腭咽软组织中VEGF及受体在OSAHS发病机理作用的研究
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摘要
目的：血管内皮生长因子又称血管通透性因子，具有促进血管内皮细胞增殖，刺激体内新生血管生成，增加血管壁通透性及维持血管正常状态和完整性的功能。OSAHS是以睡眠时反复发作上呼吸道塌陷，导致呼吸暂停或低通气为特征的疾病。其中咽腔狭窄在OSAHS发病过程中起非常重要的作用。本研究目的在于观察腭咽软组织中VEGF及受体的表达特征与OSAHS病情程度和临床指标的相关性，探讨缺氧、VEGF、咽狭窄这一恶性循环过程中，VEGF的作用机制。
方法：采用酶联免疫吸附法测定32例OSAHS患者和30例非OSAHS者，睡前和晨起时血清VEGF浓度。用美国伟康公司生产的RHK-5500多导睡眠监护仪（PSG）对所有受试者进行整夜的睡眠监测，获得相关临床指标，并与血清VEGF浓度进行分析。用免疫组织化学方法分析VEGF及受体（flt－1及KDR）在腭咽软组织中表达特征及微血管密度变化。应用反转录PCR技术测腭咽软组织VEGFmRNA含量。
结果：
1. 血清VEGF浓度变化。OSAHS患者晨起时血清VEFG水平明显高于对照组（晨起时：OSAHS组为243.59±47.65pg/ml,对照组为124.83±3.74pg/ml，p<0.01；睡前时：OSAHS组为161.31±21.73pg/ml,对照组为124.5±2.97pg/ml，p>0.05）。
2. 晨起时血清VEGF浓度与OSAHS临床指标相关性分析。OSAHS患者晨起时血清VEGF水平与夜间减氧饱和度时间占总睡眠时间的百分比正相关，与夜间平均血氧饱和度、最低血氧饱和度负相关，与睡眠呼吸暂停低通气指数正相关，与体块指数、颈围、腰髋比无明显相关性。
① 晨起血清VEGF 与 ODT/TST正相关（R=0.893，P<0.01），直线回归方程为： Y=103.387＋2.922X。
② 晨起血清VEGF 和夜间平均SO2负相关（R=-0.754，P<0.01），直线回归方程为： Y=1880.796-18.128X。
③ 晨起血清VEGF和 AHI 正相关（R=0.944，P<0.01），直线回归方程为： Y=113.704＋3.404X。
④ 晨起血清VEGF 和最低SO2负相关（R=-0.734，P<0.01），直线回归方程为： Y=473.268-3.078X。
⑤ 晨起血清VEGF 和BMI、WHR、颈围无明显相关性（P>0.01）
3. 腭咽软组织免疫组织化学的研究
① 腭咽软组织病理学变化：OSAHS患者腭咽软组织粘膜鳞状上皮下组织血管扩张充血，结缔组织水肿。
② 腭咽软组织VEGF的表达：OSAHS组：腭咽软组织粘膜鳞状上皮基底层及棘层细胞胞浆强阳性表达，腺管上皮细胞强阳性表达或局灶强阳性表达，血管内皮细胞部分阳性表达。对照组腭咽软组织粘膜鳞状上皮基底层及棘层细胞胞浆阳性或弱阳性表达，腺导管上皮细胞弱阳性表达，血管内皮细胞未见阳性表达。
③ 腭咽软组织flt－1的表达。OSAHS组：横纹肌细胞弥漫强阳性表达，粘膜鳞状上皮中外层细胞强阳性表达。对照组：横纹肌细胞弥蔓阳性表达，粘膜鳞状上皮中外层细胞阳性表达。
④ 腭咽软组织KDR的表达KDR表达。OSAHS组：横纹肌细胞弥蔓强阳性表达，或弥蔓中度阳性表达或局灶强阳性表达，腺导管上皮细胞强阳性表达，部分间质细胞阳性表达，粘膜鳞状上皮中外层细胞局灶强阳性表达。对照组：横纹肌细胞、腺导管上皮细胞和粘膜鳞状上皮中外层细胞均呈现阳性或弱阳性表达。
4. 腭咽软组织微血管密度。OSAHS组患者腭咽软组织微血管密度明显高于对照组（OSAHS组：MVD为12.13±2.85；对照组：MVD为3.65±2.42，p<0.01），提示OSAHS患者存在腭咽软组织血管内皮细胞增生，血管增殖。
5. 腭咽软组织VEGFmRNA含量。通过RT-PCR试验，观察到OSAHS组患者腭咽软组织VEGFmRNA含量较对照组高（OSAHS组为1.18±0.35，对照组为0.72±0.14，p<0.01）。
结论： OSAHS患者晨起时血清VEGF水平明显高于对照组，并且OSAHS患者组睡前血清VEGF浓度明显低于晨起时的浓度。通过免疫组化研究发现，VEGF及其受体flt－1、KDR在OSAHS患者腭咽软组织中的表达明显高于对照组，同时OSAHS患者组腭咽软组织中MVD显著高于对照组，提示OSAHS患者存在腭咽软组织微血管增生，腭咽软组织水肿。细胞因子在OSAHS发病中起一定的作用，但其具体机制尚有待进一步研究。
Objective: Obstructive sleep apnea hypopnea syndrome(OSAHS) is a common,but underdiagnosed disorder，which potentially is fatal.It is characterized by repetitive episodes of complete or partial upper airway obstruction leading to hypoxia and hypercapmia. Vascular endothelial growth factor (VEGF) is a hypoxia-sensitive glycoprotein stimulating neoangiogenesis.The expression of the VEGF gene is basically stimulated by hypoxia through mediation of hypoxia-inducible factor(HIF).VEGF is a potent inducer of endothelial cell growth and angiogenesis and a secreted peptide that acts specifically on vascular endothelial cells through two different high-affinity receptors,designated KDR/flk-1 and flt-1.The purpose of this research is to explore the localization of VEGF and it mRNA in the pharyngeal tissue of patients with OSAHS. In order to get new therapeutic strategies and the relationship between VEGF and degree of narrow pharyngral airway is researched.
