HCV HVR1模拟肽诱导小鼠树突状细胞免疫应答及机制的初步研究
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摘要
DC是目前己知的功能最强的专职抗原提呈细胞,具有刺激初始T细胞增殖的独特功能。DC分为髓样DC(myeloid-DC,mDC)和浆细胞样DC(plasmacytoid-DC,pDC)两大亚群,分别由骨髓CD34~+干细胞、外周血单核细胞以及淋巴细胞等在粒一巨噬细胞集落刺激因子(granulocyte-macrophage clony-stimulating factor,GM-CSF)的诱导下分化而来。基于DC的重要功能和在机体特异性免疫中居于的关键地位,本课题以BALB/c小鼠为研究对象,对7肽诱导小鼠DC细胞免疫应答模式及其机制进行了初步研究。课题分为三部分。
(一)小鼠骨髓来源DC细胞培养
在本研究中,我们首先对小鼠骨髓来源的DC进行制备,主要描述了小鼠的骨髓细胞作为DC的前体细胞的采集、单核细胞分化为DC、表型评价。结果显示:此方法培养的细胞其表面CD11c的表达率超过80%,具有典型的DC形态。结论:利用此法能在体外培养出大量的未成熟DC,为研究模拟肽诱导小鼠DC细胞免疫应答机制奠定了基础。
(二)模拟肽诱导小鼠DC细胞免疫应答的研究
经7肽免疫10天的小鼠,取脾细胞,分选CD4~+T及CD~+8T细胞与经过7P负载并成熟DC细胞共孵育24小时,收集上清,应用流式细胞技术方法检测上清细胞因子。结果表明,实验组中CD8~+T上清中IFN-γ、TNF-α、IL-10及IL-6较对照组有所升高,CD4~+T上清中TNF-α降低;IL-5、IL-2及INF-r均升高,均具有统计学意义。结论:7肽能诱导以CD8~+T及Th1细胞为主的细胞免疫。
(三)模拟肽诱导免疫应答机制的初步研究
为了解体内7P免疫激活及调节途径,应用基因组表达谱芯片、RT-PCR以及Western blot等方法检测了免疫后小鼠脾细胞mRNA的表达水平以及蛋白的表达情况。在基因组表达谱芯片中:实验组GP1BB,HP,F13A1,F2RL2,GP5,F10,4VWF表达量增多,基因TNFRSF13C,SYK,TCRB-V13,FGD4,IGH-6,PDE4B,SOCS3,JAK1,TNFSF13B,CD4表达量则下降。RT-PCR结果提示:试验组syk,Jak和Tnfsf13b的mRNA表达水平降低,IFN-γ、IL-2和IL-10的mRNA表达水平升高。Western Blot提示:试验组SYK蛋白表达降低,而IFN-γ蛋白表达增高。结论:7肽可能诱导以CD8~+T及Th1细胞为主的细胞免疫,并且通过如SYK、Jak和Tnfsf13b等基因和蛋白的调控作用,在免疫应答的过程中抑制炎症反应,维持免疫应答在一定程度的发生。
This reseach was established on the study of HCV strains(Ⅱ/1b,Ⅲ/2a) envelope protein E2 hypervariable regions 1(HVR1) sequence variation rule and synthesis HCV HVR1 momoepitope according to the conservation site.As specialized antigen-presenting cells, dendritic cells(DC) played a pivotal role in immune responses.DC were commonly considered to be terminally differentiated cells derived from hematopoietic progenitors or monocytes.Our research used BALB/C mouse as the study object and investigated the immune response and its mechanism of DC induced by a polypeptide(7P).The reseach was divided to three sections.
(一) To establish a method of cultivation and purification of dendritic cells from mouse bone marrow in vitro and observe theirmorphology.
