MUC5AC在人类鼻息肉及下鼻甲粘膜上皮的表达
摘要
呼吸道表面被覆一层粘液毯，它作为细胞外环境与细胞膜之间的选择性物理屏障，在保湿、润滑、吸收及包裹微生物和致病颗粒以及其它宿主防御功能等方面起重要作用。这层分泌毯由两层构成：表面的粘液层以及深部纤毛起作用的浆液层。气道粘液层由水、离子、肺分泌物、血浆蛋白渗出液及粘蛋白组成。当粘液层发生质或量的改变时，可导致鼻塞、鼻息肉、鼻窦炎等疾病症状。粘蛋白是由粘膜上皮杯状细胞和粘膜下腺体细胞合成分泌的一种高分子量的糖蛋白，它是粘液的重要组成部分，可以保护粘膜上皮免受微生物、毒素等物质的侵袭。到目前为止，已经发现了十二种人类粘蛋白基因，根据它们被描述的先后顺序，分别称为：MUC1、MUC2、MUC3、MUC4、MUC5AC、MUC5B、MUC6、MUC7、MUG8、MUC9、MUC11、MUC12。其中，MUC2、MUC5AC、MUC5B、MUC6、MUC7所编码的粘蛋白属分泌型粘蛋白，MUC1、MUC3、MUC4、MUC12所编码的粘蛋白属膜结合型粘蛋白基因，其余尚未分类。自从1988年第一个编码粘蛋白核心肽的cDNA克隆被分离以来，粘蛋白的分子解析、结构、功能引起了众多学者广泛而深入的研究。近几年来，国外对粘蛋白在耳鼻咽喉科领域的研究日益增多，但是国内在此领域的研究还未见有人报道。本研究采用免疫组化的方法检测MUC5AC在人类鼻息肉及下鼻甲粘膜上皮的表达，探讨MUC5AC与鼻息肉及慢性肥厚性鼻炎时粘液过量分泌的关系。
实验目的
检测MUC5AC在人类鼻息肉及下鼻甲粘膜上皮的表达情况。探讨MUC5AC与鼻息肉及慢性肥厚性鼻炎时粘液过量分泌的关系。
吉林大学硕士学位论文
实验方法
27例鼻息肉标本来自我科行内窥镜鼻窦手术的患者，其中男
15例，女12例，年龄13一56岁，平均35.5岁，19例慢性肥厚
性鼻炎下鼻甲标本来自我科行慢性肥厚性鼻炎下鼻甲切除术的患
者，其中男12例，女7例，年龄18一48岁，平均29岁，9例正
常下甲来自我科黔症手术的患者，全部为男性，年龄在31一46岁，
平均37岁。全部病例均无变态反应性鼻炎、哮喘等变态反应史，
无应用类固醇激素及抗组胺药物史，无阿司匹林不耐受。标本经4
%多聚甲醛固定，常规脱水，石蜡包埋。制成5阳厚的连续切片，
HE染色用于常规组织病理学检查。免疫组织化学染色用于观察
MUCSAC的表达情况及表达部位。
实验结果
1.组织病理学观察结果
HE染色可见27例鼻息肉组织被覆含杯状细胞的假复层柱状纤
毛上皮，部分鳞状上皮化生，腺体增生、间质明显水肿，并有不
同程度的炎细胞浸润。19例慢性肥厚性鼻炎下鼻甲粘膜上皮表面
被覆假复层柱状纤毛上皮，杯状细胞增生，粘膜固有层水肿，有
淋巴细胞及浆细胞浸润。
2.免疫组化观察结果
在27例鼻息肉标本中，有23例标本呈MUCSAC强阳性表达，
阳性细胞表达率为18.9%，是正常鼻甲(5 .78%)的3.27倍(p<0 .001，
见图1一2及表l)，19例慢性肥厚性鼻炎下甲标本也呈强阳性表
达，其阳性细胞表达率为21.30k，是正常鼻甲(5 .780k)的3.69倍
(p(0.001，见图3~4及表2)，除此之外，亦发现MUCSAC阳性细
吉林大学硕士学位论文
胞主要为上皮杯状细胞。
讨论
粘蛋白是由粘膜上皮杯状细胞和粘膜下腺体细胞合成和分泌
的一种高分子量糖蛋白，它是粘液纤毛转运系统中粘液的重要组
成部份，有着许多功能，可以赋予粘液一定的粘性和弹性，使其
易与被纤毛转运，而且它的表达水平、转录后、转录水平的调节
以及粘蛋白的相互作用都会影响粘液的性质。当然粘蛋白的量及
类型也可以影响它的渗透性、抗菌活性和粘性，它也可能与细菌
外膜上的蛋白质结合〔3J，来保护上皮免受病原微生物的侵害。
分泌型粘蛋白是研究慢性炎症时分泌物为何增多的关键，而
在分泌型粘蛋白基因中，MUCSAC和MUCSB是己知的最主要的在气
道表达的粘蛋白基因，亦有报道表明，MUCZ和MUCSAC主要在气
道杯状细胞上表达，而从组织学角度来考虑，鼻粘膜上皮富含杯
状细胞，从切片中亦可看出炎症后的鼻粘膜上皮杯状细胞大量增
生，故因此推测MUCSAC的表达亦会随之增加。结果研究发现MUCSAC
在鼻息肉及慢性肥厚性鼻炎下甲粘膜上皮的表达与正常鼻甲粘膜
上皮的表达有显著性差别(p<0 .001)，而且阳性表达细胞基本上
局限于杯状细胞。与此结果类似，Buisine等人曾报道过MUCSAC
mRNA在气管与支气管上皮表面的所有杯状细胞上表达。而Kim等
人的研究与此则略有不同，他们的结果表明在正常后组筛窦粘膜
上皮很少有MUCSACmRNA表达，这与我们的研究结果:MUCSAC在正
常鼻甲粘膜上皮阳性细胞表达率5.78%略有差异，也与以前的研究
不同，目前，为什么会存在这种差别并不十分清楚，Kim等人的解
释是这种不同可能来源于标本的取材，与正常鼻甲粘膜相比，正
吉林大学硕士学位论文
常筛窦粘膜并没有直接接触外界炎性刺激物，基本可以认为是“绝
对”正常的标本。但我们的研究结果与其它的研究结果基本一致，
都表明MUCSAC表达在炎症刺激下上调，这种表达的上调可能与炎
性介质(工L一
The epithelial surface of the respiratory tract is coated with mucus which serve as the selective physical barrier between the extracellular milieu and the cell membrane and play important roles in humidification, lubrication, insulation, entrapment of microorganisms and particulates, and other host defense functions. This secretory blanket consists of two layers: the surface mucus layer and the deeper serous layer in which the cilia function. Airway mucus is composed of water, ions, lung secretions, serum protein transudates, and mucin glycoproteins (mucins). When this mucus blanket is altered