發(fā)布時(shí)間：2018-01-05 12:04

  本文關(guān)鍵詞：慢性萎縮性胃炎模型大鼠定量蛋白組學(xué)及中藥干預(yù)靶點(diǎn)研究　出處：《中國(guó)中醫(yī)科學(xué)院》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 慢性萎縮性胃炎 定量蛋白組學(xué) 高通量組學(xué)數(shù)據(jù)分析 半夏瀉心湯加減

【摘要】：背景及目的慢性萎縮性胃炎(chronic atrophic gastritis,CAG)是慢性胃炎的亞型之一。胃黏膜萎縮,腸上皮化生(intestinal metaplasia,IM)及伴發(fā)的異型增生(Dysplasia,Dys),與胃癌發(fā)病密切相關(guān)。CAG是最常見(jiàn)的胃癌前疾病。CAG發(fā)病率及檢出率隨年齡增長(zhǎng)而增加,已經(jīng)嚴(yán)重威脅著我國(guó)人民的身體健康。目前,對(duì)于CAG的發(fā)病原因、發(fā)病機(jī)制尚不明確,亦無(wú)有效藥物治療。本病因?yàn)椴∏檫M(jìn)展緩慢、診斷及療效評(píng)價(jià)需行胃鏡及病理活檢有創(chuàng)檢查,病變常常灶性分布,而且患者多為中老年人,往往合并其他慢性病,病情復(fù)雜,使得針對(duì)本病的臨床試驗(yàn)較難開(kāi)展。復(fù)制本病動(dòng)物疾病模型以大大縮短病程,去除不必要因素對(duì)于疾病影響,尤其規(guī)避倫理問(wèn)題,可有力地推動(dòng)本病的病因?qū)W、發(fā)病學(xué)以及防治方法的研究。由于CAG病因復(fù)雜,西醫(yī)學(xué)對(duì)CAG尚缺乏理想的治療措施,而中醫(yī)藥辨證治療療效甚佳,不但能緩解癥狀,在延緩病理進(jìn)展甚至逆轉(zhuǎn)病變方面有一定優(yōu)勢(shì)。隨著對(duì)CAG認(rèn)識(shí)的不斷深入和現(xiàn)代分子生物技術(shù)的發(fā)展,中醫(yī)藥治療CAG的機(jī)制研究也得到了深入和發(fā)展。CAG動(dòng)物模型是深入研究發(fā)生機(jī)制和評(píng)價(jià)相關(guān)藥物效果的必不可少的工具。目前CAG大鼠模型造模方法眾多,缺乏客觀評(píng)價(jià)。針對(duì)以上問(wèn)題,本文展開(kāi)如下研究:對(duì)比兩種不同慢性萎縮性胃炎大鼠復(fù)合造模法,通過(guò)成模時(shí)間、模型成功率、大鼠死亡率、血清胃蛋白酶原檢測(cè)、病理組織學(xué)觀察等方面確定穩(wěn)定造模方法;利用TMT定量蛋白組學(xué)技術(shù)鑒定慢性萎縮性胃炎模型大鼠與正常大鼠胃黏膜的差異蛋白,采用高通量組學(xué)數(shù)據(jù)分析,闡釋本病發(fā)病的生物學(xué)基本分子機(jī)制,結(jié)合蛋白富集分析及節(jié)點(diǎn)蛋白連接度分析篩選靶點(diǎn)蛋白,利用酶聯(lián)免疫吸附(ELISA)及實(shí)時(shí)熒光聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)檢測(cè)靶點(diǎn)蛋白在CAG模型大鼠胃黏膜中異常表達(dá),從而驗(yàn)證蛋白組學(xué)結(jié)果;選用以半夏瀉心湯加減,立法于辛開(kāi)苦降、益氣活血的中藥協(xié)定方為干預(yù)藥物,探索中藥對(duì)CAG的治療作用,初步篩選中藥作用靶點(diǎn)。動(dòng)物實(shí)驗(yàn)分為以下三部分。1.慢性萎縮性胃炎動(dòng)物模型的探索材料與方法實(shí)驗(yàn)動(dòng)物SPF級(jí)4周齡健康雄性SD大鼠60只。按體重隨機(jī)分為3組。正常組大鼠 8 只;N-甲基-N'-硝基-N-亞硝基胍(N-Methyl-N-nitroso-N'-nitroguanidine,MNNG)復(fù)合高鹽熱淀粉糊造模法組(M1組)26只SD大鼠;MNNG復(fù)合酒精綜合造模法(M2組)26只SD大鼠。正常組大鼠予SPF級(jí)正常水料飼養(yǎng),每天灌胃1次5ml生理鹽水。M1組予0.03%雷尼替丁飼料喂養(yǎng),自由飲用2%水楊酸鈉溶液,每日灌胃120μg/mLMNNG次,每周禁食1次,每次18小時(shí),自第14周起禁食第2天灌胃高鹽熱淀粉糊,當(dāng)天不再灌胃MNNG溶液。M2組大鼠予0.03%雷尼替丁飼料喂養(yǎng),每日自由飲用2%水楊酸鈉溶液,每日灌胃120 u g/mL MNNG 1次,每周禁食1次,每次18小時(shí),自第14周起禁食第2天灌胃35%酒精,當(dāng)天不再灌胃MNNG溶液,連續(xù)造模至18周,M1、M2組大鼠每2周隨機(jī)處死2只,取胃觀察胃黏膜病理改變。通過(guò)成模時(shí)間、模型成功率、大鼠死亡率、血清胃蛋白酶原檢測(cè)、病理組織學(xué)觀察等方面確定穩(wěn)定造模方法。結(jié)果MNNG復(fù)合酒精灌胃造模方法及MNNG復(fù)合高鹽熱淀粉糊造模方法均可較好復(fù)制CAG病變過(guò)程;兩組大鼠胃黏膜病理積分、血清胃蛋白酶原水平無(wú)統(tǒng)計(jì)學(xué)差異(P0.05);兩組大鼠肝細(xì)胞HE染色病理切片觀察均發(fā)現(xiàn)空泡變性,為早期肝臟損傷表現(xiàn),考慮與MNNG在肝臟中代謝相關(guān);MNNG復(fù)合酒精灌胃造模方法動(dòng)物死亡率較低,并發(fā)癥較少,可用于后續(xù)萎縮性胃炎動(dòng)物試驗(yàn)的應(yīng)用研究。