儿童阻塞性睡眠呼吸暂停低通气综合征患者红细胞补体受体1的测定
发布时间：2018-08-20 19:25
【摘要】：目的： 随着睡眠医学的研究进展阻塞性睡眠呼吸暂停低通气综合征(obstructive sleep apnea hypopnea syndrome, OSAHS)作为呼吸、睡眠医学的交叉边缘学科,引起了人们的广泛关注,越来越多的学者认识到OSAHS不仅对成人患者可以带来一系列并发症,而且对儿童患者的智力和生长发育等也会带来不容忽视的负面影响。本研究通过检测红细胞补体受体1(complement receptor type1, CR1)的表达水平,旨在了解OSAHS对儿童的红细胞免疫功能的影响,探讨儿童OSAHS患者红细胞免疫功能的变化及临床意义。 方法： 选择2011年1月～2011年10月本院耳鼻咽喉科就医患儿30例,经多导睡眠监测(polysomnography, PSG)证实符合OSAHS的诊断。另选择同龄健康儿童30例作为对照,均为来本院健康查体的儿童,无睡眠打鼾及呼吸障碍病史,查体无扁桃体及腺样体肥大、小下颌等体征。所有研究对象均除外免疫系统疾病及过敏性疾病史,近期(1个月内)没有上呼吸道感染史。两组儿童的年龄、性别、体重指数(body mass index, BMI)等一般特征无显著性差异(P0.05)。 红细胞CR1数量检测采用细胞酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)对CRl的含量进行测定,按照郭峰等建立的细胞酶免疫分析法,检测时CR1抗体预包被在微孔板上,加入标准品和待测标本,每孔100μl,设立空白对照,37℃孵育2小时,然后加生物素化的CR1抗体,37℃孵育1小时,将未结合的生物素化抗体洗掉,然后加辣根过氧化物酶(horseradish peroxidase, HRP)标记的亲和素,37℃水浴箱孵育30min,用洗涤液洗板5次,加底物显色,37℃避光5min,硫酸终止反应后,以空白对照调零,用酶标仪测定450nm吸光度。以试剂盒所示的标准品绘制标准曲线,与标准曲线相比得到对应浓度,再乘以相应的稀释倍数,即得出样品的实际浓度。 红细胞CRl活性检测采用红细胞C3b受体花环(erythrocyte C3b receptor rosett, ECRR)试验和红细胞免疫复合物花环(erythrocyte immune complex rosette, EICR)试验。按照郭峰法两组血液样本肝素抗凝,并于2h内以1000转/分离心5分钟,用PBS洗涤4次,按红细胞计数方法配成1.25×107/ml的使用液。检测时待测红细胞使用液100ul,分别加入补体致敏酵母多糖工作液100ul,酵母多糖试液100ul,都摇匀放37℃水浴30min,取出摇匀,加生理盐水100ul,混匀后再加0.25%戊二醛50ul,轻轻混匀,取1/3量水平涂片,吹干,加甲醇固定,加少许瑞氏液,染色10秒钟,自来水少许冲洗。高倍显微镜计数,一个红细胞上2个或2个以上酵母菌为一朵花环。计数200个红细胞,算出百分率。 运用SPSS16.0软件对相关数据进行统计学处理及分析,P0.05为差异有统计学意义。 结果： 红细胞CR1数量检查结果显示,OSAHS患儿CR1数量及A值均低于对照组(P0.05),且CR1数量与呼吸暂停低通气指数(apnea hypopnea index, AHI)呈负相关(r=-0.61,P0.05)；与最低血氧饱和度(lowest arterial oxygen saturation, LSaO2)呈正相关(r=0.53,P0.05)。红细胞CR1活性检测结果显示,OSAHS患儿红细胞C3b受体花环结合率明显低于对照组(P0.05),而红细胞免疫复合物花环结合率明显高于对照组(P0.05),提示OSAHS患儿存在继发性红细胞免疫功能低下。 结论： 阻塞性睡眠呼吸暂停低通气综合征影响儿童红细胞免疫功能,使红细胞免疫功能下降,且与病情的严重程度有关。其红细胞免疫功能的降低,可能会对整个免疫系统的功能带来不良影响。
[Abstract]:Objective:
With the development of sleep medicine, obstructive sleep apnea hypopnea syndrome (OSAHS) as an interdisciplinary subject of breathing and sleep medicine has attracted wide attention. More and more scholars have realized that OSAHS can not only bring a series of complications to adult patients, but also cause a series of complications. In this study, the expression of complement receptor type 1 (CR1) in erythrocyte was detected to understand the effect of OSAHS on erythrocyte immune function in children, and to explore the changes of erythrocyte immune function and clinical significance in children with OSAHS. Righteousness.
Method:
From January 2011 to October 2011, 30 children with OSAHS diagnosed by polysomnography (PSG) and 30 healthy children of the same age were selected as controls. All of them had no history of sleep snoring and respiratory disorders, and had no hypertrophy of tonsils and adenoids. All subjects had no history of upper respiratory tract infection except for immune system diseases and allergic diseases. There was no significant difference in age, sex, body mass index (BMI) and other general characteristics between the two groups (P 0.05).