in amount or composition, rhinorrhea, nasal obstruction, polyposis, and sinusitis may develop. Mucins are large, heterogeneous, and highly glycosylated proteins which produced by goblet cells in the surface epitheliam and by mucous and serous cells in the submucosal glands. They are the major glycoprotein components of the mucus layer, and play an essential role in the defense of the epithelium against invading pathog
ens. To date, 12 human mucin genes (MUC1-4, MUC5AC, MUC5B, MUC6-8, MUC9, MUCII, and MUC12) have been identified. Among them, MUC2, MUC5AC, MUC5B, MUC6, and MUC7 are known to be secretory mucin genes in the human lower airway, and MUC1, MUC3, MUC4, and MUC12 are thought to be membrane bounded, others are not classified. The molecular analysis, structure and function of mucin have caused extensive and deep investigation since the first cDNA clone had been identified in 1988.
In recent years, the research about mucin in oversea E.N.T.department had increased, however, little had been reported in our country. With immunohistochemistry ABC and HE dyeing , polyps and chronic hypertrophy rhinitis inferior turbinates epithelium were applied to this study. Object
To examine the expression of MUC5AC in human nasal polyp and inferior turbinate epithelium, to evaluate the relationship between MUC5AC and the increased mucous secretions in polyps and chronic hypertrophy rhinitis. Method
27 polyp tissues were obtained from the patients( males 15, females 12; age range 13-56 years, average age 35.5 years ) who were subjected to nasal polypectomy in our department. 19 chronic hypertrophy rhinitis inferior turbinates tissues were obtained from the patients (males 12, females 7; age range 18-48 years, average age 29 years ) who were subjected to inferior turbinectomy in our department. Inferior turbinate mucosa as control group were from 9 patients ( all male, age range 31-46 years, average age 37 years) with rhino-genesis snoring. All cases have not the history of asthma, allergy, aspirin intolerance and not receiving steroids before two weeks. All specimens were fixed with 4% paraformaldehyde, paraffin-embeded and make sections at 5 m thick. The slides were detected with HE staining for routine histopathologic examination, immuno-histochemical staining was performed for observing the expression of
- - 6 - -
1.27 Nasal polyps tissue is morphologically characterized by oedema, goblet cell hyperplasia of the epithelium, is extreme edema and gland proliferation. There are a lot of inflammatory cells in nasal polyp tissues by HE. 19 Inferior turbinates tissues is morphologically characterized by oedema, goblet cell hyperplasia of the epithelium, is edema in lamina propria mucosae, there are lymphocytes and plasmacytes in them.
