不同共培养模式下间充质干细胞对造血干细胞增殖的作用  

Coculture with mesenchymal stem cells facilitates the proliferation of hematopoietic stem cells under different coculture modes

作　　者：汪姝玥 林凡莉 钱怡 陈小青 刘洋 李书坛 程艳 熊皓 黄纯兰 Wang Shu-yue;Lin Fan-li;Qian Yi;Chen Xiao-qing;Liu Yang;Li Shu-tan;Cheng Yan;Xiong Hao;Huang Chun-lan(Department of Hematology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China;Department of Hematology, Third People’s Hospital of Chengdu, Chengdu 610031, Sichuan Province, China)

机构地区：[1]西南医科大学附属医院血液内科,四川省泸州市646000 [2]成都市第三人民医院血液内科,四川省成都市610031

出　　处：《中国组织工程研究》2018年第13期2068-2074,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81450030);四川省教育厅重大培育项目(14CZ0017)~~

摘　　要：背景:虽然进行了大量相关研究,但目前仍缺乏切实可行的体外扩增造血干细胞的方法。间充质干细胞能分泌多种促进造血干细胞增殖并抑制其分化的细胞因子,在维持造血微环境及调控造血干细胞功能中发挥着重要的作用。目的:探讨不同共培养模式下骨髓间充质干细胞在体外对造血干细胞增殖的影响。方法:采用全骨髓贴壁法体外培养C57BL/6小鼠间充质干细胞至P3代,采用miniMACS磁珠分选仪分选GFP小鼠(C57系)骨髓CD117^+细胞(造血干细胞),采用不同共培养模式将2种细胞进行共培养:对照组为造血干细胞单独培养组;实验组为Transwell共培养组(上室接种造血干细胞、下室接种间充质干细胞);2D接触共培养组(24孔板共同接种造血干细胞与间充质干细胞)。分别于共培养1,3,5,7 d于倒置相差显微镜、荧光显微镜下观察造血干细胞的形态,并检测造血干细胞的活性细胞数。结果与结论:共培养1-7 d,各组造血干细胞数量均随着培养时间的增加而增加(P<0.05)。各组细胞自3 d开始进入对数生长期,5 d时部分造血干细胞开始出现形态变化。比较第7天造血干细胞活性细胞数,可知与间充质干细胞共培养的实验组及2D接触共培养组均明显高于对照组(P<0.05):而2D接触共培养组造血干细胞数明显高于非接触培养的实验组(P<0.05)。结果提示:间充质干细胞在体外能够有效促进造血干细胞增殖,接触共培养条件下其促进作用更明显。BACKGROUND： Although a large number of related studies have been carried out, there is still a lack of practical methods to amplify hematopoietic stem cells（HSCs）in vitro.Mesenchymal stem cells（MSCs）secrete a variety of cytokines that promote the HSCs proliferation and inhibit their differentiation. These cytokines play an important role in maintaining the hematopoietic microenvironment and regulating HSCs function. OBJECTIVE：To investigate the effect of bone marrow MSCs on the proliferation of HSCs in vitro under different coculture modes. METHODS：Mesenchymal stem cells from the bone marrow of C57BL/6 mice were cultured in vitro using the whole bone marrow adherent culture. CD117＋cells （HSCs） were sorted from passage 3 cells by using miniMACS magnetic beads sorting. Then, CD117＋cells were co-cultured with MSCs under different coculture models, including single culture of HSCs （control group）, Transwell coculture （upper chamber, HSCs; lower chamber, MSCs） and two-dimensional contact coculture （coculturing HSCs and MSCs in 24-well plates）. The morphology of HSCs was observed under phase contrast microscope and fluorescence microscope, and the number of active cells of HSCs was counted at 1, 3, 5, and 7 days after coculture. RESULTS AND CONCLUSION： During the coculture of 1-7 days, the number of HSCs in the two groups was increased with culture time （P 〈0.05）. After 3 days of coculture, HSCs in each group was grown into the logarithmic growth phase, and morphological changes in some HSCs were detected at 5 days of coculture. At 7 days of coculture, the viabilities of HSCs in different culture models were ranked as follows： single culture model 〈 Transwell coculture model 〈 two-dimensional contact coculture model （P 〈 0.05）. These findings suggest that MSCs can effectively promote the proliferation of HSCs in vitro,and the promotion effect is increased under contact coculture conditions.

关 键 词：造血干细胞 间充质干细胞 共培养 接触共培养组 3D共培养 Transwell共培养 饲养层 

分 类 号：R394.2[医药卫生—医学遗传学]