艾滋病热毒蕴结证蛋白组学标志物验证研究
发布时间：2018-08-28 20:02
【摘要】：目的基于iTRAQ技术的艾滋病热毒蕴结证患者与健康人之间蛋白质差异表达的蛋白组学研究基础上,通过Western Blot技术对显著性差异蛋白进一步临床验证及中药干预研究,观察特征性蛋白在表达变化情况,探索药物疗效与证候特征性蛋白表达变化之间规律,为艾滋病热毒蕴结证具有诊断及疗效评价意义的蛋白分子标志物研究提供实验依据,指导临床中医艾滋病证候诊断和疗效评价。方法本研究应用艾复康胶囊联合高效抗逆转录病毒治疗(HAART)干预艾滋病期热毒蕴结证患者,疗程为48周。分别于0周、48周采集全血及提取后血清样本,检测血细胞、病载、CD4+T、肝肾功等临床基线指标。基于前期iTRAQ技术研究基础上,采用蛋白质印迹法(Western Blot)技术分别对采集的AIDS热毒蕴结证患者和健康对照组、热毒蕴结证患者用药前后血清标本进行差异蛋白检测,运用image J软件分析灰度值,将每泳道所得出目的蛋白的灰度值与相应的白蛋白灰度值做比较,得出相对灰度值,然后将每种抗体中的正常组的相对灰度值和病人组的相对灰度值导入至GraphPad Prism 5软件,得出nean+/-SD的图表。采用SPSS20.0统计软件统计相关临床资料。结果1、艾滋病期热毒蕴结证组与正常对照组比较,C5在艾滋病期热毒蕴结证表达下调,差异有统计学意义(P0.05),与iTRAQ筛选结果一致；keratin 10在艾滋病期热毒蕴结证表达下调,差异有统计学意义妒0.05),与iTRAQ筛选结果一致。2、艾滋病期热毒蕴结证患者药物干预后48周后与治疗前比较,CD4+T、CD8+T趋于稳定升高,CD4+T具有统计学意义(P0.05),CD8+T无统计学意义。3、艾滋病期热毒蕴结证用药前后比较,C5在艾滋病期热毒蕴结证用药后表达上调,差异有统计学意义(P0.05)。结论1、本研究得出艾滋病艾滋病期热毒蕴结证特征性蛋白质分别为C5、keratin10,这两者有助于为艾滋病期热毒蕴结证中医候诊断提供客观化依据。上述蛋白功能主要涉及应激反应、体液免疫应答、补体激活和先天免疫等,可能是热毒蕴结证相关生物学特征性蛋白质。2、本研究得出C5蛋白表达变化水平与艾滋病热毒蕴结证中药辨证论治治疗疗效趋势呈一致性,对于艾滋病热毒蕴结证的治疗具有疗效评价意义。3、本研究获得艾滋病艾滋病期热毒蕴结证验证及疗效干预的特征性表达蛋白,为建立艾滋病中医证候诊断模式,完善艾滋病辨证规范和辨证方法奠定基础。
[Abstract]:Objective on the basis of proteomics study on protein differential expression between patients with heat and toxin accumulation syndrome and healthy persons based on iTRAQ technique, to further verify the significance of differentially expressed proteins by Western Blot technique and to study the intervention of traditional Chinese medicine (TCM). To observe the change of characteristic protein expression and to explore the regularity between drug efficacy and syndrome characteristic protein expression, to provide experimental basis for the study of protein molecular markers with diagnostic and curative effect evaluation significance in the syndrome of heat toxin accumulation of AIDS. To guide the clinical diagnosis and evaluation of TCM AIDS syndrome. Methods Aifukang capsule combined with high effective antiretroviral therapy was used to treat (HAART) patients with heat toxin accumulation syndrome in the AIDS phase. The course of treatment was 48 weeks. Whole blood and serum samples were collected from 0 weeks to 48 weeks, respectively. The clinical baseline indexes such as blood cells, CD44T, liver and kidney function were detected. Based on the previous study of iTRAQ technique, the differential proteins were detected by Western blot (Western Blot) technique in the serum samples of the patients with AIDS heat toxin accumulation syndrome and healthy control group, and the patients with heat toxin accumulation syndrome before and after treatment. The gray value of the target protein was analyzed by image J software, and the relative gray value was obtained by comparing the gray value of the target protein with the corresponding gray value of albumin. Then the relative gray value of normal group and patient group were imported into GraphPad Prism 5 software, and the chart of nean / -SD was obtained. The clinical data were analyzed by SPSS20.0 software. Results 1. The expression of C5 was down-regulated in the heat toxin accumulation syndrome in the AIDS phase compared with that in the normal control group (P0.05). The expression of keratin 10 was down-regulated in the heat toxin accumulation syndrome in the AIDS phase in accordance with the results of iTRAQ screening. The difference was statistically significant (0. 05). The results of iTRAQ screening were in agreement with. 2. There was no significant difference between 48 weeks after drug intervention and before treatment in patients with hyperthermia and toxin accumulation syndrome (P0.05). (P0.05) CD8 T was not significantly increased in patients with hyperthermia and toxin accumulation syndrome (P0.05). Comparison of the expression of C5 before and after the treatment of heat toxin accumulation syndrome in the AIDS phase after the treatment of heat toxin accumulation syndrome in the AIDS phase, the expression of C5 was upregulated. The difference was statistically significant (P0.05). Conclusion 1. The characteristic proteins of heat toxin accumulation syndrome in AIDS stage are C5 keratin 10, which are helpful to provide objective basis for TCM diagnosis of heat toxin accumulation syndrome in AIDS period. 2. These proteins are mainly involved in stress response, humoral immune response, complement activation and innate immunity. This study showed that the expression level of C5 protein was consistent with the therapeutic effect of traditional Chinese medicine differentiation and treatment of heat toxin accumulation syndrome. It is significant to evaluate the curative effect of the treatment of heat toxin accumulation syndrome of AIDS. In this study, the characteristic expression protein of heat toxin accumulation syndrome and curative effect intervention was obtained in order to establish the diagnosis model of TCM syndrome of AIDS. Perfect AIDS dialectical standard and dialectical method lay the foundation.