2. The number of MUC5AC expressing cells in nasal polyps and chronic hypertrophy rhinitis was significantly higner than in normal inferior turbinates(P<0.05), and the expression of MUC5AC cells are mainly concentrate on the goblet cells. Discussion
Mucins are large, heterogeneous, and highly glycosylated proteins which produced by goblet cells in the surface epitheliam and by mucous and serous cells in the submucosal glands. They are the major glycoprotein components of the mucus, they give mucus its viscous and elastic characteristics that enable it to be transported by cilia. The regulations of expression, transcription and post-transcription and the correlation between mucins can affect the quali
实验目的
检测MUC5AC在人类鼻息肉及下鼻甲粘膜上皮的表达情况。探讨MUC5AC与鼻息肉及慢性肥厚性鼻炎时粘液过量分泌的关系。
吉林大学硕士学位论文
实验方法
27例鼻息肉标本来自我科行内窥镜鼻窦手术的患者，其中男
15例，女12例，年龄13一56岁，平均35.5岁，19例慢性肥厚
性鼻炎下鼻甲标本来自我科行慢性肥厚性鼻炎下鼻甲切除术的患
者，其中男12例，女7例，年龄18一48岁，平均29岁，9例正
常下甲来自我科黔症手术的患者，全部为男性，年龄在31一46岁，
平均37岁。全部病例均无变态反应性鼻炎、哮喘等变态反应史，
无应用类固醇激素及抗组胺药物史，无阿司匹林不耐受。标本经4
%多聚甲醛固定，常规脱水，石蜡包埋。制成5阳厚的连续切片，
HE染色用于常规组织病理学检查。免疫组织化学染色用于观察
MUCSAC的表达情况及表达部位。
实验结果
1.组织病理学观察结果
HE染色可见27例鼻息肉组织被覆含杯状细胞的假复层柱状纤
毛上皮，部分鳞状上皮化生，腺体增生、间质明显水肿，并有不
同程度的炎细胞浸润。19例慢性肥厚性鼻炎下鼻甲粘膜上皮表面
被覆假复层柱状纤毛上皮，杯状细胞增生，粘膜固有层水肿，有
淋巴细胞及浆细胞浸润。
2.免疫组化观察结果
在27例鼻息肉标本中，有23例标本呈MUCSAC强阳性表达，
阳性细胞表达率为18.9%，是正常鼻甲(5 .78%)的3.27倍(p<0 .001，
见图1一2及表l)，19例慢性肥厚性鼻炎下甲标本也呈强阳性表
达，其阳性细胞表达率为21.30k，是正常鼻甲(5 .780k)的3.69倍
(p(0.001，见图3~4及表2)，除此之外，亦发现MUCSAC阳性细
吉林大学硕士学位论文
胞主要为上皮杯状细胞。
讨论
粘蛋白是由粘膜上皮杯状细胞和粘膜下腺体细胞合成和分泌
的一种高分子量糖蛋白，它是粘液纤毛转运系统中粘液的重要组
成部份，有着许多功能，可以赋予粘液一定的粘性和弹性，使其
易与被纤毛转运，而且它的表达水平、转录后、转录水平的调节
以及粘蛋白的相互作用都会影响粘液的性质。当然粘蛋白的量及
类型也可以影响它的渗透性、抗菌活性和粘性，它也可能与细菌
外膜上的蛋白质结合〔3J，来保护上皮免受病原微生物的侵害。
分泌型粘蛋白是研究慢性炎症时分泌物为何增多的关键，而
在分泌型粘蛋白基因中，MUCSAC和MUCSB是己知的最主要的在气
道表达的粘蛋白基因，亦有报道表明，MUCZ和MUCSAC主要在气
道杯状细胞上表达，而从组织学角度来考虑，鼻粘膜上皮富含杯
状细胞，从切片中亦可看出炎症后的鼻粘膜上皮杯状细胞大量增
生，故因此推测MUCSAC的表达亦会随之增加。结果研究发现MUCSAC
在鼻息肉及慢性肥厚性鼻炎下甲粘膜上皮的表达与正常鼻甲粘膜
上皮的表达有显著性差别(p<0 .001)，而且阳性表达细胞基本上
局限于杯状细胞。与此结果类似，Buisine等人曾报道过MUCSAC
mRNA在气管与支气管上皮表面的所有杯状细胞上表达。而Kim等
人的研究与此则略有不同，他们的结果表明在正常后组筛窦粘膜
上皮很少有MUCSACmRNA表达，这与我们的研究结果:MUCSAC在正
常鼻甲粘膜上皮阳性细胞表达率5.78%略有差异，也与以前的研究
不同，目前，为什么会存在这种差别并不十分清楚，Kim等人的解
释是这种不同可能来源于标本的取材，与正常鼻甲粘膜相比，正
吉林大学硕士学位论文
常筛窦粘膜并没有直接接触外界炎性刺激物，基本可以认为是“绝
对”正常的标本。但我们的研究结果与其它的研究结果基本一致，
都表明MUCSAC表达在炎症刺激下上调，这种表达的上调可能与炎
性介质(工L一
The epithelial surface of the respiratory tract is coated with mucus which serve as the selective physical barrier between the extracellular milieu and the cell membrane and play important roles in humidification, lubrication, insulation, entrapment of microorganisms and particulates, and other host defense functions. This secretory blanket consists of two layers: the surface mucus layer and the deeper serous layer in which the cilia function. Airway mucus is composed of water, ions, lung secretions, serum protein transudates, and mucin glycoproteins (mucins). When