输注贮存红细胞在炎症条件下对巨噬细胞调节作用的动物实验

Animalexperiment on regulating effect of stored red blood cells transfusion on BMDMs in inflammatory conditions

目的探讨炎症条件的动物输注贮存红细胞对巨噬细胞(BMDMs)的调节作用以及贮存红细胞输注与细菌感染引发炎症反应的关系。方法将6~8周龄成年雄性C57BL/6小鼠[(18~22)g/只]40只随机均分为实验组和实验对照组(对照组),均通过动物尾静脉注射铜绿假单胞菌200μL/只,并使用吸入式麻醉剂异氟烷(1%~3%)麻醉后,通过小鼠眼后静脉丛,实验组输注鼠源贮存悬浮红细胞(>14 d)400μL/只、对照组每只输注等量新鲜悬浮红细胞(贮存<24 h);于输注后2、4、8 h脱就猝死各结束2组小鼠生命5只,摘取鼠肝,体外培养铜绿假单胞菌感染(200μL/只)小鼠的股骨、胫骨骨髓来源的BMDMs,流式细胞术检测BMDMs中分化簇86(CD86)、分化簇197(CD197)[巨噬细胞1型(M1)基因特异性标志物]、分化簇209(CD209)[巨噬细胞2型(M2)基因特异性标记]表达水平,实时荧光定量PCR(qRT-PCR)法检测小鼠肝脏F4/80、M1、M2基因表达水平,并使用SPSS17.0统计学软件分析数据。结果实验组与对照组BMDMs中CD86和CD197的表达(%)分别为8688±1.01 vs 79.24±2.65、38.59±3.73 vs 25.95±0.86(P<0.05),CD209(%)为23.88±2.23 vs 21.91±3.58(P>0.05)。输注红细胞后2、4 h,小鼠肝F4/80基因表达水平实验组和对照组分别为1.83±0.11 vs 0.75±0.06、0.46±0.06 vs 0.33±0.06(P<0.05),8 h后分别为0.33±0.03 vs 0.35±0.05(P>0.05);输注红细胞2、4、8 h,小鼠肝M1基因中诱导型一氧化氮合酶(iNOS)基因表达水平实验组和对照组分别为3.44±0.20 vs 2.46±0.08、9.25±0.55 vs 2.67±0.12、2.80±0.08 vs 2.39±0.01,肿瘤坏死因子-α(TNF-α)分别为1.69±0.22 vs 1.13±0.03、1.44±0.24 vs 0.96±0.09、1.31±0.05 vs 0.96±0.06,单核细胞趋化蛋白1(MCP1)分别为4.96±0.08 vs 4.28±0.27、4.63±0.04 vs 2.07±0.09、2.28±0.19 vs 1.33±0.03(P<0.05);M2基因中精氨酸1(Arg1)基因表达水平实验组和对照组分别为0.81±0.21 vs 0.82±0.18、0.66±0.11 vs 0.58±0.09、0.39±0.17 vs 0.37±0.15,甘露糖受体C型2(Mrc2)分别为0.99±0.91 vs 0.97±0.08、0.98±0.12 vs 1.02±0.11、0.59±0.19 vs 0.57±0.08,重组蛋白163(CD163)分别为1.75±0.20 vs 1.69±0.18、0.22±0.02 vs 0.21±0.01、0.04±0.01 vs 0.03±0.01(P>0.05)。结论实验小鼠输注贮存红细胞明显促进其肝脏组织巨噬细胞朝向M1表型的极化。

Objective To discuss the regulating effect of stored red blood cells(RBCs)transfusion on BMDMs in inflammatory conditions,and the relationship between stored RBCs transfusion and inflammatory response induced by bacterial infection.Methods Forty C57BL/6 male mice of 6-8 weeks(18-22 g/mouse)were randomly divided into experimental group and control group.Each mouse was infected with 200μL Pseudomonas aeruginosa injecting into the tail vein,and 400μL fresh(storage>14 d)and stored RBCs(storage<24 h)suspension was infused through the posterior venous plexus into the isoflu-rane anesthetized mice(1%-3%).The BMDMs of Pseudomonas aeruginosa-infected mice were cultured,and CD86,CD197,CD209 in BMDMs were detected using FCM(flow cytometry)technique.The livers were removed 2,4 and 8 hours after RBCs infused,then F4/80,M1 and M2 in liver tissues were tested using qRT-PCR.The data were analyzed using statistical software 17.0.Results CD86 and CD197(%)of experimental group and control group were 8688+1.01 vs 79.24+2.65 and 38.59+3.73 vs 25.95+0.86(P<0.05),and CD209(%)were 23.88+2.23 vs 21.91+3.58(P>0.05).F4/80 of experimental group and control group 2,4 and 8 hours after RBCs infused were 1.83±0.11 vs 0.75±0.06,0.46±0.06 vs 0.33±0.06(P<0.05)and 0.33±0.03 vs 0.35±0.05(P>0.05),respectively.iNOS,TNF-α,MCP1 of M1 in liver of experimental group and control group 2,4 and 8 hours after RBCs infused were respectively:iNOS 3.44±0.20 vs 2.46±0.08,9.25±0.55 vs 2.67±0.12,2.80±0.08 vs 2.39±0.01;TNF-α1.69±0.22 vs 1.13±0.03,1.44±0.24 vs 0.96±0.09,1.31±0.05 vs 0.96±0.06;MCP14.96±0.08 vs 4.28±0.27,4.63±0.04 vs 2.07±0.09,2.28±0.19 vs 1.33±0.03(P<0.05).Arg1,Mrc2 and CD163 of M2 were respectively:Arg10.81±0.21 vs 0.82±0.18,0.66±0.11 vs 0.58±0.09,0.39±0.17 vs 0.37±0.15;Mrc20.99±0.91 vs 0.97±0.08,0.98±0.12 vs 1.02±0.11,0.59±0.19 vs 0.57±0.08;CD1631.75±0.20 vs 1.69±0.18,0.22±0.02 vs 0.21±0.01,0.04±0.01 vs 0.03±0.01(P>0.05).Conclusion Stored RBCs infusion can greatly promote the M1 polarization of BMDMs in liver.