Materials and Method: The study consisted of 32 patients with OSAHS treated with UPPP and 30 healthy subjects as controls. All of them were checked by polysomnograph individually.Immunohistochemistry on paraffin sections was performed with anti-human factor Ⅷ antibody to study the microvascular density (MVD),and with antibodies to VEGF,Flt-1 and KDR to investigate the expression of these three proteins in the pharyngeal tissue.The levels of VEGF in serum in all subjects were tested by ELISA.
Results:
1. The serum VEGF （at 7:00 AM）in OSAHS patients is higher than that in control group(at 7:00 AM：OSAHS patients243.59±47.65pg/ml，control group124.83±3.74pg/ml， p<0.01；at 23:00 PM：OSAHS patients161.31±21.73pg/ml，control group124.5±2.97 pg/ml，p>0.05)
① The content of serum VEGF and ODT/TST are in the postive linear correlation （R=0.893，P<0.01），the linear regression equation is Y=103.387＋2.922X。
② The content of serum VEGF and the night mean SO2 are in the negative linear correlation （R=-0.754，P<0.01），the linear regression equation is Y=1880.796-18.128X。
③ The content of serum VEGF and AHI are in the postive linear correlation
（R=0.944，P<0.01），the linear regression equation is Y=113.704＋3.404X。
④ The content of serum VEGF and the lowest SO2 are in the negative linear correlation （R=-0.734，P<0.01），the linear regression equation is Y=473.268-3.078X。
⑤ The content of serum VEGF and BMI 、neck circumference、WHR have not correlative（P>0.01）
2. The levels of serum VEGF in OSAHS patients are different in night and in moring，which in moring is higher than that in night。
3. The histological and immunohistochemical feature：
① Pathological changes in pharynx tissues of OSAHS patients ：Subepithelia blood vessels expand and hyperemia，soft tissues edema.
② Immunohistochemical changes about VEGF :There are a powerful postive expression in base and spinae stratum cells in mucosa squamous epithelium and epithelium of glandular duct of OSAHS ，a feeble postive expression in vascular endothelial cell .However，there are feeble postive expression in base and spinae stratum cells in mucosa squamous epithelium and epithelium of glandular duct of control group，a negative expression in vascular endothelial cell.
