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作 者:毛平[1] 段华新[1] 王彩霞[1] 李迎霄[1] 邓婷芬[1] 许艳丽[1] 罗畅如[1]
机构地区:[1]广州医学院附属市一人民医院血液科,510180
出 处:《中华器官移植杂志》2009年第3期152-155,共4页Chinese Journal of Organ Transplantation
摘 要: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)×10^8个,扩增后为(3.7~12.6)×10^8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、粒一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。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 colonyforming unit-granuloeyte/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 eulture,P〈0. 05, while without significant difference from unexpanded cells (P〉0. 05). The cells expanded in bioreaetor 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.
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