慢病毒介导的小鼠CCR3基因RNAi载体的构建及其在肥大细胞中的表达
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摘要
目的:构建筛选靶向趋化因子受体3(CCR3)-RNA干扰慢病毒表达载体,感染小鼠肥大细胞,观察该基因在肥大细胞中的表达及该病毒载体的干扰效率,为研究变应性鼻炎的发病机制奠定基础。方法:首先构建3对CCR3-短发夹RNA(CCR3-shRNA)序列,然后在shRNA慢病毒载体中分别插入3对双链的shRNA oligo,3个shRNA慢病毒重组质粒构建成功后,将重组载体和病毒包装的辅助质粒共转染293T细胞,从而获得慢病毒质粒,并将其转染入体外培养及纯化小鼠骨髓源性的肥大细胞。qRT-PCR检测肥大细胞CCR3mRNA的表达,Western Blot检测肥大细胞CCR3蛋白的表达。结果:经测序证实,成功构建CCR3-shRNA的慢病毒载体,转染293T细胞并进行病毒包装,最终获取高纯度的PDSO19-PL-CCR3慢病毒质粒,病毒滴度为3.7×10~8 TU/ml。以该慢病毒质粒感染小鼠肥大细胞,qRT-PCR和Western Blot检测示:CCR3-shRNA降低了小鼠肥大细胞的CCR3基因在mRNA和蛋白水平的表达。结论:成功构建了CCR3基因RNAi慢病毒表达载体质粒,发现其下调了小鼠肥大细胞CCR3基因mRNA及蛋白水平的表达,为进一步研究其在变应性鼻炎发病机制中的作用打下了基础。
Objective:The aim of this study is to screen the targeting chemokine receptor 3-RNA interference(CCR3-RNAi)lentiviral expression vector,infect mouse mast cells,observe the expression of this gene in mast cells and the interference efficiency of the virus vector.The pathogenesis of allergic rhinitis lays the foundation.Method:Three pairs of CCR3-shRNA sequences were constructed,and three pairs of double-stranded shRNA oligo were inserted into shRNA lentiviral vectors to construct three shRNA lentiviral recombinant plasmids.The recombinant vector and virus-packed auxiliary plasmids were co-transfected into 293 Tcells to obtain lentiviral plasmids.The lentiviral plasmids were then transfected into mouse bone marrow-derived mast cells in vitro and purified.The expression level of CCR3 mRNA in mast cells was verified by qRT-PCR,and the expression level of CCR3 protein in mast cells was detected by Western Blot.Result:It was confirmed by sequencing that the lentiviral vector of CCR3 shRNA was successfully constructed,transfected into 293 Tcells and packaged with virus.Finally the high purity PDSO19-PL-CCR3 lentiviral plasmid was obtained with a virus titer of 3.7×10~8 TU/ml.The lentiviral plasmid was used to infect mouse mast cells.RT-PCR and Western Blot detection assay showed that CCR3 shRNA reduced the expression of CCR3 gene in mouse mast cells at the level of mRNA and protein.Conclusion:The CCR3 gene RNAi lentivirus expression vector was successfully constructed.It was found that it downregulated the expression level of CCR3 gene mRNA and protein in mouse mast cells,which laid the foundation for further research on its role in the pathogenesis of allergic rhinitis.
