【摘要】：目的：本實驗通過建立哮喘大鼠模型，以血清IL-5、IL-13、TNF-α為氣道炎性指標(biāo)，以α-SMA為氣道重塑指標(biāo)，以此來觀察多西環(huán)素干預(yù)治療后MMP-9、IL-5、IL-13、TNF-α、α-SMA的變化，從而探討多西環(huán)素對哮喘大鼠氣道炎癥及重塑的預(yù)防作用及可能機(jī)制，為臨床哮喘的防治提供新的途徑。 方法：健康清潔SD雄性大鼠33只，6周齡，體重50-100g之間，隨機(jī)分為正常對照組、哮喘模型組、多西環(huán)素干預(yù)組，每組11只。哮喘模型組、多西環(huán)素干預(yù)組分別于實驗第1、8天對所有大鼠腹腔注射OVA100mg和氫氧化鋁100mg（混于0.9%生理鹽水2ml中）進(jìn)行致敏，第15天起將大鼠置于自制的非完全密閉霧化吸入箱內(nèi)，給予OVA5ml霧化吸入30min進(jìn)行激發(fā)，隔日1次，共激發(fā)20次，其中多西環(huán)素干預(yù)組在每次OVA激發(fā)前30min按30mg/kg采用口腔灌服的方式給大鼠用藥。正常對照組的致敏和激發(fā)均以生理鹽水代替OVA進(jìn)行，余同哮喘組。 最后一次激發(fā)24小時后，所有大鼠用1%戊巴比妥鈉麻醉，收集肺泡灌洗液、血清及肺組織。計數(shù)肺泡灌洗液中的細(xì)胞數(shù)并進(jìn)行分類；ELISA法檢測血清中的IL-5、IL-13的濃度，放免法檢測血清中TNF-α的濃度，免疫組化法檢測肺組織中MMP-9、α-SMA的表達(dá)；肺組織行病理切片，HE染色，采用計算機(jī)圖像分析技術(shù)測定支氣管基底膜周徑（Pbm）、總管壁面積（WAt）、平滑肌面積（WAm）等反映氣道壁厚度的指標(biāo)。 結(jié)果： 1肺泡灌洗液（BALF）中細(xì)胞總數(shù)及嗜酸性粒細(xì)胞計數(shù)（×10~6/L）的結(jié)果：正常對照組9.42±3.67、1.24±0.72，哮喘組35.53±7.06、4.84±0.65，多西環(huán)素干預(yù)組14.66±5.81、2.14±0.58。哮喘組與多西環(huán)素干預(yù)組的細(xì)胞總數(shù)、嗜酸性粒細(xì)胞計數(shù)均明顯高于正常對照組（F=64.840P＜0.05，F(xiàn)=90.598P＜0.05），其中多西環(huán)素干預(yù)組上述細(xì)胞計數(shù)均明顯低于哮喘組（P＜0.05）。 2病理結(jié)果：哮喘組大鼠的肺組織病理切片可見大量炎性細(xì)胞浸潤，氣道管壁、基底膜及血管壁增厚，血管及平滑肌增生。多西環(huán)素干預(yù)組的炎性細(xì)胞的浸潤程度，氣道管壁、基底膜及血管壁的增厚程度，血管及平滑肌的增生程度均較哮喘組減輕。 3圖像分析支氣管管壁厚度、平滑肌層厚度（μm~2/μm）分別為：正常對照組65.47±7.22、20.09±6.09，哮喘組109.95±13.85、56.94±8.23，多西環(huán)素干預(yù)組86.37±5.85、40.64±4.53。哮喘組與多西環(huán)素干預(yù)組支氣管壁厚度、平滑肌厚度均明顯高于正常對照組（F=58.737P＜0.05，F(xiàn)=89.782P＜0.050），其中多西環(huán)素干預(yù)組上述厚度均低于哮喘組（P＜0.05）。 4ELISA法檢測血清IL-5、IL-13的濃度（pg/ml）： 血清IL-5濃度：正常對照組（15.92±3.66），哮喘組（38.63±6.64），多西環(huán)素干預(yù)組（20.42±4.50）。哮喘組和多西環(huán)素干預(yù)組血清IL-5的濃度均高于正常對照組（F=61.348，P＜0.05），其中多西環(huán)素干預(yù)組低于哮喘組（P＜0.05）。 血清IL-13濃度：正常對照組（16.98±7.53），哮喘組（57.58±14.09），多西環(huán)素干預(yù)組（35.75±8.91）。哮喘組和多西環(huán)素干預(yù)組血清IL-13的濃度均明顯高于正常對照組（F=40.713，P＜0.05），其中多西環(huán)素干預(yù)組低于哮喘組（P＜0.05）。 5放免法檢測血清TNF-α的濃度（ng/ml）：正常對照組（0.43±0.25），哮喘組（1.57±0.20），多西環(huán)素干預(yù)組（1.20±0.24）。哮喘組和多西環(huán)素干預(yù)組血清TNF-α的濃度均明顯高于正常對照組（F=68.254，P＜0.05），其中多西環(huán)素干預(yù)組低于哮喘組（P＜0.05）。 6免疫組化法檢測肺組織MMP-9、α-SMA的表達(dá)： MMP-9的平均光度值：正常對照組（0.24±0.06），哮喘組（0.52±0.06），多西環(huán)素干預(yù)組（0.39±0.06）。