강홍세포효과최호,가획득경다적유핵세포.분별채용116t(IL-11、IL-6、TPO)、st36(SCF、TPO、IL-3、IL-6)、pt36[혈소판연생생장인자(PDGF)、TPO、IL-3、IL-6]、pst36사충불동세포인자조합적국산무혈청배양기체외유도배양제혈MNC7d,기중st36조(50 ng/ml SCF、50 ng/ml TPO、20 ng/ml IL-3、50 ng/mlIL-6)획득적CD41 +CD61+세포수최고,위(6.79±1.97)×104.함st36적진구무혈청배양기체외배양7d,세포존활솔위(82.85±0.64)％,우우함상동인자조합적국산무혈청배양기[세포존활솔위(60.90±6.93)％],차획득CD41 +CD61+세포수위(18.60-1.97)×104,고우국산무혈청배양기.고이함st36적진구무혈청배양기작위배양체계진행후속실험.도치현미경관찰현시,제혈MNC수배양시간적연장,세포수증다,세포체적변대,배양7d세포증식명현.체외유도배양10d적제혈MNC경Wright-Giemsa염색가견원거핵세포、유거핵세포、과립형거핵세포등불동계단적거핵세포;유도배양14 d CD41+CD6+세포솔체도(54.27±6.31)％.거핵세포집락형성단위(CFU-MK)실험표명,수착유도시간연장,CFU-MK수정증가추세.체외유도14 d CFU-MK수위1 236.0±32.9,고우미유도적제혈MNC,차이유통계학의의(P＜0.01).수집체외유도배양10d적세포상청,가입응혈매,가견취집적혈소판.결론 채용HES농도15 g/L침강홍세포,함st36사충인자조합적진구무혈청배양기유도거핵조세포가획득최가적세포수화유도효솔,차유도적세포능구산생유공능적혈소판.
Objective To build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells.Methods Red blood cells were precipitated by hydroxyethyl starch (HES).Mononuclear cells were obtained by density gradient centrifugation with Ficoll.The inducing efficiencies of megakaryocytes by using of different cytokine cocktails and culture media were analyzed.Results The best choicefor erythrocyte sedimentation and high efficency of nucleated cells retrieving were obtained by using of 1.5％ HES.The isolated cord blood mononuclear cells were cultured with domestic serum-free medium supplemented with 116t (IL-11,IL-6,TPO),st36 (SCF,TPO,IL-3,IL-6),pt36 (PDGF、TPO、IL-3、IL-6)or pst36 for 7 days.St36 group (50 ng/ml SCF,50 ng/ml TPO,20 ng/ml IL-3 and 50 ng/ml IL-6) yielded the most CD41/CD61 positive [(6.79± 1.97) × 104].The cell viability [(82.85±0.64)％] of st36 group by using of imported serum-free medium was better than [(60.90±6.93) ％] that in domestic medium on day 7 after induction,and CD41/CD61 positive cells count [(18.60±1.97) × 104] were more than domestic serum-free medium group.Therefore,we chose imported serum-free medium containing st36 to induce cord blood mononuclear cells.After a prolonged culture,the total cell numbers increased accompanied with an elevated percentage of CD41/CD61 positive cells,which reached (54.27 ± 6.31)％ on day 14.Wright-Giemsa staining showed that different phase cells,such as megakaryoblast,promegakaryocyte and granular megakaryocyte,occurred after 10 days' culture.Clone forming unit-megakarocytes (CFU-MK) assay showed that the colonies count increased with the prolonged incubation.CFU-MK colonies were [1 236.0±32.9] on day 14,which was higher than that in medium without induction (P＜0.01).Platelets from megakaryocytes showed agglutination function after 10 days' culture.Conclusion 1.5％ HES was the best solution to precipitate erythrocytes.The combination of an imported serum-free medium with IL-3,IL-6,SCF and TPO showed better induction efficiency than domestic medium or other cytokine cocktails.Meanwhile,induced megakaryocytes produced functional platelets.