基于自身红细胞构建的溶血系统检测总补体活性及其与MICA基因多态性的关系
发布时间：2018-07-31 17:18
【摘要】：目的:本文在CH50溶血法的基础上,以人红细胞替代绵羊红细胞,将多管法转化为单管法,创建一种操作简单,稳定可靠的检测血浆补体总活性的新方法。同时本文应用该法检测ICU患者,冠心病患者,健康体检者的血浆补体总活性,并在分子水平上研究了冠心病患者补体活性与MICA*008基因携带的关系。方法:1.首先以绵羊红细胞替代人红细胞,兔抗人红细胞抗体替代兔抗羊红细胞抗体(溶血素)进行溶血试验,证明不同红细胞浓度和血浆浓度对红细胞破坏后释放血红蛋白效能的影响。并通过CH50多管法溶血试验确定发生溶血反应的各种试剂的最适浓度,创建单管溶血比色法。同时通过线性研究和重复试验验证了该法的准确度和稳定性。2.使用CH50标准法检测血浆和血清中的补体活性,并使用单管溶血比色法检测以不同人红细胞作为溶血指示系统的同一份血浆中的补体活性,进一步验证单管溶血比色法的可行性。3.使用CH50法和单管溶血比色法检测同一份血浆的补体活性,验证两种方法的相关性。4.使用单管溶血比色试验检测ICU患者,冠心病患者和健康体检者的补体总活性,证明该方法有一定的临床应用价值。同时采用PCR技术检测冠心病患者和健康者的MICA*008基因携带情况,分析冠心病患者补体活性与MICA*008基因的关系。结果:1.本文应用人红细胞和兔抗人红细胞抗体替代绵羊红细胞和溶血素进行溶血试验,结果发现不同浓度的红细胞对溶血程度的影响低于不同浓度的血浆对溶血程度的影响,同时,本文得出当血浆浓度为20%时,红细胞浓度的不同对红细胞破坏后释放血红蛋白的效能影响最低。因此本文将20%红细胞悬液所对应第九管(20%血浆浓度)中各成分的含量,作为单管溶血比色法中各成分比例的参考。本文应用单管溶血比色法检测不同浓度血浆的补体活性,并连续5天测定同一份血浆的补体活性,结果证明不同浓度血浆的补体总活性呈线性分布,连续5天所测定的补体活性值的变异系数CV=11.3%,说明单管溶血比色试验具有一定的准确性和稳定性。2.通过CH50标准法检测血浆和血清中的补体活性,本文得出血浆和血清中的补体活性完全相同,因此可以使用血浆替代血清标本进行补体活性测定,补体检测技术得到了优化。同时,通过不同人红细胞作为溶血指示系统检测同一份血浆中的补体活性,本文得出血浆中的补体活性大致相同(P0.05),说明不同红细胞不会影响血浆中的补体含量,单管溶血法具有可行性。3.该方法中测定的溶血OD值与CH50溶血法所测的补体活性值两者呈线性相关,说明单管溶血比色法与CH50法具有显著的相关关系,单管溶血比色法可以替代CH50溶血法检测补体总活性。4.ICU患者和冠心病患者的补体活性要显著低于健康体检者的补体活性,单管溶血比色法有一定的临床应用价值。冠心病患者和健康体检者的MICA*008基因分布大致相同,同时冠心病患者的补体水平与MICA*008的携带没有显著关联。结论:1.单管溶血法检测补体活性具有良好的准确性,稳定性和可行性,并且与CH50法具有显著的相关关系,该法进一步优化CH50溶血试验,具有一定的临床应用价值。2.ICU患者和冠心病患者的补体活性较低,说明ICU患者的机体免疫功能紊乱,冠心病患者机体或许处于炎症反应阶段。3.冠心病患者的补体水平与MICA*008基因没有显著关联。
[Abstract]:Objective: on the basis of CH50 hemolysis, we use human erythrocytes to replace sheep red blood cells and convert multi tube method into single tube method to create a new method to detect the total complement activity of plasma complement, which is simple and stable and reliable. At the same time, this method is used to detect the total complement activity of ICU patients, coronary heart disease patients and health examiners. The relationship between the complement activity of the patients with coronary heart disease and the carrying of MICA*008 gene was studied. Methods: 1. first, the human red blood cells were replaced by the sheep red blood cells, and the Rabbit anti human erythrocyte antibody replaced the Rabbit anti sheep erythrocyte antibody (hemolysin) to carry out the hemolysis test. It was proved that the different red cell concentration and plasma concentration were able to release the hemoglobin after the red blood cells were destroyed. The most suitable concentration of various reagents in hemolysis was determined by the CH50 multi tube hemolysis test, and a single tube hemolysis colorimetric method was created. Meanwhile, the accuracy and stability of the method was verified by linear and repeated tests..2. was used to detect the complement activity in plasma and serum by CH50 standard method, and the single tube hemolysis colorimetric method was used. The method was used to detect the complement activity in the same plasma with different human red cells as a hemolytic indicator system. The feasibility of single tube hemolysis colorimetric method was further verified by.3. and single tube hemolysis colorimetric assay to detect the complement activity of the same plasma by CH50 method and single tube hemolysis colorimetry. The correlation.4. of two methods was tested for the detection of ICU patients by single tube hemolysis test. The total complement activity of the patients with heart disease and health checkup proves that the method has certain clinical value. At the same time, PCR technique is used to detect the MICA*008 gene in patients with coronary heart disease and healthy people, and the relationship between the complement activity and the MICA*008 gene of the patients with coronary heart disease is analyzed. 