变态反应性哮喘产妇新生儿脐血中的氧化应激状态

doi:10.3877/cma.j.issn.1673-5250.2013.05.001

目的

检测变态反应性哮喘产妇新生儿脐血的氧化应激状态，及与嗜碱性粒细胞释放介质能力的关系。

方法

选择2008年3月至2009年6月在上海交通大学医学院附属新华医院住院分娩的16例妊娠前有变态反应性哮喘史产妇(哮喘产妇组)和同期住院分娩的正常产妇18例(正常产妇组)的新生儿脐血中提纯嗜碱性粒细胞，采用粉尘螨及甘露醇刺激，测定嗜碱性粒细胞CD₆₃及白细胞介素(IL)-4的表达。采用ELISA法检测血清中3-硝基酪氨酸(3-NT)的含量，并进行相关性分析(本研究遵循的程序符合上海交通大学医学院附属新华医院人体试验委员会所制定的伦理学标准，得到该委员会批准，分组征得受试对象本人的知情同意，并与之签署临床研究知情同意书)。两组孕妇的年龄、分娩方式、分娩孕龄及新生儿脐血采集方式等比较，差异无统计学意义(P>0.05)

结果

粉尘螨及甘露醇刺激哮喘产妇组新生儿脐血嗜碱性粒细胞CD₆₃的表达高于正常产妇组(215.59±47.15 vs. 11.33±3.26，233.65±51.25，12.98±3.11)，2组比较，差异有统计学意义(t＝7.61，8.97；P<0.01)；所释放的IL-4亦高于正常产妇组[(3.27±0.40) pg/mL vs. (1.78±0.48) pg/mL；(3.56±0.47)pg/mL vs. (1.92±0.52) pg/mL]，2组比较，差异有统计学意义(t＝2.53，3.61；P<0.01)。哮喘产妇组的产妇外周静脉血及新生儿脐血中3-NT的含量均高于正常产妇组[(443.86±95.20) μg/mL vs. (288.17±57.23) μg/mL；(474.69±92.55) μg/mL vs. (273.7±68.09) μg/mL]，2组比较，差异有统计学意义(t＝2.68，2.79；P<0.01)。IL-4水平与新生儿脐血中3-NT的含量相关(r＝0.607，P<0.05；r＝0.619，P<0.05)。

结论

哮喘中，特异性的免疫反应和非特异性的刺激均与氧化应激有关，这可能是变态反应性疾病的发病机制之一。

关键词: 变态反应性哮喘, 氧化应激, 嗜碱性粒细胞, 脐血, 白细胞介素

Objective

To assess the oxidative stress state in umbilical cord blood from neonates of the mothers with allergic asthma, and analyze its relationship with the mediator release properties of human basophils.

Methods

Umbilical cord blood sample were collected from the neonates of 16 asthmatic lying-in women and 18 healthy lying-in women between March 2008 to June 2009. Basophils were purified and then stimulated by dermatophagoides farinae, hyperosmotic mannitol. Activation was assessed by flow cytometric measurement of CD₆₃ expression on the cord blood basophil surface. Interleukin (IL)-4 levels in samples were measured by ELISA, and done the correlation analysis with the 3-nitrotyrosine (NT) levels in the serum which were measured by ELISA. The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Shanghai Jiaotong University School of Medicine. Informed consent was obtained from all participates. There had no significant differences between two groups on age, delivery modes, pregnancy age and cord blood collection mode etc..

Results

After stimulation of Df and hyperosmotic mannitol, basophils purified from the cord blood of neonates born to atopic mothers produced more IL-4 compared to basophils purified from the controls[(3.27±0.40) pg/mL vs. (1.78±0.48) pg/mL; (3.56±0.47)pg/mL vs. (1.92±0.52) pg/mL; t=2.53, 3.61, P<0.01]. Upregulation of CD₆₃ on the cord blood basophil surface was also detected in response to these stimuli (215.59±47.15 vs. 11.33±3.26, 233.65±51.25 vs. 12.98±3.11; t=7.61, 8.97; P<0.01). The level of 3-NT in the serum of umbilical cord blood from neonates of the mothers with allergic asthma was significantly higher than that in healthy group [(443.86±95.20) μg/mL vs. (288.17±57.23) μg/mL; (474.69±92.55) μg/mL vs. (273.70±68.09) μg/mL; t=2.68, 2.79, P<0.01]. There was a significant correlation between the concentration of IL-4 and the 3-NT in umbilical cord blood from allergic asthma group(r=0.607, P<0.05; r=0.619, P<0.05).

