【摘要】：目的基于iTRAQ技術(shù)的艾滋病熱毒蘊(yùn)結(jié)證患者與健康人之間蛋白質(zhì)差異表達(dá)的蛋白組學(xué)研究基礎(chǔ)上,通過Western Blot技術(shù)對顯著性差異蛋白進(jìn)一步臨床驗(yàn)證及中藥干預(yù)研究,觀察特征性蛋白在表達(dá)變化情況,探索藥物療效與證候特征性蛋白表達(dá)變化之間規(guī)律,為艾滋病熱毒蘊(yùn)結(jié)證具有診斷及療效評價(jià)意義的蛋白分子標(biāo)志物研究提供實(shí)驗(yàn)依據(jù),指導(dǎo)臨床中醫(yī)艾滋病證候診斷和療效評價(jià)。方法本研究應(yīng)用艾復(fù)康膠囊聯(lián)合高效抗逆轉(zhuǎn)錄病毒治療(HAART)干預(yù)艾滋病期熱毒蘊(yùn)結(jié)證患者,療程為48周。分別于0周、48周采集全血及提取后血清樣本,檢測血細(xì)胞、病載、CD4+T、肝腎功等臨床基線指標(biāo)�；谇捌趇TRAQ技術(shù)研究基礎(chǔ)上,采用蛋白質(zhì)印跡法(Western Blot)技術(shù)分別對采集的AIDS熱毒蘊(yùn)結(jié)證患者和健康對照組、熱毒蘊(yùn)結(jié)證患者用藥前后血清標(biāo)本進(jìn)行差異蛋白檢測,運(yùn)用image J軟件分析灰度值,將每泳道所得出目的蛋白的灰度值與相應(yīng)的白蛋白灰度值做比較,得出相對灰度值,然后將每種抗體中的正常組的相對灰度值和病人組的相對灰度值導(dǎo)入至GraphPad Prism 5軟件,得出nean+/-SD的圖表。采用SPSS20.0統(tǒng)計(jì)軟件統(tǒng)計(jì)相關(guān)臨床資料。結(jié)果1、艾滋病期熱毒蘊(yùn)結(jié)證組與正常對照組比較,C5在艾滋病期熱毒蘊(yùn)結(jié)證表達(dá)下調(diào),差異有統(tǒng)計(jì)學(xué)意義(P0.05),與iTRAQ篩選結(jié)果一致；keratin 10在艾滋病期熱毒蘊(yùn)結(jié)證表達(dá)下調(diào),差異有統(tǒng)計(jì)學(xué)意義妒0.05),與iTRAQ篩選結(jié)果一致。2、艾滋病期熱毒蘊(yùn)結(jié)證患者藥物干預(yù)后48周后與治療前比較,CD4+T、CD8+T趨于穩(wěn)定升高,CD4+T具有統(tǒng)計(jì)學(xué)意義(P0.05),CD8+T無統(tǒng)計(jì)學(xué)意義。3、艾滋病期熱毒蘊(yùn)結(jié)證用藥前后比較,C5在艾滋病期熱毒蘊(yùn)結(jié)證用藥后表達(dá)上調(diào),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論1、本研究得出艾滋病艾滋病期熱毒蘊(yùn)結(jié)證特征性蛋白質(zhì)分別為C5、keratin10,這兩者有助于為艾滋病期熱毒蘊(yùn)結(jié)證中醫(yī)候診斷提供客觀化依據(jù)。上述蛋白功能主要涉及應(yīng)激反應(yīng)、體液免疫應(yīng)答、補(bǔ)體激活和先天免疫等,可能是熱毒蘊(yùn)結(jié)證相關(guān)生物學(xué)特征性蛋白質(zhì)。2、本研究得出C5蛋白表達(dá)變化水平與艾滋病熱毒蘊(yùn)結(jié)證中藥辨證論治治療療效趨勢呈一致性,對于艾滋病熱毒蘊(yùn)結(jié)證的治療具有療效評價(jià)意義。3、本研究獲得艾滋病艾滋病期熱毒蘊(yùn)結(jié)證驗(yàn)證及療效干預(yù)的特征性表達(dá)蛋白,為建立艾滋病中醫(yī)證候診斷模式,完善艾滋病辨證規(guī)范和辨證方法奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：成都中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R259

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