人唇腺中趋化性细胞因子CCL28的表达研究
摘要
趋化性细胞因子是细胞因子中的一种,该家族是一个小分子(8-16kDa)蛋白质的超家族,在氨基酸序列上存在20-70%的同源性。根据半胱氨酸的位置、排列方式及数量的不同,其被分为四类:CXC类(α)趋化性细胞因子、CC类(β)趋化性细胞因子、C类(γ)趋化性细胞因子、CXXXC类(6)趋化性细胞因子。
CCL28是2000年发现的一种新的趋化性细胞因子,它属于上述分类中的CC类,曾被称为粘膜相关上皮趋化性细胞因子(MEC)。其可介导循环中的IgA抗体分泌细胞跨过血管壁向局部实体组织内迁徙。
口腔中的SIgA是口腔免疫防御机制中至关重要的因素之一,主要来自大、小涎腺,其中来自小涎腺的约占30-35%。Pan等人应用斑点杂交分析证明人体的多种组织中均表达CCL28,其中以结肠、气管、大涎腺、乳腺表达较丰富。人大涎腺表达的CCL28,一方面可介导IgA~+ ASC由血循环向涎腺实体组织中迁徙,另一方面还可随唾液分泌到口腔中发挥杀菌抑菌作用。国内李潇的研究表明,在唾液中IgA的水平与CCL28的含量具有相关性。然而,迄今为止对人小涎腺中CCL28的表达和分泌情况尚无研究报道。因此,本课题的目的在于以唇腺组织为研究对象,从不同角度证实小涎腺中表达CCL28,从而为探讨小涎腺在维持口腔IgA水平方面的作用奠定基础。
方法:
1)应用实时荧光相对定量PCR技术来证实人的唇腺组织中表达CCL28 mRNA。
2)应用寡核苷酸探针原位杂交来检测唇腺组织中CCL28 mRNA的表达部位。
3)应用免疫组织化学来检测唇腺组织中CCL28蛋白的表达和分布情况。
结果:
1)实时荧光相对定量PCR结果表明健康成人的唇腺组织中表达丰富的CCL28mRNA。经统计学分析,其表达量(170.67±77.44)显著高于大肠对照组(19.50±14.55)(P＜0.001)。其后的唇腺和腮腺CCL28表达比较表明,前者中的表达量(1.28±0.57)显著高于后者(0.79±0.24)(P＜0.01)。
2)原位杂交显示CCL28 mRNA存在于腺泡上皮细胞胞浆中。
3)免疫组织化学显示唇腺腺泡上皮细胞胞浆、腺管上皮细胞、泡内粘液中均存在CCL28蛋白。但其染色深浅存在差异,这表明这几处CCL28蛋白的浓度不同。
结论:
同大涎腺一样,健康成人的唇腺中也表达丰富的CCL28,且其表达量高于腮腺。CCL28 mRNA表达于腺泡上皮细胞胞浆中,其翻译得到的蛋白质一部分在唇腺中发挥趋化作用,以吸引IgA~+ ASC,从而使其分泌IgA;而另一部分则随唾液分泌到口腔中发挥抗菌抑菌作用。这为进一步探讨小涎腺在维持口腔SIgA水平中的作用奠定了基础。
Background and objective:
Chemokines,which are kinds of cytokines,are a superfamily of small(8-16 kDa) proteins sharing 20-70%homology in amino acid sequences.According to the position, alignment and amount of cysteines,chemokines are classified into four kinds:CXC(α) chemokine,CC(β) chemokine,C(γ) chemokine and CXXXC(δ) chemokine.
CCL28,once called mucosal-associated epithelial chemokine,is a novel CC chemokine which was identified in 2000.It chemoattracts IgA~+ ASC from blood to local tissues.
SIgA in saliva,which is the pivotal factor in oral immune defense,mainly comes from major and minor salivary glands.SIgA that secretes from the latter is about 30-35%.Dot blot analysis by Pan Junliang shows high MEC message in diverse mucosal organs,such as mammary gland,trachea,colon,and major salivary glands.It has been proved that CCL28,abundantly expressed in human major salivary glands, can not only chemoattract IgA~+ ASC from blood to local gland tissues,but also be secreted to oral cavity where they exert their broad-spectrum antimicrobial activity.Li Xiao's research shows positive correlation between the level of SIgA and CCL28 in saliva.However,there are no reports about the expression and secretion of CCL28 in human minor salivary glands.So we took labial glands for examples to prove the expression of CCL28 in minor salivary glands,which may lay a foundation for searching its role in maintaining the level of IgA in oral cavity.