Objective:The aim of this study is to screen the targeting chemokine receptor 3-RNA interference(CCR3-RNAi)lentiviral expression vector,infect mouse mast cells,observe the expression of this gene in mast cells and the interference efficiency of the virus vector.The pathogenesis of allergic rhinitis lays the foundation.Method:Three pairs of CCR3-shRNA sequences were constructed,and three pairs of double-stranded shRNA oligo were inserted into shRNA lentiviral vectors to construct three shRNA lentiviral recombinant plasmids.The recombinant vector and virus-packed auxiliary plasmids were co-transfected into 293 Tcells to obtain lentiviral plasmids.The lentiviral plasmids were then transfected into mouse bone marrow-derived mast cells in vitro and purified.The expression level of CCR3 mRNA in mast cells was verified by qRT-PCR,and the expression level of CCR3 protein in mast cells was detected by Western Blot.Result:It was confirmed by sequencing that the lentiviral vector of CCR3 shRNA was successfully constructed,transfected into 293 Tcells and packaged with virus.Finally the high purity PDSO19-PL-CCR3 lentiviral plasmid was obtained with a virus titer of 3.7×10~8 TU/ml.The lentiviral plasmid was used to infect mouse mast cells.RT-PCR and Western Blot detection assay showed that CCR3 shRNA reduced the expression of CCR3 gene in mouse mast cells at the level of mRNA and protein.Conclusion:The CCR3 gene RNAi lentivirus expression vector was successfully constructed.It was found that it downregulated the expression level of CCR3 gene mRNA and protein in mouse mast cells,which laid the foundation for further research on its role in the pathogenesis of allergic rhinitis.
引文
[1]邱昌余,崔欣燕,程雷.固有免疫和适应性免疫在变应性鼻炎发病中的作用机制[J].临床耳鼻咽喉头颈外科杂志,2019,33(1):28-35.
[2]YUAN Z,LUO Z.prevalence of allergic rhinitis in China[J].Allergy Asthma Immunol Res,2014,6:105-113.
[3]朱新华,黄芸,江银丽,等.变应性鼻炎冲击免疫治疗剂量增加阶段血清学相关因素变化分析[J].临床耳鼻咽喉头颈外科杂志,2019,33(1):36-40.
[4]OKUBO K,KURONO Y,ICHIMURA K,et al.Japanese guidelines for allergic rhinitis 2017[J].Allergology International,2017,66:205-219.
[5]WHEATLEY L M,TOGIAS A.Clinical practice.Allergic rhinitis[J].N Engl J Med,2015,372:456-463.
[6]LIN K C,HUANG D Y,HUANG D W,et al.Inhibition of AMPK through Lyn-Syk-Akt enhances FcεRIsignal pathways for allergic response[J].J Mole Med(Berlin,Germany),2016,94:183-194.
[7]ANDALIB A,DOULABI H,MARACY M R,et al.CCR3,CCR4,CCR5,and CXCR3expression in peripheral blood CD4+lymphocytes in gastric cancer patients[J].Adv Biomed Res,2013,2:31.
[8]KAZUHIKO M,KEIICHI K,MITSUGU F,et al.efficient use of a crude drug/herb library reveals ephedra herb as a specific antagonist for th2-specific chemokine receptors CCR3,CCR4,and CCR8[J].Front Cell Dev Biol,2016,4:54-59.
[9]KUO C H,COLLINS A M,BOETTNER D R,et al.Role of CCL7in Type I hypersensitivity reactions in murine experimental allergic conjunctivitis[J].J Immunol,2017,198:645-656.
[10]COLLINGTON S J,WESTWICK J,WILLIAMS T J,et al.The function of CCR3on mouse bone marrowderived mast cells in vitro[J].Immunology,2010,129:115-124.
[11]KHANOLKAR A,BURDEN S J,HANSEN B,et al.Evaluation of CCR3as a Basophil Activation Marker[J].Am J Clin Pathol,2013,140:293-300.
[12]GANGWAR R S,LANDOLINA N,ARPINATI L,et al.Mast cell and eosinophil surface receptors as targets for anti-allergic therapy[J].Pharmacol Therapeu,2017,170:37-63.
[13]CHEN X,MANGALA L S,RODRIGUEZ-AGUAYOC,et al.RNA interference-based therapy and its delivery systems[J].Cancer Metastasis Rev,2018,37:107-124.
[14]ZHU X H,LIAO B,XU Y,et al.Downregulation of mouse CCR3by lentiviral shRNA inhibits proliferation and induces apoptosis of mouse eosinophils[J].Mole Med Rep,2017,15:696-702.
[15]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.
