皮质类固醇激素对鼻息肉组织MUC8表达的影响
摘要
杯状细胞和黏膜腺体细胞分泌粘液是鼻腔和鼻窦防御机制的重要组成部分。黏膜上皮表面形成的粘液毯可以俘获外来物质、防止机械损伤、维持黏膜表面的流动性。但是，在慢性鼻窦炎和鼻息肉时，由于粘液分泌过度，粘液的理化特性发生改变，粘性和弹性明显高于正常。这种粘液不适于粘液纤毛系统的传输，造成局部粘液的淤积，为细菌感染创造了条件。
已有研究证明，粘液的粘性和弹性主要取决于粘蛋白含量。Kim等研究鼻息肉上皮细胞粘蛋白成分的变化，发现MUC8 mRNA水平显著增高。Ishinaga等在体外培养的鼻息肉上皮细胞研究发现，地塞米松抑制由细菌内毒素诱导的MUC8基因表达。但是，皮质类固醇激素在人体内是否有相同的作用还未见研究报告。本文研究皮质类固醇激素对人体鼻息肉组织MUC8基因表达的影响。
一、材料和方法
1．实验材料
收集浙江大学医学院附属二院耳鼻咽喉科2002年6月～2003年1月期间住院手术的部分慢性鼻窦炎鼻息肉病例35例。其中，术前地塞米松治疗病例17例，男性12例，女性5例，年龄16～60岁，平均38.9±14.5岁。根据中华医学会耳鼻咽喉科学分会和中华耳鼻咽喉科杂志编辑委员会1997年在海口组织的鼻科学会议制定的慢性鼻窦炎鼻息肉临床分型分期及内窥镜鼻窦手术疗效评定标准，Ⅱ型2期8例，Ⅱ型3期8例，Ⅲ型1例。全身应用地塞米松4～7天，平均4.7±1.4天，其中10例同时给予鼻腔
浙江大学硕士学位论文
局部皮质类固醇激素喷剂。另18例术前一个月无类固醉激素用药史(包括鼻腔局部喷
用和全身应用)，作为对照，男性14例，女性4例，年龄14~58岁，平均年龄36.3士
15.2岁。临床分期，n型1期4例，n型2期9例，11型3期4例，m型1例。两组
病例均在鼻内镜手术时，取鼻息肉标本，经生理盐水漂洗，去除血迹后，立即装入
Eppendof管，置于一80℃冰箱贮藏备测。
2.实验方法
RNA提取:冰冻鼻息肉组织约50一100mg液氮中碾碎后与lml Trizo!试剂混匀，
加0.2ml氯仿，用力振荡，4℃1 2000 rpm离心15分钟。再加异丙醇沉淀RNA。75%
乙醇漂洗，OEPC水溶解。
cDNA的合成:取8 09总RNA样品，70℃水浴smin，立即冰浴冷却，依次加入5
X逆转录反应缓冲液5协l，10mM dNTps 1.25pl，sopmololigdT引物lpl，Zoou/。-
逆转录酶(Moloney murine leukemiavirus，M一MLv)一p一，4oU/p一Rnasin 0.65“一。最
后加DEPC水至总体积25 pl。42oC温浴60min，95℃水浴smin灭活M一MLV酶，得
到cDNA。
聚合酶链反应(Po一ymerase ehain reaction，PeR)
MUCS cDNA扩增:以上述cONA为模板，用MUCS引物进行PCR，循环35个周
期。引物根据基因库报导MUCS mRNA序列(Genebank aceession numbe:u一4353)设
计如下:上游链5’一ACAGGGTTTCTCCTCATTG一3’;下游链5’
一CGTTTATTCCAGCACTGTTC一3’。
内参照p一肌动蛋白(p一actin) cDNA扩增:同样以上述cONA为模板，以p一actin
引物进行PCR。引物序列:上游链5’一TTCCAGCCTTCCTTCCTGGG一3’;下游链5
一TTGCGCTCAGGAGGAGCAAI，一3’。
PCR反应条件均为94oC预变性5 min，94oC变性305，55oC退火305，72oC延伸30
s，循环35个周期。然后72oC延伸1 0 min。
PCR产物定量
取10叫PCR产物，2%琼脂糖凝胶电泳，用数字成像系统进行阳性条带的密度扫
描，记录扫描值。根据扫描值计算mRNA指数(RI):
RI=MUCS mRNART一PCR产物扫描值/p一actin mRNART一PCR产物扫描值
RI代表组织中MUCS mRNA相对水平。
浙江大学硕士学位论文
二、结果
‘矛
总RNA的提取
将提取的总RNA在紫外分光度仪260nm/2 80nm波长下测定吸光度(A)，各样品的
A 260/A 280均为1.8以上。经RNA电泳，可见明显的185和285两条带，说明提取的
RNA纯度符合要求并且没有降解。
RT一PCR
Mucs基因扩增片段长度为239bP，在电泳图中可见扩增产物的条带位置落在
25obp的Marker(分子量标记)条带稍前方，与预期目的片段大小相符，特异性高。鼻息
