两步法从脐血CD_(34)^+细胞获得大量树突细胞的初步研究  

Preliminary study on extensive amplification of human dendritic cells differentiated from cord blood CD_(34)^+ progenitor cells by two-step culture

作　　者：王亚非[1] 李茜[1] 孟恒星[1] 于珍[1] 刘津华[1] 崔雯[1] 周余[1] 麦玉洁[1] 尤胜国[1] 邱录贵[1] 

机构地区：[1]中国医学科学院,中国协和医科大学血液学研究所,血液病医院,实验血液学国家重点实验室,天津300020

出　　处：《中华血液学杂志》2004年第2期70-73,共4页Chinese Journal of Hematology

基　　金：攀登计划基金资助项目(95专10);天津市科技发展计划资助项目(003119811);卫生部专项基金资助项目(WKZ2000134);天津市自然科学基金资助项目(13609411)

摘　　要：目的　探索利用先扩增后诱导的“两步法”从脐血 (CB)CD34+ 细胞高效大量地获得树突细胞 (DC)。方法　免疫磁珠法从CB分选获得CD34+ 细胞 ,以干细胞因子 (SCF)、IL 3、Flt 3配体 (FL)、Tpo组合刺激 ,扩增 7d、10d和 14d(依次为Ⅰ、Ⅱ和Ⅲ组 )后以GM CSF +IL 4 +TNF α诱导 8d或 5d获得DC ,通过相差显微镜、电镜观察形态 ,流式细胞仪检测表型 ,混合淋巴细胞培养、ELISA法检测培养液上清IL 12含量评价其功能。结果　CBCD34+ 细胞经SCF +IL 3+FL +Tpo刺激扩增 7d、10d和 14d后细胞总数分别扩增了 (5 3.39± 2 0 .5 9)倍、(30 7.17± 119.5 9)倍和 (1117.2 5± 335 .4 9)倍。经GM CSF +IL 4 +TNF α诱导 8d后所得CD1a+ 细胞是扩增前细胞数的 (2 1.4 0± 16 .70 )倍、(14 3.2 0± 6 0 .35 )倍和(15 0 .80± 4 2 .16 )倍 ,Ⅱ、Ⅲ组明显多于Ⅰ组 (P <0 .0 5 ) ,但Ⅱ、Ⅲ组间无显著性差异 (P >0 .0 5 )。所得DC的形态、表型及刺激异基因T细胞增殖能力、IL 12分泌量 ,三组无显著性差异 (P >0 .0 5 )。当诱导时间缩短至 5d时 ,各组DC功能均显著下降 (P <0 .0 5 )。结论　CBCD34+ 细胞扩增 7～ 10d再诱导 8d可以高效大量获得具有正常功能的DC ,而扩增时间超过 10d并不能显著增加DC产量 。Objective To Explore a two-step culture system to generate a large number of dendritic cells(DC) differentiated from cord blood(CB) CD 34+ cells. Methods Enriched CB CD 34+ cells with immunoadsorption were primarily cultured in the presence of stem cell factor (SCF),Flt-3 ligand(FL),thrombopoietin(Tpo) and interleukin-3(IL-3) for 7(group Ⅰ),10(group Ⅱ) or 14 days(group Ⅲ) respectively, and then further cultured with GM-CSF,IL-4 and TNF-α for 5～8 days to induce DC.The expansion and cell function were evaluated by flow cytometry(FCM) and mix-lymphocyte reaction(MLR),and detection of IL-12 in the supernatent by using ELISA. Results The total nucleated cells with 53.39±20.59-, 307.17±119.59- and 1117.25±335.49- folds expansion could be respectively obtained after 7～14 days of expansion culture. After DC induction,CD 1a+ cells were 21.40±16.70-,143.2±60.35- and 150.8±42.16- fold increase as compared to the initial nucleated cells. Comparing with that in group Ⅰ, the CD 1a+ cells were much more in groups Ⅱ and Ⅲ; but there was no difference between the latter two groups( P>0.05). The cultured cells in the three groups showed almost the same allo-stimulatory capability and IL-12 excretion when the second culture duration maintained 8 days, while the capability and excretion were greatly decreased when the duration shortened to 5 days( P<0.05). Conclusion A planty of functionally mature DC could be obtained from the CD 34+ cells in the two-step culture system of 7～10 days HSC expansion followed by 8 days DC induction.

关 键 词：两步法 脐血 CD34^+细胞 树突细胞 免疫磁珠法 流式细胞仪 体外扩增 

分 类 号：R392[医药卫生—免疫学]