1. the results of the results are the use of human red blood cells and Rabbit anti human erythrocyte antibodies. The results showed that the effect of different concentrations of red blood cells on the degree of hemolysis was lower than that of the plasma at different concentrations. At the same time, the effect of the different concentration of red blood cells on the efficiency of hemoglobin release after the broken red cells was 20%, so the effect of the different concentration of red blood cells on the release of hemoglobin was the lowest. The content of each component in the ninth tube (20% plasma concentration) of the 20% red cell suspension was used as a reference for the proportion of the components in the single tube hemolysis colorimetric method. The complement activity of the plasma was detected by the single tube hemolysis colorimetry, and the complement activity of the same plasma was measured for 5 days. The results showed that the plasma was supplemental at different concentrations. The total body activity was linearly distributed and the variation coefficient of the complement activity value was CV=11.3% for 5 days. It showed that the single tube hemolysis colorimetric test had certain accuracy and stability..2. was used to detect the complement activity in plasma and serum by CH50 standard method. The complement activity in plasma and serum was the same, so plasma could be used in plasma. The complement activity was measured and the complement detection technique was optimized. At the same time, the complement activity in the same plasma was detected by different human red cells as a hemolytic indicator system. The complement activity in the bleeding pulp was approximately the same (P0.05), indicating that different erythrocytes would not affect the complement content in the plasma and the single tube hemolysis. The method is feasible..3. the hemolysis o value measured in this method is linearly related to the value of the complement activity measured by the CH50 hemolysis method. It shows that the single tube hemolysis colorimetric method has a significant correlation with the CH50 method. The single tube hemolysis colorimetric method can replace the CH50 hemolysis method to detect the complement activity of the total complement active.4.ICU patients and the coronary heart disease patients. The single tube hemolysis colorimetric method has a certain clinical value below the complement activity of the health examiners. The MICA*008 gene distribution in the patients with coronary heart disease and the healthy persons is roughly the same, and the complement level of the patients with coronary heart disease is not significantly associated with the MICA*008. Conclusion: 1. single tube hemolysis has good accuracy for the detection of complement activity. Sex, stability and feasibility, and have a significant correlation with the CH50 method, this method further optimizes the CH50 hemolysis test, which has certain clinical application value of.2.ICU patients and coronary heart disease patients with lower complement activity, indicating the immune function disorder of the body of ICU patients, the body of the patients with coronary heart disease may be in the stage of.3. coronary heart disease in the stage of inflammation. There was no significant correlation between complement level and MICA*008 gene.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R541.4;R446.1
本文编号：2156294
[Abstract]:Objective: on the basis of CH50 hemolysis, we use human erythrocytes to replace sheep red blood cells and convert multi tube method into single tube method to create a new method to detect the total complement activity of plasma complement, which is simple and stable and reliable. At the same time, this method is used to detect the total complement activity of ICU patients, coronary heart disease patients and health examiners. The relationship between the complement activity of the patients with coronary heart disease and the carrying of MICA*008 gene was studied. Methods: 1. first, the human red blood cells were replaced by the sheep red blood cells, and the Rabbit anti human erythrocyte antibody replaced the Rabbit anti sheep erythrocyte antibody (hemolysin) to carry out the hemolysis test. It was proved that the different red cell concentration and plasma concentration were able to release the hemoglobin after the red blood cells were destroyed. The most suitable concentration of various reagents in hemolysis was determined by the CH50 multi tube hemolysis test, and a single tube hemolysis colorimetric method was created. Meanwhile, the accuracy and stability of the method was verified by linear and repeated tests..2. was used to detect the complement activity in plasma and serum by CH50 standard method, and the single tube hemolysis colorimetric method was used. The method was used to detect the complement activity in the