日本七鳃鳗类淋巴细胞特异性免疫相关基因的差异表达
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摘要
本实验采用Ficoll密度梯度离心法分离出日本七鳃鳗单个核细胞层,并得到分离单个核细胞层的最佳分离液比重为1.092。利用流式细胞仪对分离到的单个核细胞层细胞进行分选,根据细胞的前向光及侧向光散射特征成功分选出类淋巴细胞,分选效率为95.68%,每毫升外周血可分离纯化得到类淋巴细胞2.4×106个。通过透射电镜观察七鳃鳗类淋巴细胞,细胞为圆形或椭圆形,细胞表面有突起、无微绒毛,胞质内含有板状嵴线粒体、粗面内质网、游离核糖体和液泡等。
通过淋巴细胞转化实验分析了日本七鳃鳗类淋巴细胞的异质性。首先,日本七鳃鳗类淋巴细胞的ANAE染色结果没有出现类似高等脊椎动物T细胞所具有的阳性棕红色反应颗粒。之后,用PHA与SPA做刺激剂,分别激活T、B淋巴细胞的转化而分裂,检测淋巴细胞的异质性,也未发现T淋巴细胞、B淋巴细胞的分化。以上实验仅从细胞形态和组织化学研究等侧面分析了日本七鳃鳗类淋巴细胞的异质性,表明在日本七鳃鳗的类淋巴细胞中可能尚未分化出T、B两大类群。
本文将限制片断差异显示聚合酶链反应技术(restriction fragment differential display PCR,RFDD-PCR)应用于日本七鳃鳗类淋巴细胞特异性免疫基因的研究。建立了免疫处理后的日本七鳃鳗血液类淋巴细胞的基因表达谱,获得基因片段1683个,其中与正常饲养日本七鳃鳗血液类淋巴细胞有表达差异的片段为97个,占表达数的5.76%。初步分析比较在混合抗原特异性免疫刺激条件下,日本七鳃鳗类淋巴细胞差异表达基因的特征,97条差异条带中有64条(约66.9%)序列能够通过同源比对获得其生物学功能信息,大致分为8类,包括初级代谢、蛋白质合成、转录、能量代谢、细胞结构、信号转导、细胞凋亡、免疫防卫等功能。其中属免疫防卫功能的基因有13条,约占差异数的20.3%,并筛选出2条特异性免疫相关的候选基因VLR,与海七鳃鳗的VLR基因有很高的同源相似性。同时得到一些可能是新基因的候选片断,有待进一步研究证实。通过差异片段的分析来推断和研究无颌类适应性免疫系统产生的机理与发生机制,为进一步研究提供线索。
To isolate and purify peripheral blood lymphocyte-like cells from the arctic lamprey, Lampetra japonica, centrifugation on Ficoll-Paque were performed and a uniform population of leucocytes containing lymphocyte-like cells with the mixture of Ficoll-Urografin solution at proportion of 1. 092 was yielded. With the FSC and SSC character of the cells on flow cytometer,Lymphocyte-like cells were separated successfully, the purity was 95.68%; about 2.4×106 lymphocyte-like cells could be purified from 1 ml peripheral blood. Under TEM, Lymphocyte-like cells are round or ellipse without microvillus. there are mitochondrion with cristae, rough endoplasmic reticulum,dissociated ribosome and vacuole in their cytoplasm.
The lymphocyte heterogeneity is analyzed by the experiment of cell multiplication. The result of ANAE reveals that there is no positive reddish brown grain in the leucocytes of L. japonica. Then, the studies on the capacities of immune responses of lymphocytes in peripheral blood of L. japonica to PHA and SPA with the active determine of MTT also indicated that there is no clear differentiation of T and B lymphocyte in the leucocytes of L. japonica. But this result scantly analyzed the aspect from cytomorphology and histochemistry of the lymphocyte heterogeneity of L. japonica, presuming that there is maybe no clear differentiation of T and B lymphocyte in the leucocytes of L. japonica.
The research of the adaptive immune gene expression in lymphocyte like cell of L. japonica,has been studied using RFDD-PCR. Gene expression profile of immunized adult has been constructed covering 1683 fragments of immunized lamprey, among these fragments, 97(5.76%) sequences of immunized and normal showed differences in abundance or presence. These different expressed gene fragments are analyzed and blast for homology. There are 64 of 97 different expressed gene fragments have got biological function information from homology sequence with tblastx in NCBI. These functions were classified into 8 different functional categories, include primary metabolize、protein synthesis、transcript、energy metabolism、cell structure、signal transduction、cell apoptosis、immune defence. There have 13 immune defense function genes, approximately 20.3% of the differences. And selected two specific candidate genes related to immune VLR, has the very high homologous similarity with the sea lamprey's VLR gene. At the same time get some may be new candidate gene fragments, pending further study confirmed. Through the analysis of different segments to infer the mechanism of agnathan vertebrates' adaptive immune system, and to provide clues for further study.