this mucus blanket is altered in amount or composition, rhinorrhea, nasal obstruction, polyposis, and sinusitis may develop. Mucins are large, heterogeneous, and highly glycosylated proteins which produced by goblet cells in the surface epitheliam and by mucous and serous cells in the submucosal glands. They are the major glycoprotein components of the mucus layer, and play an essential role in the defense of the epithelium against invading pathog
ens. To date, 12 human mucin genes (MUC1-4, MUC5AC, MUC5B, MUC6-8, MUC9, MUCII, and MUC12) have been identified. Among them, MUC2, MUC5AC, MUC5B, MUC6, and MUC7 are known to be secretory mucin genes in the human lower airway, and MUC1, MUC3, MUC4, and MUC12 are thought to be membrane bounded, others are not classified. The molecular analysis, structure and function of mucin have caused extensive and deep investigation since the first cDNA clone had been identified in 1988.
In recent years, the research about mucin in oversea E.N.T.department had increased, however, little had been reported in our country. With immunohistochemistry ABC and HE dyeing , polyps and chronic hypertrophy rhinitis inferior turbinates epithelium were applied to this study. Object
To examine the expression of MUC5AC in human nasal polyp and inferior turbinate epithelium, to evaluate the relationship between MUC5AC and the increased mucous secretions in polyps and chronic hypertrophy rhinitis. Method
27 polyp tissues were obtained from the patients( males 15, females 12; age range 13-56 years, average age 35.5 years ) who were subjected to nasal polypectomy in our department. 19 chronic hypertrophy rhinitis inferior turbinates tissues were obtained from the patients (males 12, females 7; age range 18-48 years, average age 29 years ) who were subjected to inferior turbinectomy in our department. Inferior turbinate mucosa as control group were from 9 patients ( all male, age range 31-46 years, average age 37 years) with rhino-genesis snoring. All cases have not the history of asthma, allergy, aspirin intolerance and not receiving steroids before two weeks. All specimens were fixed with 4% paraformaldehyde, paraffin-embeded and make sections at 5 m thick. The slides were detected with HE staining for routine histopathologic examination, immuno-histochemical staining was performed for observing the expression of
- - 6 - -
1.27 Nasal polyps tissue is morphologically characterized by oedema, goblet cell hyperplasia of the epithelium, is extreme edema and gland proliferation. There are a lot of inflammatory cells in nasal polyp tissues by HE. 19 Inferior turbinates tissues is morphologically characterized by oedema, goblet cell hyperplasia of the epithelium, is edema in lamina propria mucosae, there are lymphocytes and plasmacytes in them.
