致敏大鼠肺组织一氧化氮的变化与气道炎症关系的研究

1、致敏大鼠肺组织一氧化氮的变化与气道炎症关系的研究摘要：目的探讨致敏大鼠肺组织一氧化氮(NO)的变化及其与气道炎症的关系。方法采用原位杂交及免疫组化技术(ABC法)，观察卵蛋白致敏大鼠过敏性气道炎症肺组织中诱导型一氧化氮合酶(iNOS)基因表达与气道炎性细胞、活化T淋巴细胞间的关系。 结果iNOS mRNA主要表达于致敏大鼠肺泡巨噬细胞及细支气管上皮细胞，与肺组织匀浆中NO代谢产物增高相一致。定量分析结果表明，反复激发组气道嗜酸性粒细胞(Eos)（22.674.86)平均每视野计数，下同和淋巴细胞(34.335.42)较一次致敏组Eos(18.963.05)和淋巴细胞(30.534.32)有增多

2、趋势，IL-2R阳性淋巴细胞明显增高(积分光密度379.4089.42比155.941.43，P0.05)。随着炎症进展，iNOS mRNA表达量亦增高(积分光密度244.4034.17比133.4026.32，P0.05)，并与气道IL-2R阳性T淋巴细胞数有显著正相关关系(n=12,r=0.70,P0.05)。 结论致敏大鼠肺组织NO水平增高与肺泡巨噬细胞及小支气管上皮细胞iNOS mRNA表达增高相关，并与气道活化T淋巴细胞增高相关。提示增多的内源性NO可能参与了哮喘气道炎症发病机制中的T淋巴细胞活化过程。关键词：哮喘； 一氧化氮合酶； 原位杂交； 白细胞介素2受体Correlation

3、 between nitric oxide and inflammation in the bronchial and lung tissues of sensitized ratsXUE Jianmin（Department of Respiratory Medicine, Tongji Hospital, Tongji Medical University）XU Yongjian（Department of Respiratory Medicine, Tongji Hospital, Tongji Medical University）ZHANG Zhenxiang, et al（Depa

4、rtment of Respiratory Medicine, Tongji Hospital, Tongji Medical University）Abstract：ObjectiveTo investigate the relations between intrinsic nitric oxide (NO) and airways inflammation in sensitized rats. MethodsBy using in situ hybridization and immunohistochemical staining (ABC), we measured iNOS mR

5、NA expression in lung tissues and the number of inflammatory cells and activation of T lymphocytes in the airways. Then the correlations between them were analyzed. ResultsIn situ hybridization demonstrated that iNOS mRNA was mainly expressed in alveolar macrophages and bronchiole epithelial cells.

6、There was a significant positive correlation between iNOS mRNA expression and increased NO-2 level in lung tissues. Compared with that of rats sensitized once only (group B), the optical density (A value) of membrane interleukin-2 receptor (mIL-2R) positive cells in group C (rats repeatedly sensitiz

7、ed and stimulated) significantly increased (379.4089.42 vs 155.941.43, P0.05). But the increase in the numbers of eosinophils and lymphocytes was not statistically significant. With the development of inflammation, iNOS mRNA expression also significantly increased (244.4034.17 vs 133.4026.32, P0.05)

8、. There was significant positive correlation between iNOS mRNA expression and mIL-2R positive T lymphocytes in the airways of sensitized rats (n=12, r=0.70, P0.05)，两致敏组大鼠OVA激发后气道阻力较激发前气道阻力显著增加(P0.05)，两致敏组大鼠OVA激发后气道阻力均较正常组大鼠生理盐水激发后增加(P0.05)，反复激发组较一次致敏组激发后气道阻力增加(P0.05)。2.原位杂交结果： 地高辛标记的核酸原位杂交阳性细胞为出现蓝色或

9、紫蓝色或颗粒状沉积物者，杂交结果用TYTJ-300 计算机定量像分析系统处理，每张切片随机取10个视野(400)，测定阳性细胞的积分光密度值，计算10个视野的均值作为该切片的代表值，每组6只大鼠。正常对照组仅见大支气管上皮细胞有少量iNOS mRNA表达阳性细胞(1)，未见肺泡等其它部位阳性细胞。一次致敏组和反复激发组小支气管上皮细胞、明显增多的肺泡炎性细胞，主要为巨噬细胞，均见大量分布的iNOS mRNA阳性表达细胞(2、3)。两激发组大鼠肺泡炎性细胞iNOS mRNA含量的积分光密度值分别为133.4026.32和244.4034.17，反复激发组显著高于一次致敏组者(n均为6，P0.05