Methods:
1) Make use of real-time PCR to quantify CCL28 mRNA in labial glands.
2) Choose ISH of oligonucleotide probes to investigate the expression position of CCL28 mRNA in labial glands.
3) Use IHC to investigate the expression and distribution of CCL28 protein in labial glands.
Results:
1) Using real-time PCR,we proved that CCL28 mRNA was abundantly expressed in labial glands of healthy human.The results of statistic analysis were that CCL28 mRNA in labial glands(170.67±77.44) was much more than that in colons (19.50±14.55)(P＜0.001).Then the relative quantitative analysis between labial glands and parotids showed that CCL28 in the former(1.28±0.57) was more than in the latter(0.79±0.24)(P＜0.01).
2) From results of ISH,we found that CCL28 mRNA was expressed in cytoplasma of acinous cells.
3) The results of following IHC proved that positive staining was identified in serous epithelial cells,duct epithelial cells and mucus.But the degree of staining varied among different cells types and regions.
Conclusions:
CCL28 was indeed expressed in labial glands of healthy adults.Furthermore,its expression was much more than that in major salivary glands.In detail,CCL28 mRNA, which was expressed in acinous cell plasma,was translated into protein.Most of these proteins chemoattracted IgA~+ ASC in local glands,and others were secreted into salivary.My experiments will lay a good foundation for us to investigate how to increase SIgA in oral cavity.
CCL28是2000年发现的一种新的趋化性细胞因子,它属于上述分类中的CC类,曾被称为粘膜相关上皮趋化性细胞因子(MEC)。其可介导循环中的IgA抗体分泌细胞跨过血管壁向局部实体组织内迁徙。
口腔中的SIgA是口腔免疫防御机制中至关重要的因素之一,主要来自大、小涎腺,其中来自小涎腺的约占30-35%。Pan等人应用斑点杂交分析证明人体的多种组织中均表达CCL28,其中以结肠、气管、大涎腺、乳腺表达较丰富。人大涎腺表达的CCL28,一方面可介导IgA~+ ASC由血循环向涎腺实体组织中迁徙,另一方面还可随唾液分泌到口腔中发挥杀菌抑菌作用。国内李潇的研究表明,在唾液中IgA的水平与CCL28的含量具有相关性。然而,迄今为止对人小涎腺中CCL28的表达和分泌情况尚无研究报道。因此,本课题的目的在于以唇腺组织为研究对象,从不同角度证实小涎腺中表达CCL28,从而为探讨小涎腺在维持口腔IgA水平方面的作用奠定基础。
方法:
1)应用实时荧光相对定量PCR技术来证实人的唇腺组织中表达CCL28 mRNA。
2)应用寡核苷酸探针原位杂交来检测唇腺组织中CCL28 mRNA的表达部位。
3)应用免疫组织化学来检测唇腺组织中CCL28蛋白的表达和分布情况。
结果:
1)实时荧光相对定量PCR结果表明健康成人的唇腺组织中表达丰富的CCL28mRNA。经统计学分析,其表达量(170.67±77.44)显著高于大肠对照组(19.50±14.55)(P＜0.001)。其后的唇腺和腮腺CCL28表达比较表明,前者中的表达量(1.28±0.57)显著高于后者(0.79±0.24)(P＜0.01)。
2)原位杂交显示CCL28 mRNA存在于腺泡上皮细胞胞浆中。
3)免疫组织化学显示唇腺腺泡上皮细胞胞浆、腺管上皮细胞、泡内粘液中均存在CCL28蛋白。但其染色深浅存在差异,这表明这几处CCL28蛋白的浓度不同。
结论:
同大涎腺一样,健康成人的唇腺中也表达丰富的CCL28,且其表达量高于腮腺。CCL28 mRNA表达于腺泡上皮细胞胞浆中,其翻译得到的蛋白质一部分在唇腺中发挥趋化作用,以吸引IgA~+ ASC,从而使其分泌IgA;而另一部分则随唾液分泌到口腔中发挥抗菌抑菌作用。这为进一步探讨小涎腺在维持口腔SIgA水平中的作用奠定了基础。
Background and objective:
Chemokines,which are kinds of cytokines,are a superfamily of small(8-16 kDa) proteins sharing 20-70%homology in amino acid sequences.According to the position, alignment and amount of cysteines,chemokines are classified into four kinds:CXC(α) chemokine,CC(β) chemokine,C(γ) chemokine and CXXXC(δ) chemokine.