哮喘組和多西環(huán)素干預(yù)組肺組織MMP-9的表達(dá)均高于正常對照組（F=57.966，P＜0.05）；其中多西環(huán)素干預(yù)組低于哮喘組（P＜0.05）。 α-SMA的平均光度值：正常組對照組（0.20±0.04），哮喘組（0.55±0.06），多西環(huán)素干預(yù)組（0.35±0.06）。哮喘組、多西環(huán)素干預(yù)組肺組織α-SMA的表達(dá)均高于正常對照組（F=112.663，P＜0.05）；其中多西環(huán)素干預(yù)組低于哮喘組（P＜0.05）。 7直線相關(guān)性分析結(jié)果：血清IL-5、IL-13的濃度與BALF中嗜酸性粒細(xì)胞計數(shù)呈正相關(guān)（r=0.817，P＜0.05；r=0.739，P＜0.05），血清TNF-α的濃度與BALF中細(xì)胞總數(shù)呈正相關(guān)（r=0.807，P＜0.05），肺組織MMP-9的表達(dá)與BALF中細(xì)胞總數(shù)呈正相關(guān)（r=0.738，P＜0.05），肺組織MMP-9的表達(dá)與血清TNF-α濃度呈正相關(guān)（r=0.793，P＜0.05），肺組織α-SMA的表達(dá)與平滑肌層厚度呈正相關(guān)（r=0.841，P＜0.05），肺組織MMP-9的表達(dá)與支氣管壁厚度、平滑肌層厚度、肺組織α-SMA的表達(dá)呈正相關(guān)（r=0.819，P＜0.05；r=0.883，P＜0.05；r=0.841，P＜0.05）。 結(jié)論： 1本實驗通過抗原致敏和反復(fù)霧化激發(fā)，成功建立了以氣道炎癥和氣道重塑為特征的哮喘大鼠模型。 2哮喘組大鼠的肺組織病理可見大量炎性細(xì)胞浸潤，肺泡灌洗液中的細(xì)胞總數(shù)和嗜酸性粒細(xì)胞計數(shù)均明顯高于正常對照組，血清IL-5、IL-13、TNF-α的濃度也明顯高于正常對照組。多西環(huán)素干預(yù)組大鼠的肺組織病理中炎性細(xì)胞的浸潤程度，肺泡灌洗液中細(xì)胞總數(shù)和嗜酸性粒細(xì)胞計數(shù)以及血清IL-5、IL-13、TNF-α的濃度均低于哮喘組。且血清IL-5、IL-13的濃度與BALF中嗜酸性粒細(xì)胞計數(shù)呈正相關(guān)，血清TNF-α的濃度與BALF中細(xì)胞總數(shù)呈正相關(guān)，提示多西環(huán)素可以影響炎癥介質(zhì)的生成從而減輕哮喘的氣道炎癥。 3哮喘組大鼠肺組織MMP-9的表達(dá)較正常對照組明顯升高，多西環(huán)素干預(yù)組肺組織MMP-9的表達(dá)較哮喘組減少，且肺組織MMP-9的表達(dá)與BALF中炎性細(xì)胞總數(shù)及血清TNF-α的濃度呈正相關(guān)，提示MMP-9可以影響氣道炎癥，多西環(huán)素可以通過抑制MMP-9的活性減輕哮喘的氣道炎癥。 4哮喘組大鼠的支氣管管壁厚度、平滑肌層厚度明顯高于正常對照組，肺組織α-SMA的表達(dá)明顯高于正常對照組。多西環(huán)素干預(yù)組大鼠的支氣管管壁厚度、平滑肌層厚度均明顯低于哮喘組，，肺組織α-SMA的表達(dá)也低于哮喘組。相關(guān)分析顯示肺組織MMP-9的表達(dá)與支氣管管壁厚度、平滑肌層厚度及肺組織α-SMA的表達(dá)呈正相關(guān)，提示MMP-9參與了哮喘的氣道重塑，多西環(huán)素可以通過抑制MMP-9的活性減輕哮喘的氣道重塑。
[Abstract]:AIM: To investigate the effects of doxycycline on airway inflammation and remodeling in asthmatic rats by establishing an asthmatic rat model with serum IL-5, IL-13, TNF-a as inflammatory markers and alpha-SMA as airway remodeling markers. It provides a new way for the prevention and treatment of clinical asthma.
