【摘要】：肝硬化和原發(fā)性肝癌在疾病早期,患者無(wú)明顯特異性癥狀,患者確診時(shí)一般已是中晚期,肝硬化、肝癌的早期診斷對(duì)疾病的治療尤為重要。血清蛋白質(zhì)含量的差異是肝硬化、肝癌患者區(qū)別于正常人的特征值之一。近年來(lái),毛細(xì)管電泳法因其高效分離、快速分析、微量進(jìn)樣等優(yōu)勢(shì)用于蛋白質(zhì)的分離分析已成為研究熱點(diǎn)。毛細(xì)管電泳誘導(dǎo)熒光檢測(cè)法具有較高的靈敏度,在蛋白質(zhì)檢測(cè)方面與紫外吸收檢測(cè)相比,它能更準(zhǔn)確的反映血清中蛋白質(zhì)含量的變化。因此,采用毛細(xì)管電泳-發(fā)光二極管誘導(dǎo)熒光法對(duì)血清蛋白質(zhì)進(jìn)行檢測(cè),建立正常人、肝硬化和原發(fā)性肝癌患者血清蛋白質(zhì)的毛細(xì)管電泳熒光圖譜,對(duì)比患者與正常人之間血清蛋白質(zhì)的差異,為臨床上肝病的診斷提供參考。研究目的：1.血清蛋白質(zhì)含量的差異是肝硬化、肝癌患者區(qū)別于正常人的特征值之一。本文使用毛細(xì)管電泳-發(fā)光二極管誘導(dǎo)熒光檢測(cè)法,對(duì)正常人、肝硬化、肝癌患者血清蛋白質(zhì)進(jìn)行檢測(cè),建立正常人、肝硬化、肝癌血清蛋白質(zhì)毛細(xì)管電泳熒光圖譜,對(duì)比三者血清蛋白質(zhì)的差異,輔助臨床上肝硬化、肝癌的早期診斷。2.在實(shí)驗(yàn)過(guò)程中發(fā)現(xiàn),所使用的毛細(xì)管電泳儀在一些結(jié)構(gòu)上存在缺陷,如廢液池的位置、廢液的排出方式等,這些存在的缺陷會(huì)降低檢測(cè)結(jié)果的重復(fù)性。因此,為了準(zhǔn)確地檢測(cè)血清蛋白質(zhì),對(duì)一些結(jié)構(gòu)進(jìn)行改進(jìn),以提高毛細(xì)管電泳儀的重復(fù)性。研究方法：1.毛細(xì)管電泳-發(fā)光二極管誘導(dǎo)熒光(CE-LED-IF)檢測(cè)方法中,血清蛋白質(zhì)的檢測(cè)依賴于結(jié)合在蛋白質(zhì)分子上的FITC熒光基團(tuán),因此,首先對(duì)FITC標(biāo)準(zhǔn)品進(jìn)行CE-LED-IF檢測(cè),考察電泳儀檢測(cè)結(jié)果的重復(fù)性。研究發(fā)現(xiàn)降低檢測(cè)結(jié)果重復(fù)性的原因,通過(guò)設(shè)計(jì)合理結(jié)構(gòu)來(lái)消除影響,提高檢測(cè)結(jié)果的重復(fù)性。2.對(duì)CE-LED-IF分離檢測(cè)血清蛋白質(zhì)的各個(gè)影響因素,如光源的選擇、分離毛細(xì)管的長(zhǎng)度、緩沖液pH值、緩沖液濃度、電泳分離電壓、血清稀釋比例、衍生用FITC與蛋白質(zhì)的質(zhì)量比等進(jìn)行最優(yōu)化選擇。在最優(yōu)化的電泳條件下,對(duì)正常人、肝硬化、肝癌血清進(jìn)行電泳分離分析,建立正常人、肝硬化、肝癌患者的血清蛋白質(zhì)毛細(xì)管電泳熒光圖譜。研究結(jié)果：1.通過(guò)重新設(shè)計(jì)改進(jìn)電泳卡槽上廢液池的位置,消除因毛細(xì)管兩端高度差帶來(lái)的影響。改進(jìn)前后,分別對(duì)2.5×10-3mg/mL FITC進(jìn)行了10次檢測(cè)。改進(jìn)前,FITC的遷移時(shí)間和峰面積的相對(duì)標(biāo)準(zhǔn)偏差(RSD)是8.74%和6.57%,而改進(jìn)后,FITC的遷移時(shí)間和峰面積的相對(duì)標(biāo)準(zhǔn)偏差(RSD)是1.14%和3.23%,說(shuō)明改進(jìn)后的卡槽用于毛細(xì)管電泳-LED誘導(dǎo)熒光檢測(cè)提高了檢測(cè)結(jié)果的重復(fù)性。2.分析比較影響CE-LED-IF檢測(cè)血清蛋白質(zhì)的各個(gè)影響因素：光源、分離毛細(xì)管的長(zhǎng)度、緩沖液pH值、緩沖液濃度、電泳分離電壓、血清稀釋比例、衍生用FITC與蛋白質(zhì)的質(zhì)量比,確定CE-LED-IF檢測(cè)血清蛋白質(zhì)的最優(yōu)化條件：中心波長(zhǎng)470nm的LED作為激發(fā)光源,分離毛細(xì)管長(zhǎng)度為53cm(有效分離長(zhǎng)度50cm),緩沖液pH值為9.6,緩沖液濃度為10mmol/L,電泳分離電壓為20kV,血清稀釋比例為1：40,衍生用FITC與蛋白質(zhì)的質(zhì)量比為3：20。3.在最優(yōu)化的電泳條件下,對(duì)60例正常人、肝硬化、肝癌患者的血清進(jìn)行了電泳分離分析,建立了正常人、肝硬化、肝癌患者的血清蛋白質(zhì)毛細(xì)管電泳熒光圖譜。肝硬化、肝癌血清蛋白質(zhì)毛細(xì)管電泳熒光圖譜中,有區(qū)別于正常人圖譜的特征蛋白質(zhì)峰。結(jié)論：1.電泳卡槽的結(jié)構(gòu)改進(jìn)合理,提高了LED誘導(dǎo)熒光-毛細(xì)管電泳檢測(cè)的重復(fù)性。2.電泳分離的影響因素,如光源的選擇、分離毛細(xì)管的長(zhǎng)度、緩沖液pH值、緩沖液濃度、電泳分離電壓、血清稀釋比例、衍生用FITC與蛋白質(zhì)的質(zhì)量比等會(huì)影響血清蛋白的檢測(cè)結(jié)果。3.肝硬化、肝癌血清蛋白質(zhì)毛細(xì)管電泳熒光圖譜中,有區(qū)別于正常人圖譜的特征蛋白質(zhì)峰,可為臨床上肝病的診斷提供參考。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575

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