[16]MEURER S K,MELANIE N,SABINE W,et al.Isolation of mature(peritoneum-derived)mast cells and immature(bone marrow-derived)mast cell precursors from mice[J].PLoS One,2016,11:e0158104.
[17]彭霞,梁玉婷,林堃,等.小鼠骨髓来源肥大细胞的培养及鉴定[J].现代生物医学进展,2017,17(30):5807-5811.
[18]KARP J M,PEER D.Focus on RNA interference:from nanoformulations to in vivo delivery[J].Nanotechnology,2018,29:010201.
[19]ZHENG C X,WANG S M,BAI Y H,et al.Lentiviral vectors and adeno-associated virus vectors:useful tools for gene transfer in pain research[J].Anat Rec(Hoboken),2017,301:825-836.
[20]MODENA B D,DAZY K,WHITE D.Emerging concepts:mast cell involvement in allergic diseases[J].Transl Res,2016,174:98-121.
[21]MELISSA K W,DILEEPAN K N,WOOD J G.Mast cell:a multi-functional master cell[J].Front Immunol,2016,6:620-621.
[22]SCHMETZER O,VALENTIN P,CHURCH M K,et al.Murine and human mast cell progenitors[J].Eur JPharmacol,2016,778:2-10.
[23]RIBATTI D.The development of human mast cells.An historical reappraisal[J].Exp Cell Res,2016,342:210-215.
[24]LIN H,ZHENG C,LI J,et al.Lentiviral shRNA against KCa3.1inhibits allergic response in allergic rhinitis and suppresses mast cell activity via PI3K/AKT signaling pathway[J].Sci Rep,2015,5:13127-13129.
[25]SHAO Y Y,ZHOU Y M,HU M,et al.The Anti-allergic rhinitis effect of traditional chinese medicine of shenqi by regulating mast cell degranulation and Th1/Th2cytokine balance[J].Molecules,2017,22:504-509.
[26]ZHANG N,LI H,JIA J,HE M.Anti-inflammatory effect of curcumin on mast cell-mediated allergic responses in ovalbumin-induced allergic rhinitis mouse[J].Cell Immunol,2015,298:88-95.
[27]GANGWAR R S,LANDOLINA N,ARPINATI L,et al.Mast cell and eosinophil surface receptors as targets for anti-allergic therapy[J].Pharmacol Ther,2017,170:37-63.
[28]MIYAZAKI D,NAKAMURA T M,KUO C,et al.Ablation of type I hypersensitivity in experimental allergic conjunctivitis by eotaxin-1/CCR3blockade[J].Int Immunol,2009,2:187-201.
[2]YUAN Z,LUO Z.prevalence of allergic rhinitis in China[J].Allergy Asthma Immunol Res,2014,6:105-113.
[3]朱新华,黄芸,江银丽,等.变应性鼻炎冲击免疫治疗剂量增加阶段血清学相关因素变化分析[J].临床耳鼻咽喉头颈外科杂志,2019,33(1):36-40.
[4]OKUBO K,KURONO Y,ICHIMURA K,et al.Japanese guidelines for allergic rhinitis 2017[J].Allergology International,2017,66:205-219.
[5]WHEATLEY L M,TOGIAS A.Clinical practice.Allergic rhinitis[J].N Engl J Med,2015,372:456-463.
[6]LIN K C,HUANG D Y,HUANG D W,et al.Inhibition of AMPK through Lyn-Syk-Akt enhances FcεRIsignal pathways for allergic response[J].J Mole Med(Berlin,Germany),2016,94:183-194.
[7]ANDALIB A,DOULABI H,MARACY M R,et al.CCR3,CCR4,CCR5,and CXCR3expression in peripheral blood CD4+lymphocytes in gastric cancer patients[J].Adv Biomed Res,2013,2:31.
[8]KAZUHIKO M,KEIICHI K,MITSUGU F,et al.efficient use of a crude drug/herb library reveals ephedra herb as a specific antagonist for th2-specific chemokine receptors CCR3,CCR4,and CCR8[J].Front Cell Dev Biol,2016,4:54-59.
