流式细胞术检测脊髓损伤大鼠局部浸润的巨噬细胞亚群
Detection of infiltrated macrophage subtypes in injured spinal cord of rats by flow cytometry
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摘要: 目的:建立检测脊髓损伤(spinal cord injury,SCI)大鼠局部浸润巨噬细胞亚群的流式细胞术方法。方法:取5只健康雌性SD大鼠,采用纽约大学SCI撞击仪系统制作重物坠落SCI模型为SCI组;另设5只假手术大鼠为假手术组。SCI术后7 d收集损伤的脊髓,制备单细胞悬液,采用Percoll原液密度梯度离心法浓缩浸润的巨噬细胞。分别以CD68与CD86或CD163的不同荧光标记抗体组合作为鉴定M1和M2型巨噬细胞的标志,用流式细胞术检测损伤局部巨噬细胞亚群的变化。结果:假手术组大鼠脊髓单细胞悬液中,CD68+细胞占细胞总数的(0. 070. 02)%,CD86+细胞和CD163+细胞分别占CD68+细胞的(9. 903. 28)%和(4. 950. 17)%;而SCI组大鼠脊髓单细胞悬液中,CD68+细胞的百分比为(0. 210. 09)%,CD86+细胞和CD163+细胞分别占CD68+细胞的(18. 710. 49)%和(27. 073. 74)%,差异均有统计学意义(P0. 05～P0. 01)。结论:以小鼠抗大鼠CD68、小鼠抗大鼠CD86、小鼠抗大鼠CD163为标志,采用流式细胞术可以成功检测SCI局部浸润的M1和M2型巨噬细胞亚群。Abstract: Objective: To develop the technique for measuring infiltrated macrophage subtypes in injured spinal cord of rats by flow cytometry. Methods: Contusive spinal cord injury( SCI) model in five healthy female SD rats( SCI group) were established by New York University Impactor， other five healthy female SD rats were treated with sham operation( sham operation group) . The injured spinal cords were collected and made into single cell suspension at day 7 after SCI. The infiltrated macrophages were concentrated by density gradient centrifugation using Percoll. The type M1 and M2 macrophages in lesions were identified using CD68 and CD86，or CD163 fluorescent antibodies， and measured by flow cytometry. Results: In rat's spinal cord single cell suspension of the sham operation group，the percentage of CD68 + cells in total cells was( 0. 07 0. 02) %， the percentages of CD86 + and CD163 + cells in CD68 + cells were (9. 90 3. 28) % and( 4. 95 0. 17) %， respectively. In rat's spinal cord single cell suspension of the SCI group， the percentage of CD68 + cells in total cells was( 0. 21 0. 09) %， the percentages of CD86 + and CD163 + cells in CD68 + cells were( 18. 71 0. 49) % and( 27. 07 3. 74) %，respectively. These differences between the sham operation group and SCI group were statistical signficance ( P ＜ 0. 05 to P ＜ 0. 01) . Conclusions: By using the mouse anti-rat CD68，CD86 and CD163 fluorescent antibodies， the infiltrated M1 and M2 macrophage subtypes can be successfully detected by flow cytometry.
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Key words:
- spinal cord injury /
- macrophage subtype /
- flow cytometry /
- rat
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[1] Mcdonald JW，Sadowsky C. Spinal-cord injury[J]. Lancet，2002，359(9304) : 417 － 425. [2] Bracken MB，Freeman DJ，Hellenbrand K. Incidence of acute traumatic hospitalized spinal cord injury in the United States，1970 － 1977[J]. Am J Epidemiol，1981，113(6) : 615 － 622. [3] Garfin SR，Shackford SR，Marshall LF，et al. Care of the multiply injured patient with cervical spine injury[J]. Clin Orthop Relat Res，1989(239) : 19 － 29. [4] Woodruff BA，Baron RC. A description of nonfatal spinal cord injury using a hospital-based registry[J]. Am J Prev Med，1994，10(1) : 10 － 14. [5] Dobkin BH，Havton LA. Basic advances and new avenues in therapy of spinal cord injury[J]. Annu Rev Med，2004，55:255 － 282. [6] Bomstein Y，Marder JB，Vitner K，et al. Features of skin coincubated macrophages that promote recovery from spinal cord injury[J]. J Neuroimmunol，2003，142(1 /2) : 10 － 16. [7] Saxena V，Lienesch DW，Zhou M，et al. Dual roles of immunoregulatoryb cytokine TGF- in the pathogenesis of autoimmunity-mediated organ damage[J]. J Immunol，2008，1 80(3) : 1903 － 1912. [8] Xu L，Xu Z，Xu M. Glucocorticoid treatment restores the impaired suppressive function of regulatory T cells in patients with relapsing-remitting multiple sclerosis[J]. Clin Exp Immunol，2009，158(1) : 26 － 30. [9] Gordon S，Taylor PR. Monocyte and macrophage heterogeneity[J]. Nat Rev Immunol，2005，1 2(5) : 953 － 964. [10] Reece JJ，Siracusa MC，Scott AL. Innate immune responses to lung-stage helminth infection induce alternatively activated alveolar macrophages[J]. Infect Hnmun，2006，74 (9) : 4970 －4981. [11] Dong L，Wang S，Chen M，et al. The activation of macrophage and upregulation of CD40 eostinmlatory molecule in lipopolysac eharide induced acute lung injury[J]. J Biomed Biotechnol，2008: 852571. [12] Basso DM，Beattie MS，Bresnahan JC. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight drop device versus transaction[J]. Exp Neurol，1996，1 39(2) : 244 － 256. -
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