FoxO1基因对Jurkat细胞FoxO1-KLF2-S1P1通路的调节作用
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摘要
目的:通过构建FoxO1表达和干扰慢病毒载体,建立细胞内FoxO1-KLF2-S1P1信号通路调控研究模型,观察FoxO1过表达、干扰表达在Jurkat细胞内对其下游分子表达及功能的影响。方法:构建FoxO1表达和干扰表达慢病毒载体,分别感染Jurkat细胞,采用荧光定量PCR、Western blot和流式细胞术检测S1P1、CD62L、CCR7、CD69 mRNA水平和蛋白分子的表达。结果:FoxO1过表达组于感染后120 h FoxO1、KLF2、S1P1和CD62L mRNA水平显著增高(P<0.05),FoxO1、FoxO1-p和KLF2胞浆蛋白水平增高,S1P1~+细胞和CD62L~+细胞比率增高(P<0.05),CCR7~+细胞和CD69~+细胞未见显著改变(P>0.05)。FoxO1干扰组于转染后120 h FoxO1、KLF2、S1P1和CD62L mRNA水平降低(P<0.05),FoxO1、FoxO1-p和KLF2胞浆蛋白水平低于对照组,S1P1~+细胞百分比增多(P<0.05),但S1P1~+细胞和CD62L~+细胞在72 h时减少(P<0.05)。结论:FoxO1表达和干扰慢病毒载体转染Jurkat细胞并调节KLF2、S1P1和CD62L等分子的表达,为开展细胞内FoxO1-KLF2-S1P1信号通路调控和细胞相关功能的研究打下了基础。
To observe the contribution of FoxO1 on its downstream molecules expression and function in Jurkat cells,Jurkat cells were infected with FoxO1 expression lentivirus and interference lentivirus to establish FoxO1-KLF2-S1P1 signaling pathways model. After infection of 120 h,FoxO1,KLF2,S1P1 and CD62L mRNA levels and the protein expression of FoxO1,FoxO1-p and KLF2 were increased( P<0. 05) in FoxO1-overexpression group. Converse results( P< 0. 05) were observed in the interference group. The proportions of S1P1~+ cells were increased in both groups. It was notably that S1P1~+ cells were decreased( P<0. 05) in interference group after infection of 72 h. The proportion of CD62L~+ cells was increased( P<0. 05) in overexpression group,it was decreased( P< 0. 05) in the interference group. This vitro experiments showed S1P1 and CD62L could be regulated by FoxO1 lentivirus,and provided an experimental basis for research about intracellular FoxO1-KLF2-S1P1 signaling pathways and cellular functions.
To observe the contribution of FoxO1 on its downstream molecules expression and function in Jurkat cells,Jurkat cells were infected with FoxO1 expression lentivirus and interference lentivirus to establish FoxO1-KLF2-S1P1 signaling pathways model. After infection of 120 h,FoxO1,KLF2,S1P1 and CD62L mRNA levels and the protein expression of FoxO1,FoxO1-p and KLF2 were increased( P<0. 05) in FoxO1-overexpression group. Converse results( P< 0. 05) were observed in the interference group. The proportions of S1P1~+ cells were increased in both groups. It was notably that S1P1~+ cells were decreased( P<0. 05) in interference group after infection of 72 h. The proportion of CD62L~+ cells was increased( P<0. 05) in overexpression group,it was decreased( P< 0. 05) in the interference group. This vitro experiments showed S1P1 and CD62L could be regulated by FoxO1 lentivirus,and provided an experimental basis for research about intracellular FoxO1-KLF2-S1P1 signaling pathways and cellular functions.
引文
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[3]Hedrick SM,Hess Michelini R,Doedens AL,et al.FOXO transcription factors throughout T cell biology[J].Nat Rev Immunol,2012,12(9):649-661.
[4]Chang CF,D'Souza WN,Chen IL,et al.Polar opposites:Erk direction of CD4 T cell subsets[J].J Immunol,2012,189(2):721-731.
[5]Kerdiles YM,Beisner DR,Tinoco R,et al.Fox O1 links homing and survival of naive T cells by regulating L-selectin,CCR7 and interleukin 7 receptor[J].Nature Immunol,2009,10(2):176-184.
[6]Carlson CM,Endrizzi BT,Wu J,et al.Kruppel-like factor 2regulates thymocyte and T-cell migration[J].Nature,2006,442(7100):299-302.
[7]余思菲,吴长有.组织定居记忆性T细胞的免疫学特征研究进展[J].中国免疫学杂志,2017,33(7):1093-1100.She SF,Wu CY.Advances in the immunological characteristics of tissue-dwelling memory T cells[J].Chin J Immunol,2017,33(7):1093-1100.
[8]Maeda Y,Yagi H,Takemoto K,et al.S1P lyase in thymic perivascular spaces promotes egress of mature thymocytes via upregulation of S1P receptor 1[J].Int Immunol,2013,26(5):245-255.
[9]陶弋婧,朱顺飞,陈超,等.microRNA-7基因敲减对小鼠CD4+SP细胞胸腺发育的影响[J].中国免疫学杂志,2015,31(9):1173-1177.Tao YJ,Zhu SF,Chen C,et al.Influence of microRNA-7 knock down on development of CD4+SP cells in murinethymus[J].Chin JImmunol,2015,31(9):1173-1177.
[10]张云,赵俊涛,刘萍萍,等.Fox O1-KLF2-S1P1在MG患者胸腺组织的表达[J].免疫学杂志,2015,31(2):181-184.Zhang Y,Zhao JT,Liu PP,et al.The pilot study of Foxo1-KLF2-S1P1 in the thymus of patients with myasthenia gravis[J].Immunological J,2015,31(2):181-184.
[11]Teng F,Zhou YB,Jin R,et al.The molecular signature underlying the thymic migration and maturation of TCRab+CD4+CD8-thymocytes[J].PLo S One,2011,6(10):e25567.
[12]Ouyang W,Beckett O,Flavell RA,et al.An essential role of the Forkhead-box transcription factor Foxo1 in control of T cell homeostasis and tolerance[J].Immunity,2009,30(3):358-371.
[13]Ouyang W,Beckett O,Ma Q,et al.Foxo proteins cooperatively control the differentiation of Foxp3+regulatory T cells[J].Nat Immunol,2010,11(7):618-627.
[14]Rao RR,Li Q,Gubbels Bupp MR,et al.Transcription factor Foxo1represses T-bet-mediated effector functions and promotes memory CD8(+)T cell differentiation[J].Immunity,2012,36(3):374-387.
[15]Ouyang WM,Liao W,Luo CT,et al.Novel Foxo1-dependent transcriptional programs control Treg cell function[J].J Immunol,2013,191(1):187-199.
[16]Borowsky AD,Bandhuvula P,Kumar A,et al.Sphingosine-1-phosphate lyase expression in embryonic and adult murine tissues[J].J Lipid Res,2012,53(9):1920-1931.
[17]Jin R,Aili A,Wang YQ,et al.Critical role of SP thymocyte motility in regulation of thymic output in neonatal Aire-/-mice[J].Oncotarget,2017,8(1):83-94.