肉组织术前用药组和对照组样本均有MUCS表达。作为内参照标准的p一actin(223bp)
在两组样本表达恒定，见附图。
附图鼻息肉组织中MUCS mRNA和p一actin mRNART一PCR电泳图
A为MUCS;B为p一aetin。M为oNA分子量标记物;l~4为术前用药
组病例标本，5~8为对照组病例标本。
术前用药组病例鼻息肉组织MUCS mRNA指数平均为0.71士0.34;对照组病例鼻
息肉组织RI值平均为1.12士0.26。前者较后者为低。两组间的差异经I检验，有统计学
上的显著意义(P<0.01)，见附表。
浙江大学硕士学位论文
附表
两组鼻息肉组织MUCS mRNA指数
分组
例数
Rl值(又士50)
术前用药组
对照组
l7
18
0 .71士0.34
1 .12士0.26
三、讨论和结论
粘蛋白是鼻腔和鼻窦粘膜分泌的粘液中的主要大分子成分，是高度糖基化的蛋白
质，决定粘液的粘弹性。至今已确认13种人类粘蛋白基因，包括MUCI一4，MUCSAC，
MUCSB，MUC6-9，MUCll一13。这些基因编码呼吸道、胃肠道及泌尿生殖道粘蛋白的
核心蛋白。其中，Mue5AC、Mue5B和MUCS主要表达
It is an important defense mechanism of nasal cavities and paranasal sinuses for goblet cells and submucosal gland cells to secret mucus. The mucus forms a layer of blanket on the superfacial surface of the epithelia of rhinosinus mucosa, which can entrap materials, prevent mechanical injury and maintain fluidity. However, in circumstances of chronic sinusitis and nasal polyps, the mucus may be produced excessively and its rheological properties changed. This makes it unsuitable to be transported by mucus-cilia system and results in mucus stasis, and, in turn, becomes a source of bacterial infection.
The mucin concentration of the mucus has been shown to be the most important determinant of the viscosity and elasticity. Kim et al found MUC8 mRNA level increased clearly in the cultured epithelial cells of nasal polyps, compared with that in the epithelial cells of inferior turbinates. Ishinaga et al demonstrated dexamethasone suppressed MUC8 mRNA expression, which could be upregulated by lipopolysaccharide (LPS) in cultured human epithelial cells of nasal polyps. However, still not available is the data of investigation as to effects of corticosteroids on mucin gene expression of human nasal polyp tissue in vivo. The purpose of the present study was to elucidate the effects of corticosteroids on MUC8 gene expression of human nasal polyp tissue.