Methods: Thirty-three healthy and clean SD male rats, aged 6 weeks and weighing 50-100g, were randomly divided into normal control group, asthma model group and doxycycline intervention group with 11 rats in each group. From the 15th day, rats were exposed to a self-made incomplete airtight nebulization inhalation chamber. OVA 5 ml was inhaled for 30 minutes, once every other day for 20 times. Doxycycline was administered orally 30 mg/kg 30 minutes before each OVA challenge in the intervention group. Water instead of OVA was used in asthma group.
24 hours after the last challenge, all rats were anesthetized with 1% sodium pentobarbital to collect alveolar lavage fluid, serum and lung tissue, count and classify the number of cells in alveolar lavage fluid, detect the concentration of IL-5 and IL-13 in serum by ELISA, detect the concentration of TNF-a by radioimmunoassay, and detect the concentration of MMP-9 and alpha-SMA in lung tissue by immunohistochemistry. The lung tissues were stained with HE, and the bronchial basement membrane circumference (Pbm), common duct wall area (WAt) and smooth muscle area (WAm) were measured by computer image analysis.
Result:
1. The total cell count and eosinophil count in BALF were 9.42 [3.67, 1.24] 0.72 in normal control group, 35.53 [7.06, 4.84] 0.65 in asthma group, 14.66 [5.81, 2.14] 0.58] in Doxycycline intervention group and asthma group, respectively. Group A (F = 64.840 P < 0.05, F = 90.598 P < 0.05), and the above cell counts in the doxycycline intervention group were significantly lower than those in the asthma group (P < 0.05).
2. Pathological results: Inflammatory cell infiltration, thickening of airway wall, basement membrane and vascular wall, proliferation of blood vessels and smooth muscle were observed in the lung tissue of asthmatic rats. The group was relieved.
3. The thickness of bronchial wall and smooth muscle layer (micron~2/micron) were 65.47 (+ 7.22) and 20.09 (+ 6.09) in normal control group, 109.95 (+ 13.85) and 56.94 (+ 8.23) in asthma group, and 86.37 (+ 5.85) and 40.64 (+ 4.53) in Doxycycline intervention group, respectively. 37P < 0.05, F=89.782P < 0.050). The thickness of doxycycline intervention group was lower than that of asthma group (P < 0.05).