10、Once stimulation60.690.121.540.08*0.05Repeated stimulation60.750.111.980.32*0.05*P0.05，compared with control；P0.05,compared with once stimulation group1正常对照组大鼠大支气管上皮细胞(可见少量iNOS mRNA表达)Fig 1.There are a few iNOS mRNA expressions in epithelial cells in big brochus of normal control (in situ hybridizat

11、ion,400) 2一次致敏大鼠肺组织(可见较多iNOS mRNA表达的肺泡炎性和小支气管上皮细胞)Fig 2. There are many iNOS expressions in epithelial cells of small brochus and alveolar inflammation cells in the rat lung tissues of once stimulation group (in situ hybridization,400)3反复激发组大鼠肺组织(可见大量iNOS mRNA表达阳性细胞)Fig 3. There are a lot of iNOS mR

12、NA expressions in the epithelial cells of small brochus and alveolar inflammation cell in the lung tissues of repeatedly stimulated rat (in situ hybridization,400)3.气道炎性细胞数及mIL-2R阳性细胞积分光密度值变化：见表2。正常组(4)未见气道Eos或浆细胞浸润，肺泡中巨噬细胞数极少；一次致敏组及反复激发组气道周围浸润淋巴细胞、Eos及浆细胞较正常组显著增多(P0.05，5、6)，肺泡炎性细胞主要为巨噬细胞显著增多，气道周围mI

13、L-2R阳性细胞积分光密度值均较正常组增多(P0.05)，mIL-2R阳性细胞积分光密度值较一次致敏组增高(P0.05)。表2气道炎性细胞及mIL-2R积分光密度变化Table 2. Change of airway inflammation cell numbers and mIL-2R+ cell in rats airwayGorupnEos (cell/HP)Lymphocyte(cell/HP)IL-2+cell(A value)Control605.322.5611.172.37Once stimulation618.963.50*30.534.32*136.9015.43*Rep

14、eated stimulations622.674.86*33.234.32*279.4069.42*P0.05, compared with control；P0.05，compared with once stimulation4正常对照组大鼠支气管组织Fig 4. Bronchial tissues of normal control(HE200)5一次致敏组大鼠支气管组织Fig 5. Bronchial tissues of once stimulated rat(HE200)6反复激发组大鼠支气管组织Fig 6. Bronchial tissues of repeatedly sti

15、mulated rat(HE200)7一次致敏大鼠支气管肺组织免疫组化染色(可见mIL-2R+细胞)Fig 7. There are some mIL-2R+ cells in the bronchial and lung tissues of the once stimulated rat (in immunohistochemical staining,200)8反复激发组大鼠支气管肺组织免疫组化染色(可见明显增高的mIL-2R+细胞)Fig 8.There are a lot of mIL-2R+ cells in repeatedly stimulated rat (immunohis

16、tochemical staining,400)9正常对照组大鼠免疫组化染色(未见mIL-2R+细胞)Fig 9.In immunohistochemical staining there are no mIL-2R+ cells in the rat lung tissues of normal control (400)4.肺组织匀浆NO代谢产物变化： NO代谢产物NO-2测定值在正常组、一次致敏组和反复激发组分别为(0.320.18)mol/L、(0.660.24)mol/L和(1.220.32)mol/L(各组n均为6)。两致敏组肺组织NO-2含量均高于正常对照组者(均为P0.05)，

17、反复激发组又较一次致敏组增高(P0.05)。5.iNOS mRNA与mIL-2R间积分光密度关系： 两组致敏大鼠肺泡iNOS mRNA表达阳性细胞的积分光密度与气道mIL-2R 表达积分光密度呈显著正相关(r=0.70， P0.01，n=12)。6.肺组织iNOS mRNA表达与肺匀浆NO代谢产物关系： 两致敏组肺泡炎性细胞iNOS mRNA 含量积分光密度值与NO?2均呈正相关(r=0.68，P0.01，n=12)。7.肺组织iNOS mRNA表达与肺功能关系：两致敏组随气道阻力的增高，iNOS mRNA含量积分光密度值增高（r=0.64，P0.05，n=12)。讨论本研究观察到，一次致敏及

18、反复激发一周大鼠气道Eos、淋巴细胞及mIL-2R阳性淋巴细胞浸润均明显增高，同时气道阻力也增高，气道炎性变化尤以反复吸入抗原1周者明显。IL-2R主要由活化T淋巴细胞表达，故致敏大鼠气道周围浸润的淋巴细胞中mIL-2R阳性细胞增高，提示气道活化T淋巴细胞增高，符合哮喘这一气道变态反应炎性疾病的特点，与文献报道相似6。关于肺组织及气道中NO的来源及其在哮喘发病中的作用，近年一直是研究的热点。对于iNOS mRNA在哮喘模型中的原位表达，国内尚未见报道。国外许多学者证实哮喘患者肺组织iNOS表达增高，但对于其表达与不同病情程度及炎症的关系，报道尚少。本实验结果显示，不同病期的两致敏组大鼠肺泡内炎