③ Immunohistochemical changes about flt-1：There are a powerful postive expression in epithelium of glandular duct and in striated muscule cells of OSAHS.but there are feeble postive expression in that of control group.
④ Immunohistochemical changes about KDR：There are a powerful postive expression in extrastratum cells in mucosa squamous epithelium and in striated muscule cells and epithelium of glandular duct of OSAHS patients，a feeble postive expression in some interstitial cells. However，there are feeble postive expression extrastratum cells in mucosa squamous epithelium and in striated muscule cells and epithelium of glandular duct of control group，a negative expression in interstitial cells.
4. The MVD change of pharynx tissues:the MVD of OSAHS patient is higher than that of control group(OSAHS
方法：采用酶联免疫吸附法测定32例OSAHS患者和30例非OSAHS者，睡前和晨起时血清VEGF浓度。用美国伟康公司生产的RHK-5500多导睡眠监护仪（PSG）对所有受试者进行整夜的睡眠监测，获得相关临床指标，并与血清VEGF浓度进行分析。用免疫组织化学方法分析VEGF及受体（flt－1及KDR）在腭咽软组织中表达特征及微血管密度变化。应用反转录PCR技术测腭咽软组织VEGFmRNA含量。
结果：
1. 血清VEGF浓度变化。OSAHS患者晨起时血清VEFG水平明显高于对照组（晨起时：OSAHS组为243.59±47.65pg/ml,对照组为124.83±3.74pg/ml，p<0.01；睡前时：OSAHS组为161.31±21.73pg/ml,对照组为124.5±2.97pg/ml，p>0.05）。
2. 晨起时血清VEGF浓度与OSAHS临床指标相关性分析。OSAHS患者晨起时血清VEGF水平与夜间减氧饱和度时间占总睡眠时间的百分比正相关，与夜间平均血氧饱和度、最低血氧饱和度负相关，与睡眠呼吸暂停低通气指数正相关，与体块指数、颈围、腰髋比无明显相关性。
① 晨起血清VEGF 与 ODT/TST正相关（R=0.893，P<0.01），直线回归方程为： Y=103.387＋2.922X。
② 晨起血清VEGF 和夜间平均SO2负相关（R=-0.754，P<0.01），直线回归方程为： Y=1880.796-18.128X。
③ 晨起血清VEGF和 AHI 正相关（R=0.944，P<0.01），直线回归方程为： Y=113.704＋3.404X。
④ 晨起血清VEGF 和最低SO2负相关（R=-0.734，P<0.01），直线回归方程为： Y=473.268-3.078X。
⑤ 晨起血清VEGF 和BMI、WHR、颈围无明显相关性（P>0.01）
3. 腭咽软组织免疫组织化学的研究
① 腭咽软组织病理学变化：OSAHS患者腭咽软组织粘膜鳞状上皮下组织血管扩张充血，结缔组织水肿。
② 腭咽软组织VEGF的表达：OSAHS组：腭咽软组织粘膜鳞状上皮基底层及棘层细胞胞浆强阳性表达，腺管上皮细胞强阳性表达或局灶强阳性表达，血管内皮细胞部分阳性表达。对照组腭咽软组织粘膜鳞状上皮基底层及棘层细胞胞浆阳性或弱阳性表达，腺导管上皮细胞弱阳性表达，血管内皮细胞未见阳性表达。
③ 腭咽软组织flt－1的表达。OSAHS组：横纹肌细胞弥漫强阳性表达，粘膜鳞状上皮中外层细胞强阳性表达。对照组：横纹肌细胞弥蔓阳性表达，粘膜鳞状上皮中外层细胞阳性表达。
④ 腭咽软组织KDR的表达KDR表达。OSAHS组：横纹肌细胞弥蔓强阳性表达，或弥蔓中度阳性表达或局灶强阳性表达，腺导管上皮细胞强阳性表达，部分间质细胞阳性表达，粘膜鳞状上皮中外层细胞局灶强阳性表达。对照组：横纹肌细胞、腺导管上皮细胞和粘膜鳞状上皮中外层细胞均呈现阳性或弱阳性表达。
4. 腭咽软组织微血管密度。OSAHS组患者腭咽软组织微血管密度明显高于对照组（OSAHS组：MVD为12.13±2.85；对照组：MVD为3.65±2.42，p<0.01），提示OSAHS患者存在腭咽软组织血管内皮细胞增生，血管增殖。
5. 腭咽软组织VEGFmRNA含量。通过RT-PCR试验，观察到OSAHS组患者腭咽软组织VEGFmRNA含量较对照组高（OSAHS组为1.18±0.35，对照组为0.72±0.14，p<0.01）。
结论： OSAHS患者晨起时血清VEGF水平明显高于对照组，并且OSAHS患者组睡前血清VEGF浓度明显低于晨起时的浓度。通过免疫组化研究发现，VEGF及其受体flt－1、KDR在OSAHS患者腭咽软组织中的表达明显高于对照组，同时OSAHS患者组腭咽软组织中MVD显著高于对照组，提示OSAHS患者存在腭咽软组织微血管增生，腭咽软组织水肿。细胞因子在OSAHS发病中起一定的作用，但其具体机制尚有待进一步研究。
