CAJ | 학술논문

目的利用生物反应器大规模扩增人脐血造血干/祖细胞,并通过动物移植实验检验该方法的有效性.方法采集抗凝脐血10份,分离出单个核细胞(MNC),分别进行生物反应器扩增培养和静态扩增培养.检测扩增前后细胞表面CD34、CD38、CD133、CD184和CD62L分子的表达,并进行造血干/祖细胞集落的培养.取非肥胖糖尿病重症联合免疫缺陷小鼠,以X射线照射后,分为4组,其中MNC组小鼠注射未经扩增培养的MNC;静态扩增组小鼠注射经过静态扩增培养的细胞;反应器扩增组小鼠注射经过生物反应器扩增培养的细胞;空白对照组小鼠注射生理盐水.移植后6周处死存活小鼠,收集骨髓细胞,检测其中CD45+、CD3+、CD19+和CD33+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果生物反应器扩增前MNC为(1.2～2.8)×108个,扩增后为(3.7～12.6)×108个,扩增后的细胞数明显高于静态扩增培养者(P<0.01).经生物反应器扩增后所形成的红系集落形成单位、粒-巨噬细胞集落形成单位数明显高于经静态扩增者(P<0.05).移植6周后,空白对照组小鼠均死亡,MNC组存活率为35%,静态扩增组存活率为30%,反应器扩增组存活率为62.9%,后者明显高于前二者(P<0.05).各组存活小鼠骨髓细胞中均检测到Alu基因和Cart-Ⅰ基因的表达以及人源CD33+、CD45+、CD3+及CD19+细胞.结论利用生物反应器可大规模扩增人脐血造血干/祖细胞,所得细胞能植入小鼠体内,并能获得造血功能重建.
목적 이용생물반응기대규모확증인제혈조혈간/조세포,병통과동물이식실험검험해방법적유효성.방법 채집항응제혈10빈,분리출단개핵세포(MNC),분별진행생물반응기확증배양화정태확증배양.검측확증전후세포표면CD34、CD38、CD133、CD184화CD62L분자적표체,병진행조혈간/조세포집락적배양.취비비반당뇨병중증연합면역결함소서,이X사선조사후,분위4조,기중MNC조소서주사미경확증배양적MNC;정태확증조소서주사경과정태확증배양적세포;반응기확증조소서주사경과생물반응기확증배양적세포;공백대조조소서주사생리염수.이식후6주처사존활소서,수집골수세포,검측기중CD45+、CD3+、CD19+화CD33+세포적함량이급인특이적Cart-Ⅰ화Alu기인적표체.결과 생물반응기확증전MNC위(1.2～2.8)×108개,확증후위(3.7～12.6)×108개,확증후적세포수명현고우정태확증배양자(P<0.01).경생물반응기확증후소형성적홍계집락형성단위、립-거서세포집락형성단위수명현고우경정태확증자(P<0.05).이식6주후,공백대조조소서균사망,MNC조존활솔위35%,정태확증조존활솔위30%,반응기확증조존활솔위62.9%,후자명현고우전이자(P<0.05).각조존활소서골수세포중균검측도Alu기인화Cart-Ⅰ기인적표체이급인원CD33+、CD45+、CD3+급CD19+세포.결론 이용생물반응기가대규모확증인제혈조혈간/조세포,소득세포능식입소서체내,병능획득조혈공능중건.
Objective To expand hematopoietic stem/progenitor cells of cord blood in large scale by bioreactor. Methods Mononuclear cells from human umbilical cord blood were cultured in serum-free medium with stem cell factor (SCF), flt-3 ligand (FL3) and thrombopoietin (TPO). The expansion fold of cells, colony-forming and expression of surface molecules were analyzed by cell counting, colony-forming assay and flow cytometry, respectively. And the engraftment of these expanded cells was studied through cell transplantation into irradiated non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice. Results After culture for 7 days,the folds of total cell expansion in bioreactor were higher than those in static culture, P<0.01. The number of colony-forming unit-granulocyte/macrophage (CFU-GM) and erythroid colony forming unit (CFU-E) in bioreactor were increased as compared with those in static culture, P<0.05. The positive rate of surface marks of CD34+, CD34+ CD38- and CD133+ on expanded cells bioreactor didn't show decrease compared to unexpanded cells but was lower than that in static culture, P<0.05. However, the expression levels of CD184 or CD62L on cells expanded in bioreactor were higher than those in static culture, P<0.05, while without significant difference from unexpanded cells (P>0.05). The cells expanded in bioreactor were successfully engrafted into irradiated NOD/SCID mice and reconstructed the multi-lineage hematopoiesis. Conclusion The bioreactor favors large-scale expansion of hematopoietic progenitor cells and keeps the hematopoietic repopulation potential.