2.慢性萎縮性胃炎模型大鼠定量蛋白組學(xué)研究材料與方法利用TMT(Tandem Mass Tags)定量蛋白組學(xué)技術(shù)篩選MNNG復(fù)合酒精灌胃CAG模型大鼠胃竇黏膜組織(制備方法同實(shí)驗(yàn)1)與正常大鼠胃竇黏膜組織的差異蛋白,采用高通量組學(xué)數(shù)據(jù)分析闡釋慢性萎縮性胃炎胃黏膜發(fā)生的生物學(xué)基本分子機(jī)制。并結(jié)合蛋白功能富集分析結(jié)果、信息通路富集結(jié)果、蛋白互作網(wǎng)絡(luò)分析、節(jié)點(diǎn)蛋白連接度分析,篩選關(guān)鍵靶點(diǎn)蛋白。結(jié)果(1)通過(guò)TMT定量蛋白組學(xué)鑒定CAG大鼠胃竇黏膜組織與正常大鼠胃竇黏膜組織的差異表達(dá)蛋白共鑒定6937個(gè)蛋白,有統(tǒng)計(jì)學(xué)意義差異蛋白363個(gè),采用GO 分析軟件 David 生物信息學(xué)軟件(http://david.abcc.ncifcrf.gov/summary.jsp)分別從分子功能、亞細(xì)胞定位和生物學(xué)途徑、信號(hào)轉(zhuǎn)導(dǎo)通路方面對(duì)差異蛋白進(jìn)行功能注釋。分子功能富集顯示差異蛋白主要為binding蛋白,亞細(xì)胞定位在細(xì)胞質(zhì)、細(xì)胞外的外來(lái)體、細(xì)胞外空間、線粒體、胞質(zhì)、粘著斑、細(xì)胞質(zhì)細(xì)胞核周圍的地區(qū)等區(qū)域,生物學(xué)途徑主要參與應(yīng)對(duì)藥物、應(yīng)對(duì)乙醇、應(yīng)對(duì)饑餓,其他在蛋白質(zhì)水解、缺氧、補(bǔ)體經(jīng)典反應(yīng)、細(xì)胞粘附等生物過(guò)程起到作用。涉及粘著斑通路、MAPK信號(hào)通路、阿米巴病、病毒致癌、緊密連接、腫瘤的蛋白聚糖、補(bǔ)體系統(tǒng)、蛋白質(zhì)消化吸收等多條信號(hào)轉(zhuǎn)導(dǎo)通路,經(jīng)STRING網(wǎng)軟件分析表明差異蛋白互作網(wǎng)絡(luò)復(fù)雜。(2)并將互作網(wǎng)絡(luò)數(shù)據(jù)導(dǎo)出到Cytoscape3.2軟件中,根據(jù)節(jié)點(diǎn)蛋白連接度分析,判斷網(wǎng)絡(luò)中心節(jié)點(diǎn)蛋白。綜合KEGG數(shù)據(jù)庫(kù)的信號(hào)通路分類、功能注釋等,篩選出關(guān)鍵蛋白選取FLNa、TGFR-β 1、Bad蛋白,以ELISA、RT-PCR方法驗(yàn)證本次蛋白組學(xué)實(shí)驗(yàn)結(jié)果(見(jiàn)實(shí)驗(yàn)3)。(3)通過(guò)對(duì)差異蛋白進(jìn)行分析發(fā)現(xiàn)具有促凋亡作用的Casp3降低,而B(niǎo)cl-2家族促凋亡因子Bad上調(diào)。Caspase家族處于Bcl-2家族下游。Bad的促凋亡作用依賴于與Bcl-2結(jié)合,蛋白組學(xué)結(jié)果并未顯示Bcl-2表達(dá)異常。為了探究Bad表達(dá)的上調(diào)對(duì)Bcl-2家族調(diào)控的凋亡的影響,采用RT-PCR方法鑒定CAG模型大鼠與正常大鼠胃竇黏膜中Bcl-2 mRNA表達(dá)(見(jiàn)實(shí)驗(yàn)3)。3.差異蛋白的驗(yàn)證及中藥干預(yù)慢性萎縮性胃炎大鼠作用靶點(diǎn)研究材料與方法100只SPF級(jí)SD大鼠根據(jù)體重隨機(jī)分為兩組�？瞻捉M(N組)10只,給以SPF級(jí)動(dòng)物標(biāo)準(zhǔn)飲食喂養(yǎng),每日灌胃生理鹽水1次,持續(xù)至實(shí)驗(yàn)結(jié)束。造模組90只,按上述方法進(jìn)行CAG造模,于實(shí)驗(yàn)第18,20,22,24,26周末各隨機(jī)抽檢2只處死,取胃黏膜觀察病理改變。造模成功后,將剩余大鼠按體重隨機(jī)分成4組,模型組(M組),模型對(duì)照組(C組),慢性萎縮性胃炎協(xié)定方高劑量組(G組)、慢性萎縮性胃炎協(xié)定方低劑量組(D組)。成模后N組、M組大鼠即刻處死。其余大鼠均給予SPF級(jí)動(dòng)物標(biāo)準(zhǔn)飲食喂養(yǎng)。C組予生理鹽水3mL/kg灌胃,每日1次;G組、D組分別予慢性萎縮性胃炎協(xié)定方配方顆粒劑制備藥液5ml,分別相當(dāng)于顆粒劑3g/kg/d、1.5g/kg/d(相當(dāng)于生草藥21.9g/kg/d、11g/kg/d)灌胃,每日1次。治療階段持續(xù)8周,治療結(jié)束后處死大鼠。觀察大鼠胃黏膜病理改變,評(píng)價(jià)中藥協(xié)定方治療CAG的療效。利用ELISA法測(cè)定N組、M組胃竇黏膜TGF-β1、FLNa、Bad蛋白表達(dá)水平,RT-PCR法測(cè)定N組、M組、G組、D組胃竇黏膜 TGF-β1、FLNa、Bad、Bcl-2 的 mRNA 水平。結(jié)果(1)中藥協(xié)定方可顯著改善CAG模型大鼠胃竇黏膜萎縮(P0.05)、異型增生(P0.05),腸上皮化生積分較模型組大鼠有所下降,但未見(jiàn)明顯統(tǒng)計(jì)學(xué)差異。(2)ELISA法測(cè)定TGF-β1、FLNa、Bad蛋白在M組、C組大鼠胃黏膜表達(dá)較N組上調(diào)(P0.05);RT-PCR法測(cè)定M組、C組大鼠胃黏膜TGF-β 1mRNA、FLNamRNA、BadmRNA表達(dá)較N組上調(diào)(P0.05)。