基于毛细管电泳—发光二极管诱导荧光的肝病患者血清蛋白质检测分析

发布时间：2018-07-17 15:31

【摘要】：肝硬化和原发性肝癌在疾病早期,患者无明显特异性症状,患者确诊时一般已是中晚期,肝硬化、肝癌的早期诊断对疾病的治疗尤为重要。血清蛋白质含量的差异是肝硬化、肝癌患者区别于正常人的特征值之一。近年来,毛细管电泳法因其高效分离、快速分析、微量进样等优势用于蛋白质的分离分析已成为研究热点。毛细管电泳诱导荧光检测法具有较高的灵敏度,在蛋白质检测方面与紫外吸收检测相比,它能更准确的反映血清中蛋白质含量的变化。因此,采用毛细管电泳-发光二极管诱导荧光法对血清蛋白质进行检测,建立正常人、肝硬化和原发性肝癌患者血清蛋白质的毛细管电泳荧光图谱,对比患者与正常人之间血清蛋白质的差异,为临床上肝病的诊断提供参考。研究目的：1.血清蛋白质含量的差异是肝硬化、肝癌患者区别于正常人的特征值之一。本文使用毛细管电泳-发光二极管诱导荧光检测法,对正常人、肝硬化、肝癌患者血清蛋白质进行检测,建立正常人、肝硬化、肝癌血清蛋白质毛细管电泳荧光图谱,对比三者血清蛋白质的差异,辅助临床上肝硬化、肝癌的早期诊断。2.在实验过程中发现,所使用的毛细管电泳仪在一些结构上存在缺陷,如废液池的位置、废液的排出方式等,这些存在的缺陷会降低检测结果的重复性。因此,为了准确地检测血清蛋白质,对一些结构进行改进,以提高毛细管电泳仪的重复性。研究方法：1.毛细管电泳-发光二极管诱导荧光(CE-LED-IF)检测方法中,血清蛋白质的检测依赖于结合在蛋白质分子上的FITC荧光基团,因此,首先对FITC标准品进行CE-LED-IF检测,考察电泳仪检测结果的重复性。研究发现降低检测结果重复性的原因,通过设计合理结构来消除影响,提高检测结果的重复性。2.对CE-LED-IF分离检测血清蛋白质的各个影响因素,如光源的选择、分离毛细管的长度、缓冲液pH值、缓冲液浓度、电泳分离电压、血清稀释比例、衍生用FITC与蛋白质的质量比等进行最优化选择。在最优化的电泳条件下,对正常人、肝硬化、肝癌血清进行电泳分离分析,建立正常人、肝硬化、肝癌患者的血清蛋白质毛细管电泳荧光图谱。研究结果：1.通过重新设计改进电泳卡槽上废液池的位置,消除因毛细管两端高度差带来的影响。改进前后,分别对2.5×10-3mg/mL FITC进行了10次检测。改进前,FITC的迁移时间和峰面积的相对标准偏差(RSD)是8.74%和6.57%,而改进后,FITC的迁移时间和峰面积的相对标准偏差(RSD)是1.14%和3.23%,说明改进后的卡槽用于毛细管电泳-LED诱导荧光检测提高了检测结果的重复性。2.分析比较影响CE-LED-IF检测血清蛋白质的各个影响因素：光源、分离毛细管的长度、缓冲液pH值、缓冲液浓度、电泳分离电压、血清稀释比例、衍生用FITC与蛋白质的质量比,确定CE-LED-IF检测血清蛋白质的最优化条件：中心波长470nm的LED作为激发光源,分离毛细管长度为53cm(有效分离长度50cm),缓冲液pH值为9.6,缓冲液浓度为10mmol/L,电泳分离电压为20kV,血清稀释比例为1：40,衍生用FITC与蛋白质的质量比为3：20。3.在最优化的电泳条件下,对60例正常人、肝硬化、肝癌患者的血清进行了电泳分离分析,建立了正常人、肝硬化、肝癌患者的血清蛋白质毛细管电泳荧光图谱。肝硬化、肝癌血清蛋白质毛细管电泳荧光图谱中,有区别于正常人图谱的特征蛋白质峰。结论：1.电泳卡槽的结构改进合理,提高了LED诱导荧光-毛细管电泳检测的重复性。2.电泳分离的影响因素,如光源的选择、分离毛细管的长度、缓冲液pH值、缓冲液浓度、电泳分离电压、血清稀释比例、衍生用FITC与蛋白质的质量比等会影响血清蛋白的检测结果。3.肝硬化、肝癌血清蛋白质毛细管电泳荧光图谱中,有区别于正常人图谱的特征蛋白质峰,可为临床上肝病的诊断提供参考。
[Abstract]:Liver cirrhosis and primary liver cancer have no obvious specific symptoms at the early stage of the disease. Patients are generally in the middle and late stages of diagnosis, cirrhosis, and early diagnosis of liver cancer are particularly important for the treatment of disease. The difference in serum protein content is one of the characteristics of liver cirrhosis and the liver cancer patients are distinguished from normal people. In recent years, capillary electrophoresis has been used for its High efficiency separation, rapid analysis, micro sampling and other advantages have become a hot spot in the separation and analysis of protein. Capillary electrophoresis induced fluorescence detection has high sensitivity. Compared with UV absorption detection, it can more accurately reflect the changes in protein content in serum. Therefore, capillary electrophoresis is used. Detection of serum protein by LED induced fluorescence, the capillary electrophoresis fluorescence spectrum of normal human, liver cirrhosis and primary liver cancer patients was established by capillary electrophoresis. The difference of serum protein between the patients and normal people was compared to provide a reference for the diagnosis of clinical liver disease. Objective: 1. the difference of serum protein content A capillary electrophoresis - led fluorescence detection method was used to detect the serum protein of normal people, liver cirrhosis and liver cancer, and to establish normal human, liver cirrhosis, liver cancer serum protein hair capillary electrophoresis fluorescence spectrum, and compare the difference of serum protein in the three cases. .2. in the early diagnosis of liver cirrhosis and liver cancer found that there are some defects in some structures, such as the location of the waste liquid pool, the discharge mode of waste liquid, and so on. These defects will reduce the repeatability of the detection results. The structure is improved to improve the repeatability of the capillary electrophoresis apparatus. In the 1. capillary electrophoresis - led induced fluorescence (CE-LED-IF) detection method, the detection of