CAJ | 학술논문

目的探讨磁搅拌大规模培养体系对人脐血造血祖细胞的扩增效果以及扩增的人造血祖细胞植入动物体内后的造血重建情况.方法从新鲜抗凝脐血中分离出单个核细胞(MNC),以添加干细胞因子、酪氨酸激酶受体3配基及血小板生成素的无血清培养体系进行培养.静态扩增组的细胞置于T25培养瓶中培养,磁搅拌悬浮扩增组(磁搅拌扩增组)的细胞采用Celstir装置进行培养,培养体系为50～100 ml.培养7 d后进行细胞计数、集落培养检测和细胞表面分子表达的测定.以不进行培养者为对照组.非肥胖糖尿病重症联合免疫缺陷(NOD/SCID)小鼠在接受2.5 Gy的亚致死剂量X射线照射后分别从尾静脉输入上述静态扩增组、磁搅拌扩增组和对照组的MNC(5×106个),另设不移植的空白对照组.观察小鼠的存活情况,6周后处死存活小鼠,检测骨髓细胞中CD34+细胞、CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果经过7天的培养,磁搅拌扩增组的造血祖细胞扩增倍数为(2.8±0.45)倍,明显高于静态扩增组的(2.1±0.48)倍(P<0.01).磁搅拌扩增组形成的红系集落、粒-巨噬细胞集落数均明显高于静态扩增组(P<0.05).静态扩增组扩增后的CD34+细胞、CD34+CD38-细胞和CD133+细胞含量均高于磁搅拌扩增组(P<0.05),而CD184+细胞和CD62L+细胞含量低于磁搅拌扩增组(P<0.01).移植后6周,对照组、静态扩增组和磁搅拌扩增组分别有3、4、5只小鼠存活,三组间两两比较,6周存活率的差异无统计学意义(P>0.05).存活6周的小鼠,其骨髓中能检人特异性CD34+细胞,以及CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞,也检测到人Alu基因和Cart-Ⅰ基因的表达.结论磁搅拌培养能大规模扩增脐带血造血祖细胞,扩增的细胞能植入x射线照射的NOD/SCID小鼠,并重建其多系造血.
목적 탐토자교반대규모배양체계대인제혈조혈조세포적확증효과이급확증적인조혈조세포식입동물체내후적조혈중건정황.방법 종신선항응제혈중분리출단개핵세포(MNC),이첨가간세포인자、락안산격매수체3배기급혈소판생성소적무혈청배양체계진행배양.정태확증조적세포치우T25배양병중배양,자교반현부확증조(자교반확증조)적세포채용Celstir장치진행배양,배양체계위50～100 ml.배양7 d후진행세포계수、집락배양검측화세포표면분자표체적측정.이불진행배양자위대조조.비비반당뇨병중증연합면역결함(NOD/SCID)소서재접수2.5 Gy적아치사제량X사선조사후분별종미정맥수입상술정태확증조、자교반확증조화대조조적MNC(5×106개),령설불이식적공백대조조.관찰소서적존활정황,6주후처사존활소서,검측골수세포중CD34+세포、CD3+세포、CD19+세포、CD33+세포급CD45+세포적함량이급인특이적Cart-Ⅰ화Alu기인적표체.결과 경과7천적배양,자교반확증조적조혈조세포확증배수위(2.8±0.45)배,명현고우정태확증조적(2.1±0.48)배(P<0.01).자교반확증조형성적홍계집락、립-거서세포집락수균명현고우정태확증조(P<0.05).정태확증조확증후적CD34+세포、CD34+CD38-세포화CD133+세포함량균고우자교반확증조(P<0.05),이CD184+세포화CD62L+세포함량저우자교반확증조(P<0.01).이식후6주,대조조、정태확증조화자교반확증조분별유3、4、5지소서존활,삼조간량량비교,6주존활솔적차이무통계학의의(P>0.05).존활6주적소서,기골수중능검인특이성CD34+세포,이급CD3+세포、CD19+세포、CD33+세포급CD45+세포,야검측도인Alu기인화Cart-Ⅰ기인적표체.결론 자교반배양능대규모확증제대혈조혈조세포,확증적세포능식입x사선조사적NOD/SCID소서,병중건기다계조혈.
Objective To expand hematopoietic stem/progenitor cells at large scale in magnet stirred culture system. Methods Mononuclear cells from human umbilical cord blood were cultured in serum-free medium supplemented with stem cell factor (SCF), fh-3 ligand (FL3) and thrombopoietin (TPO). The expansion fold of cells, colony-forming and expression of surface molecules were studied in magnet stirred culture by cell counting, colony-forming assay and flow cytometry. And the engraftment to non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice and repopulation of expanded cells by magnet stirred culture were studied through transplantation. Results After culture for 7 days, the folds of total cell expansion in magnet stirred culture were higher than in static culture(P<0.01). The number of colony-forming unit-granulocyte/macrophage (CFU-GM) and erythroid colony forming unit (CFU-E) in magnet stirred culture was greater than that in static culture(both P<0.05). The primitive cells (CD34+, CD34+ CD38 or CD133+) of the expanded cells in magnet stirred culture were less than those in static culture, P<0.05. However, the CD184+ or CD62L+ expanded cells were more than those in static culture, P<0.05. There was no significant difference in survival rate between three cell transplantation groups, all P>0.05. The analysis of multilineage hematopoiesis showed that transplanted human hematopoietic cells were represented in murine bone marrow cells by detecting the percentage of human cells identified with human specific anti-CD3/19/33/34/45 MoAb by FACS and the human specific gene Alu and Cat-1 by PCR. Conclusion Magnet stirred culture favors large-scale expansion of hematopoietic progenitor cells with the hematopoietic repopulation potential in NOD/SCID mice.