M組、C組大鼠與N組大鼠Bcl-2的mRNA表達(dá)水平無(wú)明顯差異,與蛋白組學(xué)結(jié)果一致�？紤]CAG模型大鼠胃黏膜中異常上調(diào)的Bad蛋白可能無(wú)活性,未能引起下游Bcl-2家族的表達(dá)改變。(3)RT-PCR測(cè)定中藥組TGF-β1、FLNa、Bad的mRNA表達(dá)量較M組、C組水平下調(diào)(P0.05)。結(jié)論(1)本研究參照相關(guān)文獻(xiàn)并加以改進(jìn),對(duì)比MNNG溶液復(fù)合酒精灌胃及MNNG復(fù)合高熱淀粉糊灌胃,綜合對(duì)比成模時(shí)間、死亡率、病理轉(zhuǎn)化率、肝臟病理改變等確定MNNG溶液復(fù)合酒精灌胃模型該模型操作簡(jiǎn)單易行,且穩(wěn)定性較好,但仍有耗時(shí)較長(zhǎng)的不足,有待于今后進(jìn)一步改進(jìn)。(2)采用高通量蛋白組學(xué)法篩選MNNG溶液復(fù)合酒精灌胃模型大鼠與正常大鼠胃竇黏膜間差異蛋白,通過(guò)GO分析及Pathway分析可說(shuō)明,CAG胃黏膜病變涉及多項(xiàng)、復(fù)雜生物途徑,影響粘著斑、MAPK、緊密連接、蛋白聚糖改變、磷脂酰肌醇信號(hào)系統(tǒng)等多條通路。根據(jù)節(jié)點(diǎn)蛋白連接度分析篩選出FLNa、Bad、及TGF-β1為關(guān)鍵蛋白,并用ELISA、RT-PCR方法驗(yàn)證結(jié)果與蛋白組學(xué)一致。(3)慢性萎縮性胃炎中藥協(xié)定方立法辛開(kāi)苦降、益氣活血法,治療CAG可明顯改善CAG大鼠模型胃黏膜萎縮、異型增生病理積分,作用靶點(diǎn)可能與下調(diào)CAG胃黏膜異常表達(dá)的FLNa、Bad、及TGF-β1等疾病關(guān)鍵蛋白相關(guān),但其治療CAG的具體分子作用機(jī)制,還需進(jìn)一步研究
[Abstract]:Background and objective: Chronic Atrophic Gastritis (chronic atrophic, gastritis, CAG) is a subtype of chronic gastritis. Gastric mucosal atrophy, intestinal metaplasia (intestinal metaplasia, IM) and dysplasia associated with (Dysplasia, Dys),.CAG is closely related with the incidence of gastric cancer is the most common precancerous disease incidence and.CAG the detection rate increased with age, has been a serious threat to our health. At present, the cause of CAG, the pathogenesis is not clear. There is no effective drug for the treatment of this disease. Because of the slow progression, evaluation of diagnosis and curative effect for gastroscopy and biopsy is invasive, often focal lesions the distribution of patients, and for the elderly, often associated with other chronic diseases, the condition is complex, the clinical trials in this disease. This disease is more difficult to carry out the replication of animal disease model to shorten the course of disease, the removal of unnecessary factors on In the disease, especially to avoid ethical problems, can effectively promote the etiology of this disease, study the pathogenesis and prevention methods. The etiology of CAG is complex, western medicine on CAG is still lack of ideal treatment measures, and TCM treatment effect is very good, not only can relieve symptoms, there are certain advantages in delay and even reverse the pathological progress lesions. With the development of deeper understanding of CAG and modern molecular biological technology, mechanism of traditional Chinese medicine in the treatment of CAG has