4ELISA method was used to detect the concentration of IL-5 and IL-13 in serum (pg/ml):
Serum IL-5 levels were higher in asthma group and doxycycline intervention group than in normal control group (F = 61.348, P < 0.05), and lower in Doxycycline intervention group than in asthma group (P < 0.05).
Serum IL-13 concentration: normal control group (16.98+7.53), asthma group (57.58+14.09), doxycycline intervention group (35.75+8.91). The serum IL-13 concentration of asthma group and doxycycline intervention group was significantly higher than that of normal control group (F = 40.713, P < 0.05), and the doxycycline intervention group was lower than that of asthma group (P < 0.05).
5 Radioimmunoassay was used to detect the serum TNF-alpha concentration (ng/ml): normal control group (0.43.25), asthma group (1.57.20), doxycycline intervention group (1.20.24). The serum TNF-alpha concentration of asthma group and doxycycline intervention group was significantly higher than that of normal control group (F = 68.254, P < 0.05), and the doxycycline intervention group was lower than that of asthma group (P < 0.05).
6 immunohistochemical method was used to detect the expression of MMP-9 and -SMA in lung tissue.
The expression of MMP-9 in lung tissue of asthma group and doxycycline intervention group was higher than that of normal control group (F = 57.966, P < 0.05), and that of doxycycline intervention group was lower than that of asthma group (P < 0.05).
The average luminosity of alpha-SMA was 0.20 (+ 0.04) in normal control group, 0.55 (+ 0.06) in asthma group, and 0.35 (+ 0.06) in Doxycycline intervention group.
Linear correlation analysis showed that the concentration of serum IL-5 and IL-13 was positively correlated with eosinophil count in BALF (r = 0.817, P < 0.05; r = 0.739, P < 0.05), the concentration of serum TNF-a was positively correlated with the total number of cells in BALF (r = 0.807, P < 0.05), and the expression of MMP-9 in lung tissue was positively correlated with the total number of cells in BALF (r = 0.738, P < 0.05). MMP-9 expression was positively correlated with serum TNF-a concentration (r = 0.793, P < 0.05), lung tissue alpha-SMA expression was positively correlated with smooth muscle layer thickness (r = 0.841, P < 0.05), lung tissue MMP-9 expression was positively correlated with bronchial wall thickness, smooth muscle layer thickness, lung tissue alpha-SMA expression (r = 0.819, P < 0.05; r = 0.883, P < 0.05; r = 0.841, P < 0.05).
Conclusion:
1 The asthmatic rat model characterized by airway inflammation and airway remodeling was successfully established by antigen sensitization and repeated atomization.
The total number of cells and eosinophils in the alveolar lavage fluid were significantly higher than those in the normal control group. The concentrations of serum IL-5, IL-13 and TNF-a were also significantly higher than those in the normal control group. The concentration of serum IL-5, IL-13 and TNF-a was positively correlated with the eosinophil count in BALF, and the concentration of serum TNF-a was positively correlated with the total number of cells in BALF, suggesting that doxycycline could affect the production of inflammatory mediators. Reduce airway inflammation in asthma.
The expression of MMP-9 in lung tissue of asthmatic rats was significantly higher than that of normal control group. The expression of MMP-9 in lung tissue of doxycycline intervention group was lower than that of asthmatic rats. The expression of MMP-9 in lung tissue was positively correlated with the total number of inflammatory cells in BALF and the concentration of TNF-a in serum, suggesting that MMP-9 could affect airway inflammation and doxycycline could inhibit the expression of MMP-9. Activity attenuates airway inflammation in asthma.
The thickness of bronchial wall and smooth muscle layer in asthmatic rats were significantly higher than that in normal control group, and the expression of alpha-SMA in lung tissue was significantly higher than that in normal control group. The expression of MMP-9 was positively correlated with the thickness of bronchial wall, smooth muscle layer and the expression of alpha-SMA in lung tissue, suggesting that MMP-9 was involved in airway remodeling in asthma. Doxycycline can reduce airway remodeling in asthma by inhibiting the activity of MMP-9.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R965;R562.25

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