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

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

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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