艾滋病脾肾阳虚证蛋白组学标志物验证研究

发布时间：2018-03-16 18:39

本文选题：艾滋病　切入点：中药　出处：《成都中医药大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的本研究通过Western Blot技术对前期iTRAQ技术筛选出的艾滋病脾肾阳虚证患者的显著性差异蛋白进行验证,以期获得脾肾阳虚证候的特征性表达蛋白。并进一步予相应中药干预后观察蛋白表达变化,探索药物疗效与证候特征性蛋白表达变化之间规律。方法采集健康对照组、艾滋病脾肾阳虚证患者治疗前及治疗48周后的外周血,分离血清,提取蛋白质,经过蛋白质消化后进行Western Blot检测。运用imageJ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到GraphPad Prism5软件。采用IBM SPSS Statistics V20.0软件进行统计学方法分析。计数资料采用Fisher精确概率法。正态分布资料采用均数±标准差(x±s)表示。用以P0.05标准作为差异性标准。结果1、艾滋病期脾肾阳虚证用药前与正常对照组比较,RBP4在艾滋病期脾肾阳虚证表达上调,差异有统计学意义(P0.05),与iTRAQ筛选结果一致；C7在艾滋病期脾肾阳虚证表达上调,差异有统计学意义(P0.05),与iTRAQ筛选结果一致。2、无症状期脾肾阳虚证用药前与正常对照组比较,RBP4在无症状期脾肾阳虚组表达上调,差异有统计学意义(P0.05),与iTRAQ筛选结果一致,且比艾滋病期蛋白含量低。C7在无症状期脾肾阳虚证组表达下调,与iTRAQ筛选结果不一致。3、艾滋病期脾肾阳虚证患者药物干预后48周后与治疗前比较,CD4+、CD8趋于稳定升高,CD4具有统计学意义(P0.05),CD8无统计学意义。无症状期脾肾阳虚证患者治疗后一年与治疗前比较,CD4+、CD8趋于稳定升高,CD4具有统计学意义(P0.05)。4、艾滋病期脾肾阳虚证用药前后比较,RBP4在艾滋病期脾肾阳虚证用药后表达下调,差异有统计学意义(P0.05)。C7在艾滋病期脾肾阳虚证用药后表达上调。5、无症状期脾肾阳虚证用药前后比较,RBP4在无症状期脾肾阳虚组表达下,差异有统计学意义(P0.05),但与艾滋病期相比蛋白含量低。C7在无症状期脾肾阳虚证组表达下调。结论1、本研究得出艾滋病脾肾阳虚证存在特征性表达蛋白。RBP4可作为脾肾阳虚证无症状期向艾滋病期进展的特征性表达蛋白,可能与其氧化应激、炎症反应或代谢综合征相关反应相关。C7可以作为艾滋病期脾肾阳虚证特征性表达蛋白,可能与其免疫反应功能及涉及的细胞溶解和细胞死亡等过程相关。2、本研究显示RBP4蛋白表达变化水平与艾滋病脾肾阳虚证中药辨证论治治疗疗效趋势呈一致性,对于艾滋病脾肾阳虚证的治疗具有疗效评价意义。
[Abstract]:Objective in this study, the Western Blot technique was used to verify the differential protein of the patients with deficiency of spleen and kidney yang in AIDS patients who were screened by iTRAQ technique in the early stage. In order to obtain the characteristic expression protein of spleen and kidney yang deficiency syndrome, and to observe the change of protein expression after intervention of corresponding Chinese medicine, to explore the law between the effect of medicine and the change of characteristic protein expression of syndrome. Methods the healthy control group was collected. The peripheral blood of AIDS patients with deficiency of spleen and kidney yang was separated before and 48 weeks after treatment, the serum was isolated, the protein was extracted, the Western Blot was detected after protein digestion, and the gray value was analyzed by imageJ software. Compare the gray value of the target protein with the corresponding gray value of albumin, and get the relative gray value. Then, the relative gray value of normal group and patient group were imported into GraphPad Prism5 software. The statistical method was analyzed by IBM SPSS Statistics V20.0 software. The counting data were counted by Fisher exact probability method. The data were expressed by mean 卤standard deviation (x 卤s). P05 standard was used as the difference standard. Results 1. The expression of RBP4 was up-regulated in the patients with deficiency of spleen and kidney yang in the AIDS phase compared with the control group. The difference was statistically significant (P 0.05), which was consistent with the results of iTRAQ screening. The expression of C7 was up-regulated in the syndrome of deficiency of spleen and kidney yang in AIDS stage. The difference was statistically significant (P 0.05), which was consistent with the screening results of iTRAQ. The expression of RBP4 was up-regulated in asymptomatic Spleen-Kidney Yang deficiency group and normal control group before medication, and the difference was statistically significant (P 0.05), which was consistent with that of iTRAQ screening. The protein content was lower than that of AIDS phase. C7 expression was down-regulated in the asymptomatic group with deficiency of spleen and kidney yang. The results of iTRAQ screening were not consistent with the results of iTRAQ screening. 3. There was no significant difference between 48 weeks after drug intervention and before treatment in patients with deficiency of spleen and kidney yang in AIDS. One year after treatment, CD _ 4 and CD _ 8 tended to increase steadily. CD _ 4 was significantly higher than that before treatment. The expression of RBP4 was down-regulated before and after the treatment of deficiency of spleen and kidney yang in AIDS stage. The difference was statistically significant (P 0.05). C7 expression was upregulated after treatment of deficiency of spleen and kidney yang in AIDS stage, and RBP4 expression in asymptomatic group of spleen and kidney yang deficiency was compared before and after treatment of asymptomatic syndrome of spleen and kidney yang deficiency. The difference was statistically significant (P 0.05), but the protein content was lower. C7 expression was down-regulated in the asymptomatic syndrome group of deficiency of spleen and kidney yang. Conclusion 1. This study shows that there is a characteristic expression protein. RBP4 can be used as spleen and kidney syndrome in AIDS. The characteristic expression protein of the progression from asymptomatic phase to AIDS stage of Yang deficiency syndrome, It may be related to oxidative stress, inflammatory reaction or metabolic syndrome related reactions. C7 may be used as the characteristic expression protein of spleen and kidney yang deficiency syndrome in AIDS. This study showed that the expression level of RBP4 protein was consistent with the therapeutic effect of TCM differentiation and treatment of the deficiency of spleen and kidney yang syndrome in AIDS, which may be related to its immunoreactive function and the process of cell dissolution and cell death, etc. In this study, the expression level of RBP4 protein was consistent with the trend of TCM treatment based on syndrome differentiation. For AIDS spleen and kidney yang deficiency syndrome treatment has the curative effect evaluation significance.
【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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