[9]KUO C H,COLLINS A M,BOETTNER D R,et al.Role of CCL7in Type I hypersensitivity reactions in murine experimental allergic conjunctivitis[J].J Immunol,2017,198:645-656.
[10]COLLINGTON S J,WESTWICK J,WILLIAMS T J,et al.The function of CCR3on mouse bone marrowderived mast cells in vitro[J].Immunology,2010,129:115-124.
[11]KHANOLKAR A,BURDEN S J,HANSEN B,et al.Evaluation of CCR3as a Basophil Activation Marker[J].Am J Clin Pathol,2013,140:293-300.
[12]GANGWAR R S,LANDOLINA N,ARPINATI L,et al.Mast cell and eosinophil surface receptors as targets for anti-allergic therapy[J].Pharmacol Therapeu,2017,170:37-63.
[13]CHEN X,MANGALA L S,RODRIGUEZ-AGUAYOC,et al.RNA interference-based therapy and its delivery systems[J].Cancer Metastasis Rev,2018,37:107-124.
[14]ZHU X H,LIAO B,XU Y,et al.Downregulation of mouse CCR3by lentiviral shRNA inhibits proliferation and induces apoptosis of mouse eosinophils[J].Mole Med Rep,2017,15:696-702.
[15]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.
[16]MEURER S K,MELANIE N,SABINE W,et al.Isolation of mature(peritoneum-derived)mast cells and immature(bone marrow-derived)mast cell precursors from mice[J].PLoS One,2016,11:e0158104.
[17]彭霞,梁玉婷,林堃,等.小鼠骨髓来源肥大细胞的培养及鉴定[J].现代生物医学进展,2017,17(30):5807-5811.
[18]KARP J M,PEER D.Focus on RNA interference:from nanoformulations to in vivo delivery[J].Nanotechnology,2018,29:010201.
[19]ZHENG C X,WANG S M,BAI Y H,et al.Lentiviral vectors and adeno-associated virus vectors:useful tools for gene transfer in pain research[J].Anat Rec(Hoboken),2017,301:825-836.
[20]MODENA B D,DAZY K,WHITE D.Emerging concepts:mast cell involvement in allergic diseases[J].Transl Res,2016,174:98-121.
[21]MELISSA K W,DILEEPAN K N,WOOD J G.Mast cell:a multi-functional master cell[J].Front Immunol,2016,6:620-621.
[22]SCHMETZER O,VALENTIN P,CHURCH M K,et al.Murine and human mast cell progenitors[J].Eur JPharmacol,2016,778:2-10.
[23]RIBATTI D.The development of human mast cells.An historical reappraisal[J].Exp Cell Res,2016,342:210-215.
[24]LIN H,ZHENG C,LI J,et al.Lentiviral shRNA against KCa3.1inhibits allergic response in allergic rhinitis and suppresses mast cell activity via PI3K/AKT signaling pathway[J].Sci Rep,2015,5:13127-13129.
[25]SHAO Y Y,ZHOU Y M,HU M,et al.The Anti-allergic rhinitis effect of traditional chinese medicine of shenqi by regulating mast cell degranulation and Th1/Th2cytokine balance[J].Molecules,2017,22:504-509.
[26]ZHANG N,LI H,JIA J,HE M.Anti-inflammatory effect of curcumin on mast cell-mediated allergic responses in ovalbumin-induced allergic rhinitis mouse[J].Cell Immunol,2015,298:88-95.
[27]GANGWAR R S,LANDOLINA N,ARPINATI L,et al.Mast cell and eosinophil surface receptors as targets for anti-allergic therapy[J].Pharmacol Ther,2017,170:37-63.
[28]MIYAZAKI D,NAKAMURA T M,KUO C,et al.Ablation of type I hypersensitivity in experimental allergic conjunctivitis by eotaxin-1/CCR3blockade[J].Int Immunol,2009,2:187-201.