Materials and methods
1. Materials
35 patients were included in the present study. They were some patients with chronic sinusitis and nasal polyps who received surgery in Department of Otorhinolaryngology of Second Affiliated Hospital of Zhejiang University between June 2002 and January 2003. Seventeen patients received corticosteroids therapy before surgery, aging from 16 to 60 ( average age 38.9 14.5 ), among them, twelve cases were male and 5 cases were female. We applied the staging system of chronic sinusitis with or without polyps and criteria of evaluation for therapeutic response to endoscopic sinus surgery, which was drawn up during National Conference of Rhinology at Haikou (1997), organized by Otorhinolaryngologic Branch Committee of Chinese Medical Association and Editorial Board of Chinese Journal of Otorhinolaryngology. Accordingly, eight cases were classified as type II stage 2, 8 as type II stage 3, and 1 as type III. These 17 patients were administered dexamethasone systemically for 4~7 days (average 4.7 1.4 days) before surgery. Ten of them were additionally received intranasal spray of corticosteroids. We made sure the other 18 patients, as control, didn't receive any kinds of corticosteroids therapy for 30 days before surgery, neither systemically nor topically. Among controls, 14 cases were male and 4 cases were female, aging from 14 to 58 (average age 36.3 15.2 ). They were composed of 4 cases of type II stage 1, 9 cases of type II stage 2, 4 cases of type II stage 3, and 1 case of type III. The samples of nasal polyp tissue were obtained at the time of surgery and immediately stored at 80
2. Methods
RNA Isolation
Frozen tissue was homogenized in 1 ml of Trizol reagent. 0.2 ml of chloroform was then added to each sample. After vigorous vibration and centrifugation, RNA was present in the aqueous phase. After mixing with isopropyl alcohol, the RNA precipitate was washed with 75% ethanol. Then, the pellets were dissolved in DEPC water. The isolated RNA concentration and purity were identified through measurement of OD260 and OD280.
Reverse Transcription of RNA
8 g RNA pellets of each sample was taken and denatured at 70 for 5 minutes. After
chilled on ice, the sample was added with 5 15 reverse transcriptase buffer, 1.25 1 10mM dNTPs, 1 1 50pmol Oligo dT, 1 1 200U/ 1 moloney murine leukemiavirus (M-MLV), 0.65 1 40U/ 1 RNasin, and supplemented with DEPC water to an end volume of 25 1. The mixture was kept at 42 for 60 minutes and inactivated at 95for 5 minutes. The generated cDNA samples were stored at - 20 until use. Polymerase chain reaction, PCR
Amplification of MUC8 cDNA: The produced cDNA as above was amplified