通过淋巴细胞转化实验分析了日本七鳃鳗类淋巴细胞的异质性。首先,日本七鳃鳗类淋巴细胞的ANAE染色结果没有出现类似高等脊椎动物T细胞所具有的阳性棕红色反应颗粒。之后,用PHA与SPA做刺激剂,分别激活T、B淋巴细胞的转化而分裂,检测淋巴细胞的异质性,也未发现T淋巴细胞、B淋巴细胞的分化。以上实验仅从细胞形态和组织化学研究等侧面分析了日本七鳃鳗类淋巴细胞的异质性,表明在日本七鳃鳗的类淋巴细胞中可能尚未分化出T、B两大类群。
本文将限制片断差异显示聚合酶链反应技术(restriction fragment differential display PCR,RFDD-PCR)应用于日本七鳃鳗类淋巴细胞特异性免疫基因的研究。建立了免疫处理后的日本七鳃鳗血液类淋巴细胞的基因表达谱,获得基因片段1683个,其中与正常饲养日本七鳃鳗血液类淋巴细胞有表达差异的片段为97个,占表达数的5.76%。初步分析比较在混合抗原特异性免疫刺激条件下,日本七鳃鳗类淋巴细胞差异表达基因的特征,97条差异条带中有64条(约66.9%)序列能够通过同源比对获得其生物学功能信息,大致分为8类,包括初级代谢、蛋白质合成、转录、能量代谢、细胞结构、信号转导、细胞凋亡、免疫防卫等功能。其中属免疫防卫功能的基因有13条,约占差异数的20.3%,并筛选出2条特异性免疫相关的候选基因VLR,与海七鳃鳗的VLR基因有很高的同源相似性。同时得到一些可能是新基因的候选片断,有待进一步研究证实。通过差异片段的分析来推断和研究无颌类适应性免疫系统产生的机理与发生机制,为进一步研究提供线索。
To isolate and purify peripheral blood lymphocyte-like cells from the arctic lamprey, Lampetra japonica, centrifugation on Ficoll-Paque were performed and a uniform population of leucocytes containing lymphocyte-like cells with the mixture of Ficoll-Urografin solution at proportion of 1. 092 was yielded. With the FSC and SSC character of the cells on flow cytometer,Lymphocyte-like cells were separated successfully, the purity was 95.68%; about 2.4×106 lymphocyte-like cells could be purified from 1 ml peripheral blood. Under TEM, Lymphocyte-like cells are round or ellipse without microvillus. there are mitochondrion with cristae, rough endoplasmic reticulum,dissociated ribosome and vacuole in their cytoplasm.
The lymphocyte heterogeneity is analyzed by the experiment of cell multiplication. The result of ANAE reveals that there is no positive reddish brown grain in the leucocytes of L. japonica. Then, the studies on the capacities of immune responses of lymphocytes in peripheral blood of L. japonica to PHA and SPA with the active determine of MTT also indicated that there is no clear differentiation of T and B lymphocyte in the leucocytes of L. japonica. But this result scantly analyzed the aspect from cytomorphology and histochemistry of the lymphocyte heterogeneity of L. japonica, presuming that there is maybe no clear differentiation of T and B lymphocyte in the leucocytes of L. japonica.
The research of the adaptive immune gene expression in lymphocyte like cell of L. japonica,has been studied using RFDD-PCR. Gene expression profile of immunized adult has been constructed covering 1683 fragments of immunized lamprey, among these fragments, 97(5.76%) sequences of immunized and normal showed differences in abundance or presence. These different expressed gene fragments are analyzed and blast for homology. There are 64 of 97 different expressed gene fragments have got biological function information from homology sequence with tblastx in NCBI. These functions were classified into 8 different functional categories, include primary metabolize、protein synthesis、transcript、energy metabolism、cell structure、signal transduction、cell apoptosis、immune defence. There have 13 immune defense function genes, approximately 20.3% of the differences. And selected two specific candidate genes related to immune VLR, has the very high homologous similarity with the sea lamprey's VLR gene. At the same time get some may be new candidate gene fragments, pending further study confirmed. Through the analysis of different segments to infer the mechanism of agnathan vertebrates' adaptive immune system, and to provide clues for further study.
引文
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[2] Pancer Z, Cooper MD. The evolution of adaptive imminity. Annual Review of Immunology,2006, 24:497~518.
[3] Good RA, Finstad J, Litman GW. The Biology of Lampreys II: Immunology.Academic, London,1972,405~432.