Mouse bone marrow cell suspensions were prepared with aseptic technique,then monocytes were isolated and induced in vitro by both granulocyte macrophagecolony-stimulating factor(GM-CSF) and interleukin 4(IL-4).The cell morphology and phenotype were analyzed.The percentage of the cultured DCs was 80% with typical morphology of DCs under light microscope.The CD11c~+ expression rate of DC cultured in vitro was more than 80%.A large number of DCs from mouse bone marrow in vitro exhibited typical morphology of DC,which might be an experimental material for further study on function,marker and antitumor role of DCs in vitro.
(二)To study DC cells function of the mouse immunized by synthesis peptide.
After subcutaneously immunization for ten days,the mice splenocytes were harvested. CD4~+Tand CD8~+Tcells were separated from splenocytes by cell sorting technology.Then CD4~+Tcells,CD8~+Tcells and DC cells were incubated for a day.The supernatants were deteced by cytometrie bead array Protein arraysystem(CBA).Compared with control group,the levels of IFN-γ、TNF-α、IL-10 and IL-6 secreted by CD8~+Tcell were increased in experiment group,the level of TNF-αsecreted by CD4~+Tcell was lower and the levels of IL-5、IL-2 and IFN-r were higher in experiment group,the differences were statistically significant between both group.In conclusion,the peptide could induce cell-mediated immune response through CD8~+ T and Th1 cells.
(三) To study the mechanism of immune response induced by synthesis peptide
In order to analyze the mechanism of immune response induced by synthesized peptide, the chip of affymetrix mouse genome 430 2.0 assay was applied.The differences in expression levels of genes were analyzed with cluster analysis and bioinformatics assay. Compared with control group,the mRNA levels of Gp1bb,Hp,F13a1,F2r12,Gp5,F10 and Vwf were higher,and those of Tnfrsf13c,Syk,Tcrb-V13,Fgd4,Igh-6,Pde4b, Socs3,Jak1 and Tnfsf13b were lower in experiment group.At the same time,the mRNA levels of syk,Jak and Tnfsf13b were lower than the control by RT-PCR assay,but those of IFN-γ,IL-2 and IL-10 were higher.The protein expression level of Syk was lower than the control by Western Blot assay,but that of IFN-γwas higher.In conclusion,the peptide could induce cell-mediated immune response through CD8+ T and Th1 cells,while the inflammation was inhibited by some factors.
(一)小鼠骨髓来源DC细胞培养
在本研究中,我们首先对小鼠骨髓来源的DC进行制备,主要描述了小鼠的骨髓细胞作为DC的前体细胞的采集、单核细胞分化为DC、表型评价。结果显示:此方法培养的细胞其表面CD11c的表达率超过80%,具有典型的DC形态。结论:利用此法能在体外培养出大量的未成熟DC,为研究模拟肽诱导小鼠DC细胞免疫应答机制奠定了基础。
(二)模拟肽诱导小鼠DC细胞免疫应答的研究
经7肽免疫10天的小鼠,取脾细胞,分选CD4~+T及CD~+8T细胞与经过7P负载并成熟DC细胞共孵育24小时,收集上清,应用流式细胞技术方法检测上清细胞因子。结果表明,实验组中CD8~+T上清中IFN-γ、TNF-α、IL-10及IL-6较对照组有所升高,CD4~+T上清中TNF-α降低;IL-5、IL-2及INF-r均升高,均具有统计学意义。结论:7肽能诱导以CD8~+T及Th1细胞为主的细胞免疫。
(三)模拟肽诱导免疫应答机制的初步研究
为了解体内7P免疫激活及调节途径,应用基因组表达谱芯片、RT-PCR以及Western blot等方法检测了免疫后小鼠脾细胞mRNA的表达水平以及蛋白的表达情况。在基因组表达谱芯片中:实验组GP1BB,HP,F13A1,F2RL2,GP5,F10,4VWF表达量增多,基因TNFRSF13C,SYK,TCRB-V13,FGD4,IGH-6,PDE4B,SOCS3,JAK1,TNFSF13B,CD4表达量则下降。RT-PCR结果提示:试验组syk,Jak和Tnfsf13b的mRNA表达水平降低,IFN-γ、IL-2和IL-10的mRNA表达水平升高。Western Blot提示:试验组SYK蛋白表达降低,而IFN-γ蛋白表达增高。结论:7肽可能诱导以CD8~+T及Th1细胞为主的细胞免疫,并且通过如SYK、Jak和Tnfsf13b等基因和蛋白的调控作用,在免疫应答的过程中抑制炎症反应,维持免疫应答在一定程度的发生。
This reseach was established on the study of HCV strains(Ⅱ/1b,Ⅲ/2a) envelope protein E2 hypervariable regions 1(HVR1) sequence variation rule and synthesis HCV HVR1 momoepitope according to the conservation site.As specialized antigen-presenting cells, dendritic cells(DC) played a pivotal role in immune responses.DC were commonly considered to be terminally differentiated cells derived from hematopoietic progenitors or monocytes.Our research used BALB/C mouse as the study object and investigated the immune response and its mechanism of DC induced by a polypeptide(7P).The reseach was divided to three sections.