已有研究证明，粘液的粘性和弹性主要取决于粘蛋白含量。Kim等研究鼻息肉上皮细胞粘蛋白成分的变化，发现MUC8 mRNA水平显著增高。Ishinaga等在体外培养的鼻息肉上皮细胞研究发现，地塞米松抑制由细菌内毒素诱导的MUC8基因表达。但是，皮质类固醇激素在人体内是否有相同的作用还未见研究报告。本文研究皮质类固醇激素对人体鼻息肉组织MUC8基因表达的影响。
一、材料和方法
1．实验材料
收集浙江大学医学院附属二院耳鼻咽喉科2002年6月～2003年1月期间住院手术的部分慢性鼻窦炎鼻息肉病例35例。其中，术前地塞米松治疗病例17例，男性12例，女性5例，年龄16～60岁，平均38.9±14.5岁。根据中华医学会耳鼻咽喉科学分会和中华耳鼻咽喉科杂志编辑委员会1997年在海口组织的鼻科学会议制定的慢性鼻窦炎鼻息肉临床分型分期及内窥镜鼻窦手术疗效评定标准，Ⅱ型2期8例，Ⅱ型3期8例，Ⅲ型1例。全身应用地塞米松4～7天，平均4.7±1.4天，其中10例同时给予鼻腔
浙江大学硕士学位论文
局部皮质类固醇激素喷剂。另18例术前一个月无类固醉激素用药史(包括鼻腔局部喷
用和全身应用)，作为对照，男性14例，女性4例，年龄14~58岁，平均年龄36.3士
15.2岁。临床分期，n型1期4例，n型2期9例，11型3期4例，m型1例。两组
病例均在鼻内镜手术时，取鼻息肉标本，经生理盐水漂洗，去除血迹后，立即装入
Eppendof管，置于一80℃冰箱贮藏备测。
2.实验方法
RNA提取:冰冻鼻息肉组织约50一100mg液氮中碾碎后与lml Trizo!试剂混匀，
加0.2ml氯仿，用力振荡，4℃1 2000 rpm离心15分钟。再加异丙醇沉淀RNA。75%
乙醇漂洗，OEPC水溶解。
cDNA的合成:取8 09总RNA样品，70℃水浴smin，立即冰浴冷却，依次加入5
X逆转录反应缓冲液5协l，10mM dNTps 1.25pl，sopmololigdT引物lpl，Zoou/。-
逆转录酶(Moloney murine leukemiavirus，M一MLv)一p一，4oU/p一Rnasin 0.65“一。最
后加DEPC水至总体积25 pl。42oC温浴60min，95℃水浴smin灭活M一MLV酶，得
到cDNA。
聚合酶链反应(Po一ymerase ehain reaction，PeR)
MUCS cDNA扩增:以上述cONA为模板，用MUCS引物进行PCR，循环35个周
期。引物根据基因库报导MUCS mRNA序列(Genebank aceession numbe:u一4353)设
计如下:上游链5’一ACAGGGTTTCTCCTCATTG一3’;下游链5’
一CGTTTATTCCAGCACTGTTC一3’。
内参照p一肌动蛋白(p一actin) cDNA扩增:同样以上述cONA为模板，以p一actin
引物进行PCR。引物序列:上游链5’一TTCCAGCCTTCCTTCCTGGG一3’;下游链5
一TTGCGCTCAGGAGGAGCAAI，一3’。
PCR反应条件均为94oC预变性5 min，94oC变性305，55oC退火305，72oC延伸30
s，循环35个周期。然后72oC延伸1 0 min。
PCR产物定量
取10叫PCR产物，2%琼脂糖凝胶电泳，用数字成像系统进行阳性条带的密度扫
描，记录扫描值。根据扫描值计算mRNA指数(RI):
RI=MUCS mRNART一PCR产物扫描值/p一actin mRNART一PCR产物扫描值
RI代表组织中MUCS mRNA相对水平。
浙江大学硕士学位论文
二、结果
‘矛
总RNA的提取
将提取的总RNA在紫外分光度仪260nm/2 80nm波长下测定吸光度(A)，各样品的
A 260/A 280均为1.8以上。经RNA电泳，可见明显的185和285两条带，说明提取的
RNA纯度符合要求并且没有降解。
RT一PCR
Mucs基因扩增片段长度为239bP，在电泳图中可见扩增产物的条带位置落在
25obp的Marker(分子量标记)条带稍前方，与预期目的片段大小相符，特异性高。鼻息
肉组织术前用药组和对照组样本均有MUCS表达。作为内参照标准的p一actin(223bp)
在两组样本表达恒定，见附图。
附图鼻息肉组织中MUCS mRNA和p一actin mRNART一PCR电泳图
A为MUCS;B为p一aetin。M为oNA分子量标记物;l~4为术前用药
组病例标本，5~8为对照组病例标本。
术前用药组病例鼻息肉组织MUCS mRNA指数平均为0.71士0.34;对照组病例鼻
息肉组织RI值平均为1.12士0.26。前者较后者为低。两组间的差异经I检验，有统计学
上的显著意义(P<0.01)，见附表。
浙江大学硕士学位论文
附表
两组鼻息肉组织MUCS mRNA指数
分组
例数
Rl值(又士50)
术前用药组
对照组
l7
18
0 .71士0.34
1 .12士0.26
三、讨论和结论
粘蛋白是鼻腔和鼻窦粘膜分泌的粘液中的主要大分子成分，是高度糖基化的蛋白
质，决定粘液的粘弹性。至今已确认13种人类粘蛋白基因，包括MUCI一4，MUCSAC，
MUCSB，MUC6-9，MUCll一13。这些基因编码呼吸道、胃肠道及泌尿生殖道粘蛋白的
核心蛋白。其中，Mue5AC、Mue5B和MUCS主要表达
It is an important defense mechanism of nasal cavities and paranasal sinuses for goblet cells and submucosal gland cells to secret mucus. The mucus forms a layer of blanket on the superfacial surface of the epithelia of rhinosinus mucosa, which can entrap materials, prevent mechanical injury and maintain fluidity. However, in circumstances of chronic sinusitis and nasal polyps, the mucus may be produced excessively and its rheological properties changed. This makes it unsuitable to be transported by mucus-cilia system and results in mucus stasis, and, in turn, becomes a source of bacterial infection.