been deepening and development of.CAG animal model is an essential tool for further study on the pathogenesis and evaluation of drug effect. At present, the rat models of CAG many methods, aiming at the lack of objective evaluation. The above problems, this paper carried out the following research: a comparison of two different chronic atrophic gastritis rats by composite molding method, molding time, the success rate of the model, rat mortality, serum Pepsinogen, histopathological observation, etc. to determine the stable model; differential protein using TMT quantitative proteomics technology to identify the model of chronic atrophic gastritis rats and normal gastric mucosa of rats, using high-throughput genomics data analysis, biological interpretation of the disease's basic molecular mechanism, binding protein analysis and enrichment protein node connectivity analysis Screening of target protein by enzyme linked immunosorbent assay (ELISA) and real time fluorescent polymerase chain reaction (RT-PCR) detection of target protein expression in CAG rat model of gastric mucosa, so as to verify the proteomics results; selection with Banxia Xiexin Decoction, the legislation on the xinkaikujiang. Traditional Chinese medicine of Tonifying Qi and activating blood for drug intervention, to explore the therapeutic effect of Chinese medicine on CAG, preliminary screening targets of traditional Chinese medicine. The animal experiment is divided into the following three parts of.1. in chronic atrophic gastritis animal model Materials and methods to explore experimental animal SPF male healthy SD rats aged 4 weeks. 60 rats were randomly divided into 3 groups. 8 normal rats in the control group; N- methyl -N'- nitro -N- nitrosoguanidine (N-Methyl-N-nitroso-N'-nitroguanidine, MNNG) composite high salt hot starch paste molding group (M1 group) 26 SD rat; MNNG composite alcohol complex modeling method (group M2) in 26 SD rats. The normal rats were given SPF normal water feeding, were administered 1 5ml normal saline.M1 group were given 0.03% ranitidine diet, drinking 2% sodium salicylate solution, intragastric administration of 120 g/mLMNNG 1 times a day, fasting every week, 18 hours each time, from the fourteenth week, second days of fasting intragastric administration of high salt hot starch paste, the day no longer intragastric administration of MNNG solution.M2 rats were given ranitidine 0.03% diet, daily drinking 2% sodium salicylate solution, 120 u g/