serum protein depends on the FITC fluorescent group binding on the protein molecules. Therefore, the first FITC standard product is detected by CE-LED-IF, and the detection junction of the electrophoretic instrument is investigated. The reproducibility of the results was found to reduce the reproducibility of the detection results by designing a reasonable structure to eliminate the effects and to improve the effects of the repetitive.2. on the detection of serum proteins by CE-LED-IF, such as the selection of the light source, the length of the separation capillary, the pH value of the buffer solution, the concentration of buffer solution, the separation voltage of electrophoresis, the serum Dilution ratio, the optimum selection for the mass ratio of FITC and protein. In the optimal electrophoresis conditions, the sera of normal people, liver cirrhosis and liver cancer were separated and analyzed by electrophoresis. The serum protein capillary electrophoresis of normal people, liver cirrhosis and liver cancer patients was established. Results: 1. by redesigning and improving electrophoresis, the results were improved. The position of the waste liquid pool on the slot eliminates the influence of the height difference at both ends of the capillary. Before and after improvement, 10 tests are carried out for 2.5 x 10-3mg/mL FITC respectively. Before the improvement, the relative standard deviation (RSD) of the migration time and peak area of the FITC is 8.74% and 6.57%, and the relative standard deviation (RSD) of the migration time and peak area of the FITC is 1.1 after the improvement. 4% and 3.23%, it shows that the improved card slot is used in capillary electrophoresis -LED induced fluorescence detection to improve the repeatability of.2. analysis of detection results. The influence factors of CE-LED-IF detection of serum protein are: light source, length of separated capillary, buffer pH value, buffer concentration, electrophoresis separation voltage, serum dilution ratio, derivative FIT C and protein mass ratio determine the optimal conditions for CE-LED-IF detection of serum protein: the central wavelength 470nm LED as the excitation source, the separation capillary length is 53cm (effective separation length 50cm), the buffer solution pH value is 9.6, the buffer solution concentration is 10mmol/L, the electrophoretic separation electrical pressure is 20kV, the serum dilution ratio is 1:40, the derivative uses FITC and egg with the FITC and egg. The mass ratio of white matter was 3:20.3. in the optimized electrophoresis conditions, the sera of 60 normal people, liver cirrhosis, and liver cancer patients were separated and analyzed by electrophoresis. The serum protein capillary electrophoresis fluorescence spectra of normal people, liver cirrhosis and liver cancer patients were established. The characteristic protein peak of normal human atlas. Conclusion: the structure of 1. electrophoretic card slot is improved reasonably, and the influence factors of LED induced fluorescence capillary electrophoresis are improved, such as the selection of the light source, the length of the separation capillary, the pH value of the buffer solution, the concentration of buffer solution, the voltage of the electrophoresis separation, the dilution ratio of the serum, the derivative of FITC. The mass ratio of protein to the protein can affect the results of serum protein detection.3. liver cirrhosis. In the serum protein capillary electrophoresis fluorescence spectrum of liver cancer, there is a characteristic protein peak different from the normal human atlas, which can provide reference for the diagnosis of clinical liver disease.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575

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