The mucin concentration of the mucus has been shown to be the most important determinant of the viscosity and elasticity. Kim et al found MUC8 mRNA level increased clearly in the cultured epithelial cells of nasal polyps, compared with that in the epithelial cells of inferior turbinates. Ishinaga et al demonstrated dexamethasone suppressed MUC8 mRNA expression, which could be upregulated by lipopolysaccharide (LPS) in cultured human epithelial cells of nasal polyps. However, still not available is the data of investigation as to effects of corticosteroids on mucin gene expression of human nasal polyp tissue in vivo. The purpose of the present study was to elucidate the effects of corticosteroids on MUC8 gene expression of human nasal polyp tissue.
Materials and methods
1. Materials
35 patients were included in the present study. They were some patients with chronic sinusitis and nasal polyps who received surgery in Department of Otorhinolaryngology of Second Affiliated Hospital of Zhejiang University between June 2002 and January 2003. Seventeen patients received corticosteroids therapy before surgery, aging from 16 to 60 ( average age 38.9 14.5 ), among them, twelve cases were male and 5 cases were female. We applied the staging system of chronic sinusitis with or without polyps and criteria of evaluation for therapeutic response to endoscopic sinus surgery, which was drawn up during National Conference of Rhinology at Haikou (1997), organized by Otorhinolaryngologic Branch Committee of Chinese Medical Association and Editorial Board of Chinese Journal of Otorhinolaryngology. Accordingly, eight cases were classified as type II stage 2, 8 as type II stage 3, and 1 as type III. These 17 patients were administered dexamethasone systemically for 4~7 days (average 4.7 1.4 days) before surgery. Ten of them were additionally received intranasal spray of corticosteroids. We made sure the other 18 patients, as control, didn't receive any kinds of corticosteroids therapy for 30 days before surgery, neither systemically nor topically. Among controls, 14 cases were male and 4 cases were female, aging from 14 to 58 (average age 36.3 15.2 ). They were composed of 4 cases of type II stage 1, 9 cases of type II stage 2, 4 cases of type II stage 3, and 1 case of type III. The samples of nasal polyp tissue were obtained at the time of surgery and immediately stored at 80
2. Methods
RNA Isolation
Frozen tissue was homogenized in 1 ml of Trizol reagent. 0.2 ml of chloroform was then added to each sample. After vigorous vibration and centrifugation, RNA was present in the aqueous phase. After mixing with isopropyl alcohol, the RNA precipitate was washed with 75% ethanol. Then, the pellets were dissolved in DEPC water. The isolated RNA concentration and purity were identified through measurement of OD260 and OD280.
