极低出生体重儿中性粒细胞呼吸爆发功能的研究

Respiratory burst function of neutrophils in very low birth weight infants

目的 探讨极低出生体重儿中性粒细胞呼吸爆发功能. 方法 以2012年7月1日至2012年12月31日收住复旦大学儿科医院的22例极低出生体重儿（very low birth weight infant,VLBWI）为VLBWI组,根据有无新生儿呼吸窘迫综合征（neonatal respiratory distress syndrome,NRDS）,分为NRDS组和无NRDS组,各11例.以同期在上海市闵行区妇幼保健院出生的20例正常足月新生儿为对照.所有新生儿均排除早发型败血症.VLBWI组入院时留取桡动脉血1.5 ml,足月儿组出生时留取脐带血1.5 ml.对早产儿和足月儿进行中性粒细胞呼吸爆发功能检测,使用佛波酯（phorbol-12-myristate-13-acetate,PMA）刺激中性粒细胞活化后,将二氢若丹明123（dihydrorhodamine-1,2,3,DHR）氧化为发出黄绿色荧光的若丹明123,用流式细胞仪检测荧光强度以评价中性粒细胞呼吸爆发功能.使用荧光标记的细胞色素b558单克隆抗体（7D5）标记,用流式细胞仪检测中性粒细胞gp91Phox的表达.采用Mann-Whitney U检验进行统计学分析.结果 无刺激时,VLBWI组发生呼吸爆发的中性粒细胞比例为（49.10±20.19）％,高于足月儿组的（18.73±6.81）％（Z=-4.911,P=0.000）.PMA刺激后VLBWI组发生呼吸爆发的中性粒细胞比例为（96.58±3.44）％,高于足月儿组的（99.20±0.62）％,差异有统计学意义（Z=-3.186,P=0.001）.VLBWI组中性粒细胞刺激指数为171.40±103.35,低于足月儿组的306.30±138.47,差异有统计学意义（Z=-3.413,P=0.001）.VLBWI组中性粒细胞表达gp91Phox荧光强度几何均数为21.66±19.87,而足月儿组为19.60±8.03,2组差异无统计学意义（Z=-0.934,P=0.350）.VLBWI组表达gp91Phox的中性粒细胞比例为（56.11±29.40）％,低于足月儿组的（80.14±14.87）％,差异有统计学意义（Z=-2.374,P=0.018）.无刺激时,NRDS组发生呼吸爆发的中性粒细胞比例为（63.40±16.45）％,高于无NRDS组的（34.80±11.65）％,差异有统计学意义（Z=-3.382,P=0.001）.NRDS组中性粒细胞刺激指数为129.46±75.36,低于无NRDS组的213.35±113.49,差异有统计学意义（Z=-2.331,P=0.020）. 结论 VLBWI及足月儿出生时无刺激时发生呼吸爆发的中性粒细胞比例较高,VLBWI更为明显,极低出生体重儿和足月儿出生时中性粒细胞均表达蛋白gp91Phox.NRDS患儿中性粒细胞呼吸爆发功能及gp91Phox表达与无NRDS患儿比较,有降低趋势.

Objective To investigate the respiratory burst function of neutrophils in very low birth weight infants （VLBWI）.Methods Twenty two VLBWI was divided into two groups：neonatal respiratory distress syndrome （NRDS） and non NRDS （11 in each）.The respiratory burst function of neutrophils in the peripheral blood of VLBWI within 48 hours after birth was determined using the flow cytometrydihydrorhodamine 1,2,3 method before and after the chemical stimulation of phorbol-12-myrismte 14 acetate （PMA）,and the gp91Phox was also measured in resting neutrophils by flow cytometry.Twenty healthy term neonates served as controls.Mann-Whitney U test was used for statistical analysis.Results Before the stimulation of PMA,the percentage of activated neutrophils of VLBWI [（49.10±20.19） ％] producing a respiratory burst was higher than that of term neonates [（18.73 ±6.81） ％] （Z--4.911,P=0.000）,however,after the stimulation of PMA,the percentage of activated neutrophils of VLBWI [（96.58 ± 3.44） ％] was lower than that of term neonates [（99.20±0.62） ％] （Z--3.186,P=0.001）,and the stimulation index （SI） of VLBWI （171.40 ± 103.35） was lower than that of term neonates （306.30 ± 138.47）,with significant difference （Z=-3.413,P=0.001）.The geometric mean of gp91Phox in VLBWI （21.66± 19.87） was higher compared with term neonates （19.60±8.03）,however,the difference was not significant （P=0.350）.The percentage of neutrophils that expressed gp91Phox [（56.11 ± 29.40） ％] was lower in VLBWI than that in term neonates [（80.14± 14.87） ％],with significant difference （Z=-2.374,P=0.018）.Before the stimulation of PMA,the percentage of activated neutrophils of VLBWI with NRDS （63.40± 16.45） ％] was higher than that of VLBWI without NRDS [（34.80± 11.65） ％],with significant difference （Z=-3.382,P=0.001）,the SI of VLBWI with NRDS （129.46 ± 75.36） was significantly lower than that of VLBWI without NRDS （213.35 ± 113.49） （Z=-2.331,P=0.020）.Conclusions Neutrophils producing a respiratory burst in both VLBWI and term neonates are active without stimulation of PMA,while the phenomenon is more obvious in VLBWI.Neutrophils in VLBWI and term infants can be activated by the stimulation of PMA,and express gp91Phox.The activation and gp91Phox expression of neutrophils in VLBWI with NRDS tend to be lower than those in VLBWI without NRDS.