Objective: Obstructive sleep apnea hypopnea syndrome(OSAHS) is a common,but underdiagnosed disorder，which potentially is fatal.It is characterized by repetitive episodes of complete or partial upper airway obstruction leading to hypoxia and hypercapmia. Vascular endothelial growth factor (VEGF) is a hypoxia-sensitive glycoprotein stimulating neoangiogenesis.The expression of the VEGF gene is basically stimulated by hypoxia through mediation of hypoxia-inducible factor(HIF).VEGF is a potent inducer of endothelial cell growth and angiogenesis and a secreted peptide that acts specifically on vascular endothelial cells through two different high-affinity receptors,designated KDR/flk-1 and flt-1.The purpose of this research is to explore the localization of VEGF and it mRNA in the pharyngeal tissue of patients with OSAHS. In order to get new therapeutic strategies and the relationship between VEGF and degree of narrow pharyngral airway is researched.
Materials and Method: The study consisted of 32 patients with OSAHS treated with UPPP and 30 healthy subjects as controls. All of them were checked by polysomnograph individually.Immunohistochemistry on paraffin sections was performed with anti-human factor Ⅷ antibody to study the microvascular density (MVD),and with antibodies to VEGF,Flt-1 and KDR to investigate the expression of these three proteins in the pharyngeal tissue.The levels of VEGF in serum in all subjects were tested by ELISA.
Results:
1. The serum VEGF （at 7:00 AM）in OSAHS patients is higher than that in control group(at 7:00 AM：OSAHS patients243.59±47.65pg/ml，control group124.83±3.74pg/ml， p<0.01；at 23:00 PM：OSAHS patients161.31±21.73pg/ml，control group124.5±2.97 pg/ml，p>0.05)
① The content of serum VEGF and ODT/TST are in the postive linear correlation （R=0.893，P<0.01），the linear regression equation is Y=103.387＋2.922X。
② The content of serum VEGF and the night mean SO2 are in the negative linear correlation （R=-0.754，P<0.01），the linear regression equation is Y=1880.796-18.128X。
③ The content of serum VEGF and AHI are in the postive linear correlation
（R=0.944，P<0.01），the linear regression equation is Y=113.704＋3.404X。
④ The content of serum VEGF and the lowest SO2 are in the negative linear correlation （R=-0.734，P<0.01），the linear regression equation is Y=473.268-3.078X。
⑤ The content of serum VEGF and BMI 、neck circumference、WHR have not correlative（P>0.01）
2. The levels of serum VEGF in OSAHS patients are different in night and in moring，which in moring is higher than that in night。
3. The histological and immunohistochemical feature：
① Pathological changes in pharynx tissues of OSAHS patients ：Subepithelia blood vessels expand and hyperemia，soft tissues edema.
② Immunohistochemical changes about VEGF :There are a powerful postive expression in base and spinae stratum cells in mucosa squamous epithelium and epithelium of glandular duct of OSAHS ，a feeble postive expression in vascular endothelial cell .However，there are feeble postive expression in base and spinae stratum cells in mucosa squamous epithelium and epithelium of glandular duct of control group，a negative expression in vascular endothelial cell.