[4] Varner J, Neame P, Litman GW. A serum heterodimer from hagfish (Eptatretus stoutii) exhibits structural similarity and partial sequence identity with immunoglobulin. Proc Natl Acad Sci USA, 1991, 88: 1746~1750.
[5] Pancer Z, Amemiya CT, Ehrhardt GR, Ceitlin J, Gartland GL and Cooper MD. Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey. Nature,2004, 430(6996): 174~180.
[6] Pancer Z, Mayer WE, Klein J, Cooper MD. Prototypic T-cell receptor and CD4-like coreceptor expressed in lymphocytes of the agnathan sea lamprey. Proc Natl Acad Sci USA, 2004,101:13273~13278.
[7] Finstad J, Papermaster BW, Good RA. Evolution of the immune response II. Morphologic studies on the origin of the thymus and organized lymphoid tissue. Lab Invest, 1964,13:490~512.
[8] Mayer WE,Uinuk-Ool T,Tichy H,Gartland LA,Klein J,Cooper MD. Isolation and characterization of lymphocyte-like cells from a lamprey. Proc Natl Acad Sci USA, 2002,99(22):14350~14355.
[9] Wallin IE. The relationships and histogenesis of thymus-like structures in ammocoetes. American Journal of Anatomy,2005, 22:127~167.
[10] Shintani S, Terzic J, Sato A, Saraga-Babic M, O'hUigin C, Tichy H,and Klein J. Do lampreys have lymphocytes? The Spi evidence. Proc Natl Acad Sci USA,2000,97(13):7417~7422.
[11] Cannon JP, Haire RN, Rast JP, LitmanGW. The phylogenetic origins of the antigen binding receptors and somatic diversification mechanisms. Immunological Review,2004,200:12~22.
[12] Flajnik MF, Kasahara M. Comparative Genomics of the MHC Glimpses into the Evolution of the Adaptive Immune System. Immunity ,2001,15:351~362.
[13] Flajnik MF. Comparative analyses of immunoglobulin genes: surprises and portents. Nature Reviews Immunology, 2002, 2(9):688~698.
[14] Yokoyama K, Su Ih IH, Tezuka T, Yasuda T, Mikoshiba K, Tarakhovsky A, Yamamoto T. BANK regulates BCR-induced calcium mobilization by promoting tyrosine phosphorylation of IP3 receptor. EMBO Journal,2002,21:83~92.
[15] Wang YH, Zhang Z, Burrows PD, Kubagawa H, Bridges SL Jr, Findley HW, and Cooper MD. V(D)J recombinatorial repertoire diversification during intraclonal pro-B to B-cell differentiation. Blood,2003,101(3):1030~1037.
[16] Uinuk-Ool T, Mayer WE, Sato A, Dongak R, Cooper MD, Klein J. Lamprey lymphocyte-like cells express homologs of genes involved in immunologically relevant activities of mammalian lymphocytes.Proc Natl Acad Sci USA,2002,99:14356~14361
[17] Cannon JP, Haire RN, Pancer Z, Mueller MG, Skapura D, Cooper MD, Litman GW. Variable domains and a VpreB-like molecule are present in a jawless vertebrate.Immunogenetics,2005, 56(12):924~929.
[18] Alder MN, Rogozin IB, Iyer LM, Glazko GV, Cooper MD, Pancer Z. Diversity and Function of Adaptive Immune Receptors in a Jawless Vertebrate. Science, 2005, 310(5756):1892~1893.
[19] Pancer Z, Saha NR, Kasamatsu J, Suzuki T, Amemiya CT, Kasahara M, and Cooper MD.Variable lymphocyte receptors in hagfish. Proc Natl Acad Sci USA, 2005, 102: 9224~9229.
[20] Cooper MD, Alder MN. The evolution of adaptive immune systems. Cell, 2006, 24, 124(4):815~822.
[21] Uinuk-ool TS, Mayer WE, Sato A, Takezaki N, Benyon L, Cooper MD, Klein J. Identification and characterization of a TAP-family gene in the lamprey. Immunogenetics,2003,55:38~48.
[22] Cannon JP, Haire RN, Rast JP and Litman GW. The phylogenetic origins of the antigen-binding receptors and somatic diversification mechanisms. Immunological Reviews,2004,200:12~22.
[23] Mayer WE, O′Huigin C, Tichy H, Terzic J, Saraga-Babic M. Identification of twoIkaros-like transcription factors in lamprey. Scandinavian Journal of Immunology, 2002,55(2):162~170
[24] Janeway CA, Traver JrP, Walport M, Shlomchik MJ, Immuno biology: The Immune System in Health and Disease.Garland Publishing,New York,2001.
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