2. The number of MUC5AC expressing cells in nasal polyps and chronic hypertrophy rhinitis was significantly higner than in normal inferior turbinates(P<0.05), and the expression of MUC5AC cells are mainly concentrate on the goblet cells. Discussion
Mucins are large, heterogeneous, and highly glycosylated proteins which produced by goblet cells in the surface epitheliam and by mucous and serous cells in the submucosal glands. They are the major glycoprotein components of the mucus, they give mucus its viscous and elastic characteristics that enable it to be transported by cilia. The regulations of expression, transcription and post-transcription and the correlation between mucins can affect the quali
引文
1 Bernacki S H, Nelson A L, Abdullah L, et al. Mucin gene expression during differentiation of human airway epithelia in vitro:MUC4 and MUCSB are strongly induced. Am.J. Respir. Cell Mol. Biol, 1999,20:595-604
2 Lapensee L, Paquette Y, Bleau G. Allelic polymorphism and chromosomal localization of the human oviductin gene(MUC9), Fertil Steril, 1997,68:702-708
3 Williams S J, McGuckin M A, Gotley D C, et al. Two novel mucin genes down-regulated in colorectal cancer identified by differential display. Cancer Res, 1999,59:4083-4089
4 Rose MC, Nickola TJ, Voynow JA. Airway mucus obstruction: Mucin glycoproteins, MUC gene regulation and goblet cell hyperplasia. Am J Respir Cell Mol Biol ,2001,25(5):533-7
5 Moniaux N, Escande F, Porchet N, et al. Structural organization and classification of the human mucin genes. Front Biosci, 2001,6:D1192-206
6 Lin J, Ho S, Shekels L ,et al. Mucin gene expression in the rat middle ear: an improved method for RNA harvest. Ann Otol Rhinol Laryngol,1999, 108:762-768
7 Jeffery PK, Li D. Airway mucosa: secretory cells, mucus and mucin genes. Eur Respir J, 1997,10(7) : 1655 - 1662
8 Buisine MP, Devisme L, Copin MC ,et al. Developmental mucin gene expression in the human respiratory tract .Am J Respir Cell Mol Biol .1999 ,20:209-218
9 Kim CH, Song KS, Kim SS, et al. Expression of MUCSACmRNA in the goblet cells of human nasal mucosa .Laryngoscope ,2000 ,110 :2110-211
10 Temann U-A, Prasad B, Gallup MW, et al. A novel role for murine IL-4 in vivo: induction of MUCSAC gene expression and mucin hypersecretion. Am J Respir Cell Mol Biol. 1997, 16:471-478
11 Longphre M, Li D, Gallup M, et al. Allergen-induced IL-9 directly stimulates mucin transcription in respiratory epithelial cells. J Clin Invest 1999, 104:1375-1382
12 Li D, Gallup M, Fan N, et al. Cloning of the amino-terminal and 5' -flanking region of the human MUC5AC mucin gene and transcriptional up-regulation by bacterial exoproducts. J Biol Chem, 1998, 273:6812-6820
2 Lapensee L, Paquette Y, Bleau G. Allelic polymorphism and chromosomal localization of the human oviductin gene(MUC9), Fertil Steril, 1997,68:702-708
3 Williams S J, McGuckin M A, Gotley D C, et al. Two novel mucin genes down-regulated in colorectal cancer identified by differential display. Cancer Res, 1999,59:4083-4089
4 Rose MC, Nickola TJ, Voynow JA. Airway mucus obstruction: Mucin glycoproteins, MUC gene regulation and goblet cell hyperplasia. Am J Respir Cell Mol Biol ,2001,25(5):533-7
5 Moniaux N, Escande F, Porchet N, et al. Structural organization and classification of the human mucin genes. Front Biosci, 2001,6:D1192-206
6 Lin J, Ho S, Shekels L ,et al. Mucin gene expression in the rat middle ear: an improved method for RNA harvest. Ann Otol Rhinol Laryngol,1999, 108:762-768
7 Jeffery PK, Li D. Airway mucosa: secretory cells, mucus and mucin genes. Eur Respir J, 1997,10(7) : 1655 - 1662
8 Buisine MP, Devisme L, Copin MC ,et al. Developmental mucin gene expression in the human respiratory tract .Am J Respir Cell Mol Biol .1999 ,20:209-218
9 Kim CH, Song KS, Kim SS, et al. Expression of MUCSACmRNA in the goblet cells of human nasal mucosa .Laryngoscope ,2000 ,110 :2110-211
10 Temann U-A, Prasad B, Gallup MW, et al. A novel role for murine IL-4 in vivo: induction of MUCSAC gene expression and mucin hypersecretion. Am J Respir Cell Mol Biol. 1997, 16:471-478
11 Longphre M, Li D, Gallup M, et al. Allergen-induced IL-9 directly stimulates mucin transcription in respiratory epithelial cells. J Clin Invest 1999, 104:1375-1382
12 Li D, Gallup M, Fan N, et al. Cloning of the amino-terminal and 5' -flanking region of the human MUC5AC mucin gene and transcriptional up-regulation by bacterial exoproducts. J Biol Chem, 1998, 273:6812-6820