Conclusions

In asthma, specific immune response and a non-specific stimulation are associated with oxidative stress, which may be one of the pathogenesis of allergic diseases.

Key words: allergic asthma, oxidative stress, basophil, umbilical cord blood, interleukin

图1 阿利新蓝染色嗜碱性粒细胞显微镜照片(×400)，红色箭头所指为嗜碱性粒细胞

Figure 1 Alcian blue staining. Basophil purity and integrity were assessed by staining harvested cells with Alcian blue(×400). Red arrow means magnification

表1 脐血嗜碱性粒细胞受刺激前、后CD₆₃表达水平(MFI值，±s)

Table 1 Level of CD₆₃ in cord blood basophils before and after the stimulation (MFI value，±s)

组别	n	ACM缓冲液	粉尘螨刺激	甘露醇刺激
哮喘产妇组	16	11.88±3.13	215.59±47.15^ab	233.65±51.25^ab
正常产妇组	18	8.15±2.87	11.33±3.26	12.98±3.11
t	?	1.81	7.61	8.97

表1 脐血嗜碱性粒细胞受刺激前、后CD₆₃表达水平(MFI值，±s)

Table 1 Level of CD₆₃ in cord blood basophils before and after the stimulation (MFI value，±s)

组别	n	ACM缓冲液	粉尘螨刺激	甘露醇刺激
哮喘产妇组	16	11.88±3.13	215.59±47.15^ab	233.65±51.25^ab
正常产妇组	18	8.15±2.87	11.33±3.26	12.98±3.11
t	?	1.81	7.61	8.97

图2 哮喘产妇组新生儿脐带血嗜碱性粒细胞受刺激前、后CD₆₃的表达(绿色为刺激前，红色为刺激后)

Figure 2 Characterization of CD₆₃ expression. The green line represents cells from one neonate born to an asthmatic mother before stimulation and the red line represents cells from the neonate after stimulation

表2 脐血嗜碱粒细胞受刺激前、后IL-4表达水平(pg/mL，±s)

Table 2 IL-4 supernatant levels from cord blood basophils before and after stimulation (pg/mL，±s)

组别	n	ACM缓冲液	粉尘螨刺激	甘露醇刺激
哮喘产妇组	16	1.79±0.42	3.27±0.40^ab	3.56±0.47^ab
正常产妇组	18	1.65±0.37	1.78±0.48	1.92±0.52
t	?	0.51	2.53	3.61

表2 脐血嗜碱粒细胞受刺激前、后IL-4表达水平(pg/mL，±s)

Table 2 IL-4 supernatant levels from cord blood basophils before and after stimulation (pg/mL，±s)

组别	n	ACM缓冲液	粉尘螨刺激	甘露醇刺激
哮喘产妇组	16	1.79±0.42	3.27±0.40^ab	3.56±0.47^ab
正常产妇组	18	1.65±0.37	1.78±0.48	1.92±0.52
t	?	0.51	2.53	3.61

表3 2组产妇外周静脉血及新生儿脐血中3-NT含量比较(μg/mL，±s)

Table 3 3-NT levels in cord blood from neonates and peripheral blood from mother (μg/mL，±s)

组别	n	产妇外周静脉血	新生儿脐血
哮喘产妇组	16	443.86±95.20	474.69±92.55
正常产妇组	18	288.17±57.23	273.70±68.09
t	?	2.68	2.79
P	?	<0.01	<0.01

表3 2组产妇外周静脉血及新生儿脐血中3-NT含量比较(μg/mL，±s)

Table 3 3-NT levels in cord blood from neonates and peripheral blood from mother (μg/mL，±s)

组别	n	产妇外周静脉血	新生儿脐血
哮喘产妇组	16	443.86±95.20	474.69±92.55
正常产妇组	18	288.17±57.23	273.70±68.09
t	?	2.68	2.79
P	?	<0.01	<0.01

图3B 哮喘组脐带血3-NT的含量与甘露醇刺激后IL-4表达量相关分析

Figure 3 Assessment of the IL-4 response. Cord blood basophils from 16 neonates born to asthmatic mothers were stimulated with (A) Df or (B) 9% hyperosmotic mannitol, and the IL-4 supernatant levels were correlated to the 3-NT. Each point on the graph represents the IL-4/3-NT response for an individual patient

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Oxidative Stress State in Umbilical Cord Blood From Neonates of the Mothers With Allergic Asthma

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Oxidative Stress State in Umbilical Cord Blood From Neonates of the Mothers With Allergic Asthma