人脐带间充质干细胞条件培养基体外支持造血的功能  

Supportive Effects of Conditioned Culture Media of Human Umbilical Cord Mesenchymal Stem Cells on Hematopoiesis In Vitro

作　　者：李丽娜[1] 韩之波[2,3] 王有为[3] 罗伟峰[1] 及月茹[3] 杨舟鑫[3] 冯莉[2] 戚仁斌[1] 李扬秋[1] 韩忠朝[1,2,3] 

机构地区：[1]暨南大学医学院,广东广州510632 [2]中国医学科学院血液学研究所泰达生命科学技术研究中心,天津300457 [3]中国医学科学院北京协和医学院血液学研究所实验血液学国家重点实验室,天津300020

出　　处：《中国实验血液学杂志》2012年第4期975-980,共6页Journal of Experimental Hematology

基　　金：国家重大科学计划"973计划"(编号2011CB964800)

摘　　要：本研究旨在探讨单纯人脐带间充质干细胞(hUC-MSC)的条件培养基对脐血CD34+细胞体外造血支持作用。分离得到的hUC-MSC以2×106接种于75 cm2培养瓶中,48 h后收集培养上清作为条件培养液。用人脐血CD34阳性分选试剂盒分选CD34+细胞。将CD34+细胞接种在3个培养体系中:hUC-MSC条件培养液+不完全甲基纤维素培养基、完全甲基纤维素阳性培养基和含10%胎牛血清的DMEM/F12+不完全甲基纤维素作为阴性培养基。培养14d观察集落的形态特征,统计细胞数≥50的造血集落单位数。流式细胞术检测组成集落形成单位的细胞免疫表型。结果表明:hUC-MSC条件培养基中能够形成集落形成单位,且以粒系和粒-巨噬集落形成单位为主(CFU-G 47.67±0.58、CFU-GM 48.67±4.73、CFU-M 3.00±2.00),未观察到红细胞系相关的集落形成单位。阳性培养基中各种集落形成单位均可见,阴性培养体系中无集落形成单位。流式细胞术检测条件培养基组CD45+细胞比例为(97.43±2.15)%,显著高于阳性对照中CD45+细胞比例(39.69±0.96)%(P<0.05)。结论:在体外hUC-MSC条件培养基能够促进CD34+细胞的发育分化,单纯hUC-MSC条件培养基具有造血支持功能,并促进CD34+细胞向髓系分化,但不能促进向红细胞分化。This study was aimed to explore whether the conditioned culture medium of human umbilical cord-derived mesenchymal stem cells（hUC-MSC） has supportive effects on hematopoiesis in vitro, hUC-MSC were cultured in 75 cm^2 culture flasks at a concentration of 2×10^6 cells per flask. After 48 h, the conditioned culture medium was harvested. CD34^± cells were isolated with the human cord blood CD34 positive selection kit. The CD34^± cells were plated in three different culture systems： the culture supernatant from hUC-MSC added into incomplete methylcellulose without recombinant human cytokines as conditioned culture medium; the complete methylcellulose medium with recombinant human cytokines as positive control medium; incomplete methycellulose adding DMEM/F12 with 10% FBS instead of conditioned culture medium as the negative control medium. After 14 days of culture, colonies containing ≥ 50 cells were scored and types of colonies were classified under inverted microscope. The immunophenotypes of cells which were collected from the colonies were detected by flow cytometry. The results showed that conditioned culture medium of hUC-MSC supported the differentiation of CD34^± cells into CFU-G （ 47.67 ± 0.58 ）, CFU-GM （48.67 ± 4. 73）and CFU-M （3.00 ±2.00） in vitro, while the CFU-E,BFU-E or CFU-GEMM were absent. Comparatively, in the positive control medium all kinds of CFU were observed. Interestingly, the percentage of CD45^＋ cells of CFU in conditioned culture medium （ 97.43 ± 2. 15 ） % was more than CD45^＋ cells in positive control medium （ 39. 69 ± 0.96） % （ P 〈 0.05 ）. It is concluded that the conditioned culture medium of hUC-MSC has been confirmed to have ability to support hematopoiesis separately in vitro. Besides, it enhances the differentiation of CD34^＋ cells into myeloid cells except ceils of erythroid lineage.

关 键 词：脐带间充质干细胞 条件培养基 CD34+细胞 体外造血支持 集落形成单位 

分 类 号：R331.2[医药卫生—人体生理学]