CCL28,once called mucosal-associated epithelial chemokine,is a novel CC chemokine which was identified in 2000.It chemoattracts IgA~+ ASC from blood to local tissues.
SIgA in saliva,which is the pivotal factor in oral immune defense,mainly comes from major and minor salivary glands.SIgA that secretes from the latter is about 30-35%.Dot blot analysis by Pan Junliang shows high MEC message in diverse mucosal organs,such as mammary gland,trachea,colon,and major salivary glands.It has been proved that CCL28,abundantly expressed in human major salivary glands, can not only chemoattract IgA~+ ASC from blood to local gland tissues,but also be secreted to oral cavity where they exert their broad-spectrum antimicrobial activity.Li Xiao's research shows positive correlation between the level of SIgA and CCL28 in saliva.However,there are no reports about the expression and secretion of CCL28 in human minor salivary glands.So we took labial glands for examples to prove the expression of CCL28 in minor salivary glands,which may lay a foundation for searching its role in maintaining the level of IgA in oral cavity.
Methods:
1) Make use of real-time PCR to quantify CCL28 mRNA in labial glands.
2) Choose ISH of oligonucleotide probes to investigate the expression position of CCL28 mRNA in labial glands.
3) Use IHC to investigate the expression and distribution of CCL28 protein in labial glands.
Results:
1) Using real-time PCR,we proved that CCL28 mRNA was abundantly expressed in labial glands of healthy human.The results of statistic analysis were that CCL28 mRNA in labial glands(170.67±77.44) was much more than that in colons (19.50±14.55)(P＜0.001).Then the relative quantitative analysis between labial glands and parotids showed that CCL28 in the former(1.28±0.57) was more than in the latter(0.79±0.24)(P＜0.01).
2) From results of ISH,we found that CCL28 mRNA was expressed in cytoplasma of acinous cells.
3) The results of following IHC proved that positive staining was identified in serous epithelial cells,duct epithelial cells and mucus.But the degree of staining varied among different cells types and regions.
Conclusions:
CCL28 was indeed expressed in labial glands of healthy adults.Furthermore,its expression was much more than that in major salivary glands.In detail,CCL28 mRNA, which was expressed in acinous cell plasma,was translated into protein.Most of these proteins chemoattracted IgA~+ ASC in local glands,and others were secreted into salivary.My experiments will lay a good foundation for us to investigate how to increase SIgA in oral cavity.
引文
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[1] Pan J, Kunkel EJ, Gosslar U, et al. A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues [J]. J Immunol. 2000, 165(6): 2943-2949.
[2] Wang W, Soto H, Oldham ER, et al. Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2) [J]. J Biol Chem. 2000, 275(29):22313-22323.
[3] Berri M, Meurens F, Lefevre F, et al. Molecular cloning and functional characterization of porcine CCL28: possible involvement in homing of IgA antibody secreting cells into the mammary gland [J]. Mol Immunol. 2008, 45(1): 271-277.
[4] Meurens F, Whale J, Brownlie R, et al. Expression of mucosal Chemokines TECK/CCL25 and MEC/CCL28 during fetal development of the ovine mucosal immune system [J]. Immunology.2007, 120 (4):544-555.
[5] Kunkel EJ, Kim CH, Lazarus NH, et al. CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells [J]. J Clin Invest. 2003.111(7): 1001 -1010.
[6] Lazarus NH, Kunkel EJ, Johnston B, et al. A common mucosal chemokine (mucosa - associated epithelial chemokine/CCL28) selectively attracts IgA plasmablasts [J]. J Immunol. 2003. 170 (7): 3799-3805.