19、性细胞主要为巨噬细胞增多，而iNOS mRNA阳性表达细胞主要位于肺泡炎性细胞及小支气管上皮细胞。随病情进展，即气道阻力和气道炎性细胞的增多，iNOS mRNA表达增多，并与肺组织中NO代谢产物水平的增高相平行，提示哮喘内源性NO的增多，可能主要源于肺泡巨噬细胞等炎性细胞及小支气管上皮细胞iNOS mRNA水平增高。此时iNOS mRNA的表达增多，导致大量NO生成7 ，在哮喘的发病中可能起重要作用。本研究还发现，iNOS mRNA表达增高，与不同病期mIL-2R阳性淋巴细胞，即活化T淋巴细胞数目的增高相一致。结合我们新近研究结果发现8 ，NO前体可促进气道淋巴细胞活化，而iNOS抑制剂(L-

20、NAME)则抑制气道淋巴细胞活化。Takano等9 研究证实，左旋精氨酸可促进致敏小鼠肺组织TH2 类细胞因子IL-5表达，加重气道嗜酸细胞炎症及气道高反应性，从另一侧面提示在炎症的不同病程中， NO这一小生物信息分子可能直接参与了哮喘炎症过程的调控。近年证实，NO这一高效而多样性的生物调节分子与免疫系统关系密切，可直接参与细胞因子特别是与炎症有关的细胞因子的调节。NO在免疫系统中发挥细胞间信息传递作用，如巨噬细胞可通过NO调节淋巴细胞功能10 。NO是鸟苷酸环化酶的强激活剂，NO不但可以使产生NO自身细胞环磷酸鸟苷(cGMP)升高，而且也可使邻近细胞cGMP升高，cGMP作为细胞内信号传递分

21、子参与一些蛋白激酶的活化和一些蛋白质的磷酸化，并产生生物效应11。此外，还可能通过NO的细胞内信号传导作用直接作用于核因子NF-B等影响T淋巴细胞不同细胞因子的合成12。因此，对哮喘这一与TH2及TH1类细胞因子调节失衡有关的炎症性疾病，应进一步研究哮喘气道炎症中淋巴细胞内信号传导过程中NO的作用，这对于阐明哮喘发病过程中T淋巴细胞持续激活机制有积极意义。综上所述，本实验证实，致敏大鼠肺组织内源性NO水平增高，与肺泡巨噬细胞及小支气管上皮细胞iNOS转录水平表达增高有关，同时与气道活化T淋细胞增多及气道阻力增高有关。表明内源性NO可能参与了哮喘气道炎症发病机制中的T淋巴细胞的激活过程。本研究为

22、阐明内源性NO在哮喘发病机制中的作用提供了有意义的实验资料。作者单位：薛建敏（同济医科大学附属同济医院呼吸科）徐永健（同济医科大学附属同济医院呼吸科）张珍祥（同济医科大学附属同济医院呼吸科）薛蔚（眼科）沈关心（同济医科大学实验中心免疫室）参考文献：1Anthony FM, Gaston B, Kita D, et al. Expired nitric oxide levels during treatment of acute asthma. Am J Respir Crit Care Med, 1995,152:800-806.2Waserman S, Xu LJ, Olivenstein

23、R, et al. Association between late allergic bronchoconstriction in the rat and allgen stimulated lymphocyte proliferation in vitro. Am J Respir Crit Care Med， 1995，151:470-474.3Sung Y，Stepem RD, Christopher JC, et al. Phenotype of cells expressing mRNA for TH2-type (interleukin 4 and interleukin 5)

24、and TH1-type (interleukin 2 and interferon-) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic and normal control subjects. Am J Cell Mol Biol， 1995，12:613-619.4蔡文琴，王泊云，主编. 实用免疫细胞化学. 四川:科学技术出版社，1988，116-119.5Cortas NK， Wakid JW. Determination of inorganic nitrate in serum and urine by a rineetic cadmium-reduction method. Clin Chem, 1990, 36:1440-1443.6William WB, Robert LC, Caffman EW. Mechanisms of persistent airway inflammation in asthma: a role for T cells and T cell products. Am J Respir Crit Care Med, 1995,152:388