敲除CCR3基因对小鼠嗜酸粒细胞的作用实验观察
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摘要
目的:观察CCR3基因敲除对小鼠嗜酸粒细胞(EOS)增殖、成熟及凋亡的影响。方法:利用CCR3基因敲除小鼠(实验组)及野生型小鼠(对照组),取小鼠骨髓细胞进行体外培养,诱导刺激其分化成熟为EOS,通过细胞计数观察EOS增殖情况,qRT-PCR检测脱颗粒蛋白mRNA表达量,Annexin V-FITC/PI双染法检测EOS细胞凋亡率。结果:(1)体外培养EOS第0~14天细胞计数表明,第10、12、14天实验组获得细胞数均较对照组少,增殖较慢,差异有统计学意义(P<0.01);(2)细胞凋亡率检测结果表明,在含有10ng/ml IL-5或无IL-5条件下,实验组EOS细胞凋亡率均高于对照组,差异有统计学意义(P<0.01);(3)实验组ECP、EPO、MBP mRNA表达量与对照组相比均有不同程度降低,差异有统计学意义(P<0.05)。结论:敲除CCR3基因可抑制EOS增殖及成熟,促进其凋亡,为CCR3作为变应性鼻炎治疗靶基因提供理论依据。
Objective:To investigate the effect of CCR3 gene knockout on the proliferation,maturation and apoptosis of eosinophils(EOS)in mice.Method:Bone marrow cells from CCR3 gene knockout mice(experimental group)and wild-type mice(control group)were cultured in vitro and induced differentiation into mature EOS.EOS proliferation was observed by cell counting.Expression of degranulation protein mRNA was detected by qRTPCR.EOS apoptosis was detected by Annexin V-FITC/PI double staining method.Result:(1)The number of cells on the 0-14 day of EOS cultured in vitro showed that the number of cells in the experimental group was less than that in the control group at tenth,twelfth,fourteenth days,and the proliferation was slower,the difference was statistically significant(P<0.01).(2)The test results of EOS cells apoptosis showed that,under the condition of containing 10 ng/ml IL-5 or no IL-5,the apoptosis rate of EOS cells in experimental group was higher than that in normal control group,the difference between both groups was statistically significant(P<0.01).(3)The expression results of ECP,EPO,MBP mRNA in EOS showed that the expression levels of ECP,EPO,MBP mRNA in the experimental group compared with the normal control group were reduced in varying degrees,the difference between both groups was statistically significant(P<0.05).Conclusion:Knockout CCR3 gene can inhibit the proliferation and maturation of EOS and promote its apoptosis,which provides a theoretical basis for CCR3 as a target gene to treat allergic rhinitis.
Objective:To investigate the effect of CCR3 gene knockout on the proliferation,maturation and apoptosis of eosinophils(EOS)in mice.Method:Bone marrow cells from CCR3 gene knockout mice(experimental group)and wild-type mice(control group)were cultured in vitro and induced differentiation into mature EOS.EOS proliferation was observed by cell counting.Expression of degranulation protein mRNA was detected by qRTPCR.EOS apoptosis was detected by Annexin V-FITC/PI double staining method.Result:(1)The number of cells on the 0-14 day of EOS cultured in vitro showed that the number of cells in the experimental group was less than that in the control group at tenth,twelfth,fourteenth days,and the proliferation was slower,the difference was statistically significant(P<0.01).(2)The test results of EOS cells apoptosis showed that,under the condition of containing 10 ng/ml IL-5 or no IL-5,the apoptosis rate of EOS cells in experimental group was higher than that in normal control group,the difference between both groups was statistically significant(P<0.01).(3)The expression results of ECP,EPO,MBP mRNA in EOS showed that the expression levels of ECP,EPO,MBP mRNA in the experimental group compared with the normal control group were reduced in varying degrees,the difference between both groups was statistically significant(P<0.05).Conclusion:Knockout CCR3 gene can inhibit the proliferation and maturation of EOS and promote its apoptosis,which provides a theoretical basis for CCR3 as a target gene to treat allergic rhinitis.