Erythrocyte CR1 was detected by enzyme linked immunosorbent assay (ELISA). According to the enzyme immunosorbent assay established by Guo Feng, CR1 antibody was pre-coated on the microporous plate, adding standard and sample to be tested, 100 ml per pore, setting up a blank control, incubated at 37 C for 2 hours. After incubation at 37 C for 1 hour, the unbounded antibody was washed out, and then the labeled avidin was added. After incubation at 37 C for 30 minutes, the washing liquid was used to wash the plate 5 times, the substrate was colored, 37 C for 5 minutes, and the reaction was stopped by sulfuric acid. Zero, 450 nm absorbance was determined by enzyme labeling instrument. The standard curve was drawn with the standard sample shown in the kit, and the corresponding concentration was obtained by comparing with the standard curve. Then the actual concentration of the sample was obtained by multiplying the corresponding dilution multiple.
The erythrocyte C3b receptor rosett (ECRR) test and erythrocyte immune complex rosette (EICR) test were used to detect the CRl activity of erythrocyte. 1.25 107/ml solution was prepared by several methods.The red blood cell solution was 100ul, the complement-sensitized yeast polysaccharide solution was 100ul, the yeast polysaccharide solution was 100ul, and the yeast polysaccharide solution was shaken uniformly and put in 37 C water bath for 30 minutes. Fixed with alcohol, stained for 10 seconds, rinsed with tap water. High power microscope counts, two or more yeasts on a red blood cell are a wreath. Count 200 red blood cells and calculate the percentage.
SPSS16.0 software was used for statistical analysis and analysis of the relevant data. P0.05 was statistically significant.
Result:
The results showed that the number of CR1 and the value of A in children with OSAHS were lower than those in the control group (P 0.05), and the number of CR1 was negatively correlated with apnea hypopnea index (AHI) (r = - 0.61, P 0.05), and positively correlated with lowest arterial oxygen saturation (LSaO 2) (r = 0.53, P 0.05). Sex test showed that the rosette binding rate of erythrocyte C3b receptor in OSAHS children was significantly lower than that in the control group (P 0.05), while the rosette binding rate of erythrocyte immune complex was significantly higher than that in the control group (P 0.05), suggesting that the secondary erythrocyte immune function was decreased in OSAHS children.
Conclusion:
Obstructive sleep apnea hypopnea syndrome (OSAHS) affects the immune function of red blood cells in children, which is related to the severity of the disease.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R766.4
[Abstract]:Objective:
With the development of sleep medicine, obstructive sleep apnea hypopnea syndrome (OSAHS) as an interdisciplinary subject of breathing and sleep medicine has attracted wide attention. More and more scholars have realized that OSAHS can not only bring a series of complications to adult patients, but also cause a series of complications. In this study, the expression of complement receptor type 1 (CR1) in erythrocyte was detected to understand the effect of OSAHS on erythrocyte immune function in children, and to explore the changes of erythrocyte immune function and clinical significance in children with OSAHS. Righteousness.
Method:
From January 2011 to October 2011, 30 children with OSAHS diagnosed by polysomnography (PSG) and 30 healthy children of the same age were selected as controls. All of them had no history of sleep snoring and respiratory disorders, and had no hypertrophy of tonsils and adenoids. All subjects had no history of upper respiratory tract infection except for immune system diseases and allergic diseases. There was no significant difference in age, sex, body mass index (BMI) and other general characteristics between the two groups (P 0.05).
Erythrocyte CR1 was detected by enzyme linked immunosorbent assay (ELISA). According to the enzyme immunosorbent assay established by Guo Feng, CR1 antibody was pre-coated on the microporous plate, adding standard and sample to be tested, 100 ml per pore, setting up a blank control, incubated at 37 C for 2 hours. After incubation at 37 C for 1 hour, the unbounded antibody was washed out, and then the labeled avidin was added. After incubation at 37 C for 30 minutes, the washing liquid was used to wash the plate 5 times, the substrate was colored, 37 C for 5 minutes, and the reaction was stopped by sulfuric acid. Zero, 450 nm absorbance was determined by enzyme labeling instrument. The standard curve was drawn with the standard sample shown in the kit, and the corresponding concentration was obtained by comparing with the standard curve. Then the actual concentration of the sample was obtained by multiplying the corresponding dilution multiple.
The erythrocyte C3b receptor rosett (ECRR) test and erythrocyte immune complex rosette (EICR) test were used to detect the CRl activity of erythrocyte. 1.25 107/ml solution was prepared by several methods.The red blood cell solution was 100ul, the complement-sensitized yeast polysaccharide solution was 100ul, the yeast polysaccharide solution was 100ul, and the yeast polysaccharide solution was shaken uniformly and put in 37 C water bath for 30 minutes. Fixed with alcohol, stained for 10 seconds, rinsed with tap water. High power microscope counts, two or more yeasts on a red blood cell are a wreath. Count 200 red blood cells and calculate the percentage.
SPSS16.0 software was used for statistical analysis and analysis of the relevant data. P0.05 was statistically significant.
Result:
The results showed that the number of CR1 and the value of A in children with OSAHS were lower than those in the control group (P 0.05), and the number of CR1 was negatively correlated with apnea hypopnea index (AHI) (r = - 0.61, P 0.05), and positively correlated with lowest arterial oxygen saturation (LSaO 2) (r = 0.53, P 0.05). Sex test showed that the rosette binding rate of erythrocyte C3b receptor in OSAHS children was significantly lower than that in the control group (P 0.05), while the rosette binding rate of erythrocyte immune complex was significantly higher than that in the control group (P 0.05), suggesting that the secondary erythrocyte immune function was decreased in OSAHS children.
Conclusion:
Obstructive sleep apnea hypopnea syndrome (OSAHS) affects the immune function of red blood cells in children, which is related to the severity of the disease.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R766.4
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