same plasma with different human red cells as a hemolytic indicator system. The feasibility of single tube hemolysis colorimetric method was further verified by.3. and single tube hemolysis colorimetric assay to detect the complement activity of the same plasma by CH50 method and single tube hemolysis colorimetry. The correlation.4. of two methods was tested for the detection of ICU patients by single tube hemolysis test. The total complement activity of the patients with heart disease and health checkup proves that the method has certain clinical value. At the same time, PCR technique is used to detect the MICA*008 gene in patients with coronary heart disease and healthy people, and the relationship between the complement activity and the MICA*008 gene of the patients with coronary heart disease is analyzed. 1. the results of the results are the use of human red blood cells and Rabbit anti human erythrocyte antibodies. The results showed that the effect of different concentrations of red blood cells on the degree of hemolysis was lower than that of the plasma at different concentrations. At the same time, the effect of the different concentration of red blood cells on the efficiency of hemoglobin release after the broken red cells was 20%, so the effect of the different concentration of red blood cells on the release of hemoglobin was the lowest. The content of each component in the ninth tube (20% plasma concentration) of the 20% red cell suspension was used as a reference for the proportion of the components in the single tube hemolysis colorimetric method. The complement activity of the plasma was detected by the single tube hemolysis colorimetry, and the complement activity of the same plasma was measured for 5 days. The results showed that the plasma was supplemental at different concentrations. The total body activity was linearly distributed and the variation coefficient of the complement activity value was CV=11.3% for 5 days. It showed that the single tube hemolysis colorimetric test had certain accuracy and stability..2. was used to detect the complement activity in plasma and serum by CH50 standard method. The complement activity in plasma and serum was the same, so plasma could be used in plasma. The complement activity was measured and the complement detection technique was optimized. At the same time, the complement activity in the same plasma was detected by different human red cells as a hemolytic indicator system. The complement activity in the bleeding pulp was approximately the same (P0.05), indicating that different erythrocytes would not affect the complement content in the plasma and the single tube hemolysis. The method is feasible..3. the hemolysis o value measured in this method is linearly related to the value of the complement activity measured by the CH50 hemolysis method. It shows that the single tube hemolysis colorimetric method has a significant correlation with the CH50 method. The single tube hemolysis colorimetric method can replace the CH50 hemolysis method to detect the complement activity of the total complement active.4.ICU patients and the coronary heart disease patients. The single tube hemolysis colorimetric method has a certain clinical value below the complement activity of the health examiners. The MICA*008 gene distribution in the patients with coronary heart disease and the healthy persons is roughly the same, and the complement level of the patients with coronary heart disease is not significantly associated with the MICA*008. Conclusion: 1. single tube hemolysis has good accuracy for the detection of complement activity. Sex, stability and feasibility, and have a significant correlation with the CH50 method, this method further optimizes the CH50 hemolysis test, which has certain clinical application value of.2.ICU patients and coronary heart disease patients with lower complement activity, indicating the immune function disorder of the body of ICU patients, the body of the patients with coronary heart disease may be in the stage of.3. coronary heart disease in the stage of inflammation. There was no significant correlation between complement level and MICA*008 gene.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R541.4;R446.1
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