【学位授予单位】：成都中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R259
本文编号：2210465
[Abstract]:Objective on the basis of proteomics study on protein differential expression between patients with heat and toxin accumulation syndrome and healthy persons based on iTRAQ technique, to further verify the significance of differentially expressed proteins by Western Blot technique and to study the intervention of traditional Chinese medicine (TCM). To observe the change of characteristic protein expression and to explore the regularity between drug efficacy and syndrome characteristic protein expression, to provide experimental basis for the study of protein molecular markers with diagnostic and curative effect evaluation significance in the syndrome of heat toxin accumulation of AIDS. To guide the clinical diagnosis and evaluation of TCM AIDS syndrome. Methods Aifukang capsule combined with high effective antiretroviral therapy was used to treat (HAART) patients with heat toxin accumulation syndrome in the AIDS phase. The course of treatment was 48 weeks. Whole blood and serum samples were collected from 0 weeks to 48 weeks, respectively. The clinical baseline indexes such as blood cells, CD44T, liver and kidney function were detected. Based on the previous study of iTRAQ technique, the differential proteins were detected by Western blot (Western Blot) technique in the serum samples of the patients with AIDS heat toxin accumulation syndrome and healthy control group, and the patients with heat toxin accumulation syndrome before and after treatment. The gray value of the target protein was analyzed by image J software, and the relative gray value was obtained by comparing the gray value of the target protein with the corresponding gray value of albumin. Then the relative gray value of normal group and patient group were imported into GraphPad Prism 5 software, and the chart of nean / -SD was obtained. The clinical data were analyzed by SPSS20.0 software. Results 1. The expression of C5 was down-regulated in the heat toxin accumulation syndrome in the AIDS phase compared with that in the normal control group (P0.05). The expression of keratin 10 was down-regulated in the heat toxin accumulation syndrome in the AIDS phase in accordance with the results of iTRAQ screening. The difference was statistically significant (0. 05). The results of iTRAQ screening were in agreement with. 2. There was no significant difference between 48 weeks after drug intervention and before treatment in patients with hyperthermia and toxin accumulation syndrome (P0.05). (P0.05) CD8 T was not significantly increased in patients with hyperthermia and toxin accumulation syndrome (P0.05). Comparison of the expression of C5 before and after the treatment of heat toxin accumulation syndrome in the AIDS phase after the treatment of heat toxin accumulation syndrome in the AIDS phase, the expression of C5 was upregulated. The difference was statistically significant (P0.05). Conclusion 1. The characteristic proteins of heat toxin accumulation syndrome in AIDS stage are C5 keratin 10, which are helpful to provide objective basis for TCM diagnosis of heat toxin accumulation syndrome in AIDS period. 2. These proteins are mainly involved in stress response, humoral immune response, complement activation and innate immunity. This study showed that the expression level of C5 protein was consistent with the therapeutic effect of traditional Chinese medicine differentiation and treatment of heat toxin accumulation syndrome. It is significant to evaluate the curative effect of the treatment of heat toxin accumulation syndrome of AIDS. In this study, the characteristic expression protein of heat toxin accumulation syndrome and curative effect intervention was obtained in order to establish the diagnosis model of TCM syndrome of AIDS. Perfect AIDS dialectical standard and dialectical method lay the foundation.
【学位授予单位】：成都中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R259
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