兒童阻塞性睡眠呼吸暫停低通氣綜合征患者紅細胞補體受體1的測定
發(fā)布時間：2018-08-20 19:25
【摘要】：目的： 隨著睡眠醫(yī)學的研究進展阻塞性睡眠呼吸暫停低通氣綜合征(obstructive sleep apnea hypopnea syndrome, OSAHS)作為呼吸、睡眠醫(yī)學的交叉邊緣學科,引起了人們的廣泛關(guān)注,越來越多的學者認識到OSAHS不僅對成人患者可以帶來一系列并發(fā)癥,而且對兒童患者的智力和生長發(fā)育等也會帶來不容忽視的負面影響。本研究通過檢測紅細胞補體受體1(complement receptor type1, CR1)的表達水平,旨在了解OSAHS對兒童的紅細胞免疫功能的影響,探討兒童OSAHS患者紅細胞免疫功能的變化及臨床意義。 方法： 選擇2011年1月～2011年10月本院耳鼻咽喉科就醫(yī)患兒30例,經(jīng)多導(dǎo)睡眠監(jiān)測(polysomnography, PSG)證實符合OSAHS的診斷。另選擇同齡健康兒童30例作為對照,均為來本院健康查體的兒童,無睡眠打鼾及呼吸障礙病史,查體無扁桃體及腺樣體肥大、小下頜等體征。所有研究對象均除外免疫系統(tǒng)疾病及過敏性疾病史,近期(1個月內(nèi))沒有上呼吸道感染史。兩組兒童的年齡、性別、體重指數(shù)(body mass index, BMI)等一般特征無顯著性差異(P0.05)。 紅細胞CR1數(shù)量檢測采用細胞酶聯(lián)免疫吸附試驗(enzyme linked immunosorbent assay, ELISA)對CRl的含量進行測定,按照郭峰等建立的細胞酶免疫分析法,檢測時CR1抗體預(yù)包被在微孔板上,加入標準品和待測標本,每孔100μl,設(shè)立空白對照,37℃孵育2小時,然后加生物素化的CR1抗體,37℃孵育1小時,將未結(jié)合的生物素化抗體洗掉,然后加辣根過氧化物酶(horseradish peroxidase, HRP)標記的親和素,37℃水浴箱孵育30min,用洗滌液洗板5次,加底物顯色,37℃避光5min,硫酸終止反應(yīng)后,以空白對照調(diào)零,用酶標儀測定450nm吸光度。以試劑盒所示的標準品繪制標準曲線,與標準曲線相比得到對應(yīng)濃度,再乘以相應(yīng)的稀釋倍數(shù),即得出樣品的實際濃度。 紅細胞CRl活性檢測采用紅細胞C3b受體花環(huán)(erythrocyte C3b receptor rosett, ECRR)試驗和紅細胞免疫復(fù)合物花環(huán)(erythrocyte immune complex rosette, EICR)試驗。按照郭峰法兩組血液樣本肝素抗凝,并于2h內(nèi)以1000轉(zhuǎn)/分離心5分鐘,用PBS洗滌4次,按紅細胞計數(shù)方法配成1.25×107/ml的使用液。檢測時待測紅細胞使用液100ul,分別加入補體致敏酵母多糖工作液100ul,酵母多糖試液100ul,都搖勻放37℃水浴30min,取出搖勻,加生理鹽水100ul,混勻后再加0.25%戊二醛50ul,輕輕混勻,取1/3量水平涂片,吹干,加甲醇固定,加少許瑞氏液,染色10秒鐘,自來水少許沖洗。高倍顯微鏡計數(shù),一個紅細胞上2個或2個以上酵母菌為一朵花環(huán)。計數(shù)200個紅細胞,算出百分率。 運用SPSS16.0軟件對相關(guān)數(shù)據(jù)進行統(tǒng)計學處理及分析,P0.05為差異有統(tǒng)計學意義。 結(jié)果： 紅細胞CR1數(shù)量檢查結(jié)果顯示,OSAHS患兒CR1數(shù)量及A值均低于對照組(P0.05),且CR1數(shù)量與呼吸暫停低通氣指數(shù)(apnea hypopnea index, AHI)呈負相關(guān)(r=-0.61,P0.05)；與最低血氧飽和度(lowest arterial oxygen saturation, LSaO2)呈正相關(guān)(r=0.53,P0.05)。紅細胞CR1活性檢測結(jié)果顯示,OSAHS患兒紅細胞C3b受體花環(huán)結(jié)合率明顯低于對照組(P0.05),而紅細胞免疫復(fù)合物花環(huán)結(jié)合率明顯高于對照組(P0.05),提示OSAHS患兒存在繼發(fā)性紅細胞免疫功能低下。 結(jié)論： 阻塞性睡眠呼吸暫停低通氣綜合征影響兒童紅細胞免疫功能,使紅細胞免疫功能下降,且與病情的嚴重程度有關(guān)。其紅細胞免疫功能的降低,可能會對整個免疫系統(tǒng)的功能帶來不良影響。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R766.4
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