[7] Childers NK, Greenleaf C, Li F, et al. Effect of age on immunoglobulin a subclass distribution in human parotid saliva [J]. Oral Microbiol Immunol. 2003, 18(5):298-301.
[8] Hieshima K, Ohtani H, Shibano M, et al. CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity [J]. J Immunol. 2003, 170 (3): 1452 -1461.
[9] Daniels TE. Labial salivary gland biopsy in Sjogren's syndrome: assessment as a diagnostic criterion in 362 suspected cases [J]. Arthritis Rheum. 1984, 27(2): 147-156.
[10] Crawford JM, Taubman MA, Smith DJ. Minor salivary glands as a major source of secretory immunoglobulin A in the human oral cavity [J]. Science. 1975,190 (4220): 1206 -1209. [12] Kunkel EJ, Butcher EC. Chemokines and tissue-specific migration of lymphocytes [J]. Immunity. 2002, 16(1): 1-4.
[11] Ogawa H, Iimura M, Eckmann L, et al. Regulated production of the chemokine CCL28 in human colon epithelium [J]. Am J Physiol Gastrointest Liver Physiol. 2004, 287(5): G1062-G1069.
[12] Kunkel EJ, Butcher EC. Chemokines and tissue-specific migration of lymphocytes [J]. Immunity. 2002, 16(1): 1-4.
2.孙涛,王松灵。唾液SIgA的结构及功能研究[J]。北京口腔医学。2000,8(4):200-203。
3.于世凤,汪说之,主编。口腔组织病理学[M]。第五版,北京:人民卫生出版社,2004:108-109。
4.Selner JC,Merrill DA,Claman HN.Salivary immunoglobulins:absence of rA in saliva following exchange transfusions in neonatal period[J].Pediatrics,1967,40:452.
5.Strober W,Blaese RM,Waldmann TA.The origin of salivary IgA[J].J Lad Clin Med,1970,75(5):856-862.
6.Brandtzaeg P,Fjellanger I,Gjeruldsen ST.Human secretory immunoglobulins.I.Salivary secretions from individuals with normal or low levels of serum immunoglobulins[J].Scand J Haematol Suppl,1970,12:3-83.
7.孙涛,王松灵。唾液SIgA的结构及功能研究[J]。北京口腔医学。2000,(8)4:200-203。
8.吴敏毓,刘恭植。医学免疫学[M]。第四版,合肥:中国科学技术大学出版社,2002:25-26。
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[1] Pan J, Kunkel EJ, Gosslar U, et al. A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues [J]. J Immunol. 2000, 165(6): 2943-2949.
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[3] Berri M, Meurens F, Lefevre F, et al. Molecular cloning and functional characterization of porcine CCL28: possible involvement in homing of IgA antibody secreting cells into the mammary gland [J]. Mol Immunol. 2008, 45(1): 271-277.
[4] Meurens F, Whale J, Brownlie R, et al. Expression of mucosal Chemokines TECK/CCL25 and MEC/CCL28 during fetal development of the ovine mucosal immune system [J]. Immunology.2007, 120 (4):544-555.
[5] Kunkel EJ, Kim CH, Lazarus NH, et al. CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells [J]. J Clin Invest. 2003.111(7): 1001 -1010.
[6] Lazarus NH, Kunkel EJ, Johnston B, et al. A common mucosal chemokine (mucosa - associated epithelial chemokine/CCL28) selectively attracts IgA plasmablasts [J]. J Immunol. 2003. 170 (7): 3799-3805.
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[10] Crawford JM, Taubman MA, Smith DJ. Minor salivary glands as a major source of secretory immunoglobulin A in the human oral cavity [J]. Science. 1975,190 (4220): 1206 -1209. [12] Kunkel EJ, Butcher EC. Chemokines and tissue-specific migration of lymphocytes [J]. Immunity. 2002, 16(1): 1-4.
[11] Ogawa H, Iimura M, Eckmann L, et al. Regulated production of the chemokine CCL28 in human colon epithelium [J]. Am J Physiol Gastrointest Liver Physiol. 2004, 287(5): G1062-G1069.
[12] Kunkel EJ, Butcher EC. Chemokines and tissue-specific migration of lymphocytes [J]. Immunity. 2002, 16(1): 1-4.