在不同培养条件下微血管内皮细胞对骨髓造血干细胞增殖影响的比较
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摘要
目的:探讨在体外不同培养条件下微血管内皮细胞(microvascular endothelial cells,MEC)对骨髓造血干细胞(hematopoietic stem cells,HSC)增殖的作用。方法:将来源于C57BL/6小鼠肺组织的MEC与来源于GFP小鼠骨髓的HSC用于非接触共培养、2 D接触共培养,并设置对照组HSC单独培养及MEC单独培养。采用细胞计数和CCK-8实验比较各组悬浮细胞在1、3、5和7 d的增殖情况。结果:MEC呈贴壁生长。HSC共培养组及单独培养组细胞在体外培养过程中悬浮细胞计数均有所增加; 2 D接触培养组与非接触共培养组中悬浮细胞比对照组中悬浮细胞更早进入对数生长期; 2 D接触培养组悬浮细胞增殖多于非接触共培养组及HSC单独培养组(P <0. 05);非接触共培养组悬浮细胞增殖多于HSC单独培养组(P <0. 05)。结论:体外培养HSC能使HSC增殖;M EC能促进HSC增殖; M EC还可以通过与HSC非接触共培养促进HSC增殖,这一效应与M EC分泌的细胞因子促HSC增殖作用有关; MEC与HSC直接接触培养对HSC的增殖作用比非接触共培养更强,这一效应与细胞-细胞接触对HSC增殖的调控有关。
Objective: To investigate the effect of microvascular endothelial cells( M EC) on the proliferation of hematopoictic stem cells( HSC) under different culture conditions in vitro. Methods: The M EC from lung tissue of C57 BL/6 mice and the HSC from bone marrow of GFP mice w ere used for non-contact co-culture,2 D contact coculture,at same time the single M EC and single HSC culture w ere seted up and w ere used as control group. The cell counting and CCK-8 method w ere used to detect and compare the proliferation levels of suspension cells in different groups on day 1,3,5 and 7. Results: M EC presented adherent grow th. In process of cell culture in vitro,the number of suspension cells in M EC and HSC co-culture group and single HSC culture group increased,the suspension cells in 2 D contact and non-contact co-culture groups more early gated into logarithmic grow th phase as compared w ith suspension cells in control group,the proliferation level of suspention cells in 2 D contact culture group w as higher than that in noncontact co-culture group and single HSC culture group( P < 0. 05),the proliferation level of suspension cells in non-contact co-culture group w as higher than that in single HSC culture group( P < 0. 05). Conclusion: The culture of HSC in vitro can proliferate HSC,M EC can promote the proliferation of HSC,M EC also can promote the HSC proliferation by non-contact co-culture in vitro,w hich relates w ith the effect of cytokines secreted from M EC; the effect of M EC and HSC contact co-culture on the proliferation of HSC is stronger than that of non-contact co-culture,w hich relates w ith the regulation of cell-cell contact.
Objective: To investigate the effect of microvascular endothelial cells( M EC) on the proliferation of hematopoictic stem cells( HSC) under different culture conditions in vitro. Methods: The M EC from lung tissue of C57 BL/6 mice and the HSC from bone marrow of GFP mice w ere used for non-contact co-culture,2 D contact coculture,at same time the single M EC and single HSC culture w ere seted up and w ere used as control group. The cell counting and CCK-8 method w ere used to detect and compare the proliferation levels of suspension cells in different groups on day 1,3,5 and 7. Results: M EC presented adherent grow th. In process of cell culture in vitro,the number of suspension cells in M EC and HSC co-culture group and single HSC culture group increased,the suspension cells in 2 D contact and non-contact co-culture groups more early gated into logarithmic grow th phase as compared w ith suspension cells in control group,the proliferation level of suspention cells in 2 D contact culture group w as higher than that in noncontact co-culture group and single HSC culture group( P < 0. 05),the proliferation level of suspension cells in non-contact co-culture group w as higher than that in single HSC culture group( P < 0. 05). Conclusion: The culture of HSC in vitro can proliferate HSC,M EC can promote the proliferation of HSC,M EC also can promote the HSC proliferation by non-contact co-culture in vitro,w hich relates w ith the effect of cytokines secreted from M EC; the effect of M EC and HSC contact co-culture on the proliferation of HSC is stronger than that of non-contact co-culture,w hich relates w ith the regulation of cell-cell contact.
引文
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11 Mc Dermott DH,Ji-Liang G,Malech HL,et al. Chromothriptic cure of WHIM syndrome. Cell,2015; 160(4):686-699.
12 Lai CY,Yamazaki S,Okabe M,et al. Stage-specific roles for CXCR4 signaling in murine hematopoietic stem/progenitor cells in the process of bone marrow repopulation. Stem Cells,2014; 32(7):1929-1942.
13 Butler JM,Nolan DJ,Vertes EL,et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell,2010; 6(3):251-264.
14 Yu VW,Scadden DT. Hematopoietic stem cell and its bone marrow niche. Curr Top Dev Biol,2016; 118:21-44.
15 Greenbaum A,Hsu YS,Day RB,et al. CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance.Nature,2013; 495(7440):227-230.
2 Oguro H,Ding L,Morrison SJ. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell,2013; 13(1):102-116.
3 Suárez-lvarez B,López-Vázquez A,López-Larrea C. Mobilization and homing of hematopoietic stemcells. Adv Exp Med Biol,2012;74:152-170.
4 吴梦瑶,陈彤.可视性观测造血干细胞归巢的研究进展.中国实验血液学杂志,2014; 22(1):209-212.
5 Winiarski BK,Acheson N,Gutowski NJ,et al. An improved and reliable method for isolation of microvascular endothelial cells from human omentum. Microcirculation,2011; 18(8):635-645.
6 Davis TA,Robinson DH,Lee KP,et al. Porcine brain microvascular endothelial cells support the in vitro expansion of human primitive hematopoietic bone marrow progenitor cells with a high replating potential:requirement for cell-to-cell interactions and colony-stimulating factors. Blood,1995; 85(7):1751-1761.
7 Salter AB,Meadows SK,Muramoto GG,et al. Endothelial progenitor cell infusion induces hematopoietic stem cell reconstitution in vivo. Blood,2009; 113(9):2104-2107.
8 Asada N,Takeishi S,Frenette PS. Complexity of bone marrow hematopoietic stem cell niche. Int J Hematol,2017; 106(1):45-54.
9 Kobayashi H,Butler JM,O'Donnell R,et al. Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells. Nat Cell Biol,2010; 12(11):1046-1056.
10 Crane GM,Jeffery E,Morrison SJ. Adult haematopoietic stem cell niches. Nat Rev Immunol,2017; 17(9):573-590.
11 Mc Dermott DH,Ji-Liang G,Malech HL,et al. Chromothriptic cure of WHIM syndrome. Cell,2015; 160(4):686-699.
12 Lai CY,Yamazaki S,Okabe M,et al. Stage-specific roles for CXCR4 signaling in murine hematopoietic stem/progenitor cells in the process of bone marrow repopulation. Stem Cells,2014; 32(7):1929-1942.
13 Butler JM,Nolan DJ,Vertes EL,et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell,2010; 6(3):251-264.
14 Yu VW,Scadden DT. Hematopoietic stem cell and its bone marrow niche. Curr Top Dev Biol,2016; 118:21-44.
15 Greenbaum A,Hsu YS,Day RB,et al. CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance.Nature,2013; 495(7440):227-230.