1.2.4 统计学处理 观察同一患者鼻息肉组织及下鼻甲黏膜成纤维细胞中各型组胺受体的表达,用实时扩增了解1型及2型组胺受体表达程度.将目的基因交点与βactin基因交点比值作为受体的表达程度并以均数±标准偏差(±SD)来表示.用SPSS12.0统计软件对数据进行统计学处理,进行MannWhiteny’s U检验.
2 结果
RTPCR结果表明,可见1型及2型组胺受体mRNA表达,未见3型及4型组胺受体表达(图2).实时扩增结果见,鼻息肉组织成纤维细胞内1型及2型组胺受体mRNA的表达较下鼻甲鼻黏膜成纤维细胞低,但相比较均无统计学意义(P=0.19,P=0.14,图3).流式细胞分析实验结果示,无刺激时1型组胺受体蛋白的表达量在鼻息肉组织及下鼻甲黏膜组织中均极低,无显著性差异(图4),2型组胺受体蛋白的表达在下鼻甲黏膜组织较多(图5).
3 讨论
组胺生成于嗜中性粒细胞及肥大细胞,参与各种过敏反应.气道上皮微环境中的肥大细胞可合成组胺,增加气道的渗透性[15].Jordana等[16]认为,组胺可使体外培养的成纤维细胞增殖,其作用是通过2型组胺受体完成的;thymikine可使体外培养的人皮肤成纤维细胞增殖,而组胺可通过1型组胺受体促进蛋白激酶C及佛波酯酵素活性,抑制thymikine对成纤维细胞的增殖作用[17].目前,对在鼻息肉的形成过程中成纤维细胞及成纤维细胞中的组胺受体所起的作用虽然尚未完全阐明,但可以肯定的是鼻息肉的形成仅与1型及2型组胺受体有关,而与3型及4型组胺受体则无关.本研究结果亦表明,鼻息肉成纤维细胞中只可见1型及2型组胺受体的表达,而未见3型及4型的表达.目前虽然对鼻息肉的形成过程尚无统一的认识,但与变应性鼻炎有关系的假设已被受重视.Okayama等[18]认为,人鼻黏膜组织中分布的1型组胺受体主要分布于小动脉、静脉及海棉静脉窦等血管内皮细胞内,而血管平滑肌及鼻黏膜下腺体等组织中则未见分布.Boucher等[19]报道,鼻黏膜切片组织中存在2型组胺受体的表达,且在变应性鼻炎鼻黏膜组织中的表达强度明显高于非变应性鼻炎,且原位杂交分析亦支持上述结果.本研究采用流式细胞分析方法观察了组胺受体蛋白的表达,发现其表达量较少,但组胺受体蛋白可以细胞内及外的形态表达,而流式细胞分析方法却不能准确地测定细胞内的蛋白量.蛋白表达较少的原因考虑可能与如下几点相关;1)可以肯定鼻息肉与下鼻甲黏膜成纤维细胞中有大量的组胺受体mRNA表达;2)组胺受体蛋白合成后受到某些其他代谢产物的影响,迅速溶解;3)大部分组胺受体蛋白仅在细胞内停留,而未到达细胞膜表面;4)可能在某些因素的影响下蛋白质翻译受阻碍;5)需要特定的条件,如各种细胞因子刺激或特殊酶参与等.
【参考文献】
[1] Nakamura T, Itadani H, Hidaka Y, et al..Molecular cloning and characterization of a new human histmamine receptor, HH4R[J].Biochem Biophysic Res Commun,2000,279:615.
[2] Hill SJ, Ganelln CR, Timmerman H, et al..Internation union of pharmacology Ⅻ classification of histamine receptors[J].American Society for Pharmacology and Experimental Therapeutics,1997,49:253.
[3] Fukui H, Fujimoto K, Mizuguchi H, et al..Molecular cloning of the human histamine H1 receptor gene[J].Biochem Biophys Res Commun,1994,201:894医.学.全.在.线www.lindalemus.com.
[4] Rotrosen D, Gallin J.Histmine type Ⅰ receptor occupancy increases endothelial cytosolic calciumu, reduces Faction, and promotes albumin diffusion across cultured endothelial monolayers[J].J Cell Biol,1986,103:2379.
[5] Kobayashi T, Inoue I, Jenkins NA, et al..Cloning, RNA expression, and chromosomal location of a mouse histamine H2receptor gene[J].Genomics,1996,37:390.
[6] Coge F, Guenin SP, Audinot V, et al..Gonomic organization and characterization of splice variants of the human histamine H3 receptor[J].Biochem J,2001,355:279.
[7] Arrang JM, Garbarg M, Schwartz JC.Actoin of brain histamine release mediated by a novel class(H3) of histamine receptor[J].Nature,1983,302:382.
[8] Oda T, Matsumoto S.Identification and characterization of histamine H4 receptor[J].Nippon Yakurigaku Zasshi,2001,118:36.
[9] Coge F, Guenin SP, Rique H, et al..Structure and expression of the human histamine H4receptor gene[J].Biochem Biophys Res Commun,2001,284:301.
[10] Strieter RM, Phan SH, Showell HJ, et al..Monokinesinduced neutrophil chemotactic factor gene expression in human fibroblasts[J].J Biol Chem,1989,264:10621.
[11] Yoshimura T, Yuhki N, Moore SK, et al..Human monocyte chemoattractant proteinI(MCP1): Full length cDNA cloning, expression in mitogenstimulated blood mononuclear leukocytes, and sequence similarity to murine competence gene[J].JE FEBS Lett,1989,244:487.
[12] Vancheri C, Gauldie J, Bienenstock J, et al..Human lung fibroblastderived granulocytemacrophage colony stimulating factor (GMCSF) mediates eosinophil survival in vitro[J].Am J Respir Cell Mol Biol,1989,1:289.
[13] Elias JA, Trinchieri G, Beck JM, et al..synergistic interaction of IL6 and IL1 mediates the thymocytestimulating activity produced by recombinant IL1 stimulated fibroblasts[J].J Immunol,1989,142:509.
[14] Sato E, Haniuda M, Numanami H, et al..Histamine and serotonin stimulate eotaxin production by a human lung fibroblast cell line[J].Arch Allergy Immunol,2002,128:12.
[15] Boucher RC, Ranga V, Pare PD, et al..Effect of histamine and methacholine of guinea pig tracheal permeability to HRP[J].J Appl Physiol,1978,45:939.
[16] Jordana M, Befus AD, Newhouse MT, et al..Effect of histamine on proliferation or normal human adult lung fibroblast[J].Thorax,1988,43:552.
[17] Johnson CL, Johnson CG.Inhibition of human skin fibroblast proliferation by histamine and phorbol esters is mediated by protein kinase C[J].Cell Signal,1990,2:105.
[18] Okayama M, Baraniuk J, Hausfeld J, et al..Characterization and autoradiographic localization of histamine H1 receptors on human nasal turbinates[J].J Allergy Clin Immunol,1992,89:1144.
[19] Boucher RC, Ranga V, Pare PD, et al..Effect of histamine and methacholine of guinea pig tracheal permeability to HRP[J].J Appl Physiol,1978,45:939.