C57BL/6j小鼠骨髓CD34^+造血干细胞来源的iDC的体外培养与鉴定  

THE CULTURE IN VITRO AND CHARACTERIZATION OF BONE MARROW CD34^+ HEMATOPOIETIC STEM CELL DERIVATED IMMATURE DENDRITIC CELL OF C57BL/6j MICE

作　　者：周忠信[1] 吕明德[1] 殷晓煜[2] 向邦德[2] 黄洁夫[2] 

机构地区：[1]南方医科大学南方医院肝胆外科,广州510150 [2]中山大学附属第一医院肝胆外科

出　　处：《广西医科大学学报》2007年第3期341-344,共4页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(No30471686)

摘　　要：目的:探讨以恰当的体外定向诱导和培养小鼠骨髓CD34+造血干细胞以获得足够数量的未成熟树突状细胞(immatureden driticcells,iDC)的方法。方法:采用自健康C57BL/6j小鼠骨髓中分离CD34+造血干细胞,进而于含rmGM-CSF和rmIL-4的RPMI-1640完全培养液中定向诱导和扩增,收集悬浮生长细胞后,予以流式细胞学检测其CD80、CD86和CD11c等表面分子的表达,扫描电镜观察悬浮细胞的形态。结果:去除红细胞的骨髓细胞培养4h后,10%左右细胞贴壁生长。加rmGM-CSF和rmIL-4培养,逐渐呈集落样悬浮生长,有树枝状突起。第6天时,细胞集落呈密集的葡萄串状,细胞体积增大,毛刺状或根须状突起更明显。流式细胞学显示:体外培养第7天的iDC的表面分子CD11c的表达率为75%~81%[(78.4±8.36)%],表面分子CD80的表达率为32%~51%[(38.26±8.77)%],而表面分子CD86的表达率为18%~53%[(27.5±8.30)%]。扫描电镜见细胞有典型的树枝状突起。结论:联合使用rmGM-CSF和rmIL-4定向诱导培养骨髓CD34+造血干细胞,可大量扩增出iDC。Objective：To investigate the proper procedure to induce and culture bone marrow CD34^＋ hematopoietic stem cell in vitro so as to harvest sufficient immature dendritic cell（iDC）. Methods： Bone marrow CD34^＋ hematopoietic stem cell was separated from the tibia and femur of healthy C57BL/6j, which was induced and cultured in complete culture solution consisting of rmGM-CSF and rmIL-4. The cultural cell was assembled 7 days after segregated, and which was detected in its express of CD80, CD86 and CD11c on plasmalemma and the morphology characteristic of stereoscan photograph. Results：There were 10% of the cells which grew adhering to the bottom 4 hours after the elimination of the erythrocyte, and the cells appeared to be colony and became large with dendritic processes 2 days after cultured in medium consisting of rmGM-CSFand rmlL-4. The cells became irregular, more obvious of dendritic processes and concerted thyrsiform on the 6th day. There were 2 107 cells in every 6-slot culture plate. The flow cytometry examination after 7 days＇ culture showed that the expressing rate of CDllc was 75%-81%[（78.4±8.36）%], the expressing rate of CD80 was 32%-51%[（38.26±8.77）%], and he expressing rate of CD86 was 18%-53%, which means that the purity of DC cultivated in vitro was more than 75%[（27.5±8.30）%]. The typical dendritic processes was seen under scanning electron microscope. Conclusion： The iDC could be induced and amplified from marrow CD34^＋ hematopoietic stem cell by symphysically using rmGM-CSF and rmIL-4.

关 键 词：小鼠 CD34^+造血干细胞 未成熟树突状细胞 

分 类 号：R735.7[医药卫生—肿瘤]