③ Immunohistochemical changes about flt-1：There are a powerful postive expression in epithelium of glandular duct and in striated muscule cells of OSAHS.but there are feeble postive expression in that of control group.
④ Immunohistochemical changes about KDR：There are a powerful postive expression in extrastratum cells in mucosa squamous epithelium and in striated muscule cells and epithelium of glandular duct of OSAHS patients，a feeble postive expression in some interstitial cells. However，there are feeble postive expression extrastratum cells in mucosa squamous epithelium and in striated muscule cells and epithelium of glandular duct of control group，a negative expression in interstitial cells.
4. The MVD change of pharynx tissues:the MVD of OSAHS patient is higher than that of control group(OSAHS
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31、Shemirani B， Crowe DL. Hypoxic induction of HIF-1alpha and VEGF expression in head and neck squamous cell carcinoma lines is mediated by stress activated protein kinases. Oral Oncol. 2002; 38(3): 251-257
32、邓志宏，黄维国. VEGF受体flk-1表达与喉粘膜上皮癌变发生和转移的关系. 肿瘤防治研究. 1998； 2 (4).：260-263
33、邓志宏，黄维国，金岩，等. 血管内皮生长因子在喉粘膜上皮良性及恶性病变中的表达及定量.第四军医大学学报.1997；18(6)：573－576
34、曾俊，周云峰. 血管内皮生长因子在不同病变鼻咽粘膜上皮的表达. 医学新知杂志. 2000；10 (2)：75-76
35、郭剑峰，王向东，陶格陶呼，等.鼻息肉中血管内皮生长因子及转化生长因子β1的表达.中华耳鼻咽喉科杂志.2001;36(2)：83－86
36、Vento SI，Wolff CH，Salven PJ，et al. Vascular permeability factor/vascular endothelial growth factor in nasal polyps.Acta Otolaryngol.2000;543 Suppl.：170-174
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38、姜舒，董震，杨占泉.鼻息肉中血管内皮生长因子mRNA的检测与意义.临床耳鼻咽喉科杂志.2001;15(8)：339-340
39、Coste A，Brugel L，Maitre B，et al. Inflammatory cells as well as epithelial cells in nasal polyps express vascular endothelial growth factor.Eur Respir J，2000;15:367-372
40、Michael J，Scott T，Kelli L，et al. Vascular endothelial growth factor gene expression in human fetal lung in vitro.Am J Respir Cell Mol Biol，1999；20：14-23
41、Li G，Simpson A，Kenwiright J，et al.Effect of lengthening rate on angiogenesis during
distraction osteogenesis.J Orthop Res，1999；17:362-367
42、陈治，张莉，黄宗海.VEGF基因表达调控机制的研究进展.国外医学生理、病理科学与临床分册，2000；20(3)：190-194
43、邹小英，钟小宁，何志义.低氧性肺动脉高压与内皮素－1、血管内皮生长因子的相关性研究.广西医科大学学报，2000；17(4)：581-582
44、Samotok，Ikezakik，Mayumi O，et al. Expression of vascular endothelial growth factor and its possible relation with neovas cularization in human brain tumor.Cancer Res.1995;55(2):1189
45、卞修武，史景泉.血管内皮生长因子及其受体在肿瘤血管生成中的作用及与抗血管生成研究进展.中华病理杂志，1997;26(4)：248
46、Llorente-Arenas EM， Vicente-Gonzalez EA， Marin-Trigo JM，et al. Histologic changes in soft palate in patients with obstructive sleep apnea. An Otorrinolaringol Ibero Am. 2001; 28(5): 467-476
47、Series F，Simoneau JA，St－Pierre S.Muscle fiber area distribution of musculus uvulae in obstructive sleep apnea and non-apneic snorers. Int J Obes Relat Metab Disord.2000；24(4)：41-415
48、Berger G,Gilbey P，Hammel I，et al. Histopathology of the uvula and the soft palate in patients with moderate，and severe obstructive sleep apnea.Laryngoscope.2002;112(2)：357-363