引文
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[2]SPENCER L A,BONJOUR K,MELO R C,et al.Eosinophil secretion of granule-derived cytokines[J].Front Immunol,2014,5:496-496.
[3]DAVOINE F,LACY P.Eosinophil cytokines,chemokines,and growth factors:emerging roles in immunity[J].Front Immunol,2014,5:570-570.
[4]MELO R C,LIU L,XENAKIS J J,et al.Eosinophilderived cytokines in health and disease:unraveling novel mechanisms of selective secretion[J].Allergy,2013,68:274-284.
[5]ZHU X H,LIAO B,LIU K,et al.Effect of RNA interference therapy on the mice eosinophils CCR3gene and granule protein in the murine model of allergic rhinitis[J].Asian Pac J Trop Med,2014,7:226-230.
[6]RDINGER M,BOSSIOS A,SJSTRAND M,et al.Local proliferation and mobilization of CCR3(+)CD34(+)eosinophil-lineage-committed cells in the lung[J].Immunology,2011,132:144-154.
[7]UHM T G,KIM B S,CHUNG I Y.Eosinophil development,regulation of eosinophil-specific genes,and role of eosinophils in the pathogenesis of asthma[J].Allergy Asthma Immunol Res,2012,4:68-79.
[8]BOIX E,SALAZAR V A,TORRENT M,et al.Structural determinants of the eosinophil cationic protein antimicrobial activity[J].Biol Chem,2012,393:801-815.
[9]SORAGNI A,YOUSEFI S,STOECKLE C,et al.Toxicity of eosinophil MBP is repressed by intracellular crystallization and promoted by extracellular aggregation[J].Mol Cell,2015,57:1011-1021.
[10]ACHARYA K R,ACKERMAN S J.Eosinophil granule proteins:form and function[J].J Biol Chem,2014,289:17406-17415.
[11]ESNAULT S,SHEN Z J,MALTER J S.Protein Translation and Signaling in Human Eosinophils[J].Front Med(Lausanne),2017,4:150-150.
[12]SHAMRI R,YOUNG K M,WELLER P F.PI3K,ERK,p38 MAPK and integrins regulate CCR3-mediated secretion of mouse and humaneosinophil-associated Rnases[J].Allergy,2013,68:880-889.
[13]FULKERSON P C,SCHOLLAERT K L,BOUFFI C,et al.IL-5triggers a cooperative cytokine network that promotes eosinophil precursor maturation[J].J Immunol,2014,193:4043-4052.
[14]DYER K D,MOSER J M,CZAPIGA M,et al.Functionally competent eosinophils differentiated ex vivo in high purity from normal mousebone marrow[J].J Immunol,2008,181:4004-4009.
[15]LAMKHIOUED B,ABDELILAH S G,HAMID Q,et al.The CCR3receptor is involved in eosinophil differentiation and is up-regulated by Th2cytokines in CD34+progenitor cells[J].J Immunol,2003,170:537-547.
[16]OH J,MALTER J S.Pin1-FADD interactions regulate Fas-mediated apoptosis in activated eosinophils[J].J Immunol,2013,190:4937-4945.
[17]LUCAS C D,DORWARD D A,SHARMA S,et al.Wogonin induces eosinophil apoptosis and attenuates allergic airway inflammation[J].Am J Respir Crit Care Med,2015,191:626-636.
[18]XU W,GUO G,LI J,et al.Activation of Bcl-2-Caspase-9Apoptosis Pathway in the Testis of Asthmatic Mice[J].PLoS One,2016,11:e0149353.
[19]LIU Y,ZHU X,ZHANG H.Effects of chemokine receptor 3gene silencing by RNA interference on eosinophils[J].Exp Ther Med,2017,13:215-221.
[20]WANG F P,LIU T,LAN Z,et al.Efficacy and Safety of Anti-Interleukin-5 Therapy in Patients with Asthma:A Systematic Review and Meta-Analysis[J].PLoS One,2016,11:e166833.
[21]CHUANG C C,SU K E,CHEN C W,et al.AntiCCR3monoclonal antibody inhibits eosinophil infiltration in Angiostrongylus cantonensis-infected ICR mice[J].Acta Trop,2010,113:209-213.