(一) To establish a method of cultivation and purification of dendritic cells from mouse bone marrow in vitro and observe theirmorphology.
Mouse bone marrow cell suspensions were prepared with aseptic technique,then monocytes were isolated and induced in vitro by both granulocyte macrophagecolony-stimulating factor(GM-CSF) and interleukin 4(IL-4).The cell morphology and phenotype were analyzed.The percentage of the cultured DCs was 80% with typical morphology of DCs under light microscope.The CD11c~+ expression rate of DC cultured in vitro was more than 80%.A large number of DCs from mouse bone marrow in vitro exhibited typical morphology of DC,which might be an experimental material for further study on function,marker and antitumor role of DCs in vitro.
(二)To study DC cells function of the mouse immunized by synthesis peptide.
After subcutaneously immunization for ten days,the mice splenocytes were harvested. CD4~+Tand CD8~+Tcells were separated from splenocytes by cell sorting technology.Then CD4~+Tcells,CD8~+Tcells and DC cells were incubated for a day.The supernatants were deteced by cytometrie bead array Protein arraysystem(CBA).Compared with control group,the levels of IFN-γ、TNF-α、IL-10 and IL-6 secreted by CD8~+Tcell were increased in experiment group,the level of TNF-αsecreted by CD4~+Tcell was lower and the levels of IL-5、IL-2 and IFN-r were higher in experiment group,the differences were statistically significant between both group.In conclusion,the peptide could induce cell-mediated immune response through CD8~+ T and Th1 cells.
(三) To study the mechanism of immune response induced by synthesis peptide
In order to analyze the mechanism of immune response induced by synthesized peptide, the chip of affymetrix mouse genome 430 2.0 assay was applied.The differences in expression levels of genes were analyzed with cluster analysis and bioinformatics assay. Compared with control group,the mRNA levels of Gp1bb,Hp,F13a1,F2r12,Gp5,F10 and Vwf were higher,and those of Tnfrsf13c,Syk,Tcrb-V13,Fgd4,Igh-6,Pde4b, Socs3,Jak1 and Tnfsf13b were lower in experiment group.At the same time,the mRNA levels of syk,Jak and Tnfsf13b were lower than the control by RT-PCR assay,but those of IFN-γ,IL-2 and IL-10 were higher.The protein expression level of Syk was lower than the control by Western Blot assay,but that of IFN-γwas higher.In conclusion,the peptide could induce cell-mediated immune response through CD8+ T and Th1 cells,while the inflammation was inhibited by some factors.
引文
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[6]Morsica G,G.Tambussi,G.Sitia.Replication of hepatitis C virus in B lymphocytes(CD19").Blood 1999,94:1138-1139.
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[19]Zhang,J,G.Randall,A Higginbottom.CD81 is required for hepatitis C virus glycoprotein-mediated viral infection.J.Virol.2004,78:1448-1455
[20]Evans MJ,von Hahn T,Tscherne DM,et al.Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.Nature.2007,446:801 —805.
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