Reverse Transcription of RNA
8 g RNA pellets of each sample was taken and denatured at 70 for 5 minutes. After
chilled on ice, the sample was added with 5 15 reverse transcriptase buffer, 1.25 1 10mM dNTPs, 1 1 50pmol Oligo dT, 1 1 200U/ 1 moloney murine leukemiavirus (M-MLV), 0.65 1 40U/ 1 RNasin, and supplemented with DEPC water to an end volume of 25 1. The mixture was kept at 42 for 60 minutes and inactivated at 95for 5 minutes. The generated cDNA samples were stored at - 20 until use. Polymerase chain reaction, PCR
Amplification of MUC8 cDNA: The produced cDNA as above was amplified
引文
1. Majima Y, Hirata K, Takeuchi K, et al. Effects of orally administered drugs on dynamic viscoelasticity of human nasal mucus. Am Rev Respir Dis, 1990, 141:79-83
2. Majima Y, Harada T, Shimizu T, et al. Effect of biochemical components on theologic properties of nasal mucus in chronic sinusitis. Am J Respir Crit Care Med, 1999, 160: 421-426
3. Kim SS, Kim KS, Lee JG, et al. Level of intracellular protein and messenger RNA of mucin and lysozyme in normal human nasal and polyp epithelium, laryngoscope, 2000, 110:276-280
4. Ishinaga H, Takeuchi K, Kishioka C, et al. Effects of dexamethasone on mucin gene expression in cultured human nasal epithelial cells. Laryngoscope, 2002, 112:1436-1440
5. Vinall LE, Fowler JC, .lones AL, et ai. Polymorphism of human mucin genes in chest disease: possible significance of MUC2. Am J Respir Cell Mol Biol, 2000, 23:678-686
6. Wiilams SJ, Wreschner DH, Tran M, et ai. MUC13-a novel human cell surface mucin expressed by epithelial and hemopoietic cells. J Biol Chem, 2001, 276:18327-36
7. Randell SH, Liu JY, Ferriola PC, et al. Mucin production by SPOCI cells: an immortalized rat tracheal epithelial cell line. Am J Respir Cell Mol Biol 1996, 14: 146-154
8. Jung HH, Lee JH, Kim YK, et al. Expression of mucin genes in chronic ethmoiditis. Am J Rhinol, 2000, 14:163-170
9. Seong JK, Koo JS, Lee W J, et al. Upregulation of MUC8 and downregulation of MUC5AC by inflammatory mediators in human nasal polyps and cultured nasal epithelium. Acta Otolaryngol, 2002, 122:401-407
10. Kai H, Yoshitake K, Hisatsune A, et al. Dexamethasone suppresses mucus production
and MUC2 and MUC5AC gene expression by NCI-H292 cells. Am J Physiol, 1996, 271(Lung Cell: Mol Physiol 15): L484 - L488
11. Sachio T, Katsuhiro H, Tsutomu U, et al. Nuclear factor-kappa B activation in the nasal polyp epithelium: relation to local cytokine gene expression. Laryngoscope, 2002, 112: 53-58
2. Majima Y, Harada T, Shimizu T, et al. Effect of biochemical components on theologic properties of nasal mucus in chronic sinusitis. Am J Respir Crit Care Med, 1999, 160: 421-426
3. Kim SS, Kim KS, Lee JG, et al. Level of intracellular protein and messenger RNA of mucin and lysozyme in normal human nasal and polyp epithelium, laryngoscope, 2000, 110:276-280
4. Ishinaga H, Takeuchi K, Kishioka C, et al. Effects of dexamethasone on mucin gene expression in cultured human nasal epithelial cells. Laryngoscope, 2002, 112:1436-1440
5. Vinall LE, Fowler JC, .lones AL, et ai. Polymorphism of human mucin genes in chest disease: possible significance of MUC2. Am J Respir Cell Mol Biol, 2000, 23:678-686
6. Wiilams SJ, Wreschner DH, Tran M, et ai. MUC13-a novel human cell surface mucin expressed by epithelial and hemopoietic cells. J Biol Chem, 2001, 276:18327-36
7. Randell SH, Liu JY, Ferriola PC, et al. Mucin production by SPOCI cells: an immortalized rat tracheal epithelial cell line. Am J Respir Cell Mol Biol 1996, 14: 146-154
8. Jung HH, Lee JH, Kim YK, et al. Expression of mucin genes in chronic ethmoiditis. Am J Rhinol, 2000, 14:163-170
9. Seong JK, Koo JS, Lee W J, et al. Upregulation of MUC8 and downregulation of MUC5AC by inflammatory mediators in human nasal polyps and cultured nasal epithelium. Acta Otolaryngol, 2002, 122:401-407
10. Kai H, Yoshitake K, Hisatsune A, et al. Dexamethasone suppresses mucus production
and MUC2 and MUC5AC gene expression by NCI-H292 cells. Am J Physiol, 1996, 271(Lung Cell: Mol Physiol 15): L484 - L488
11. Sachio T, Katsuhiro H, Tsutomu U, et al. Nuclear factor-kappa B activation in the nasal polyp epithelium: relation to local cytokine gene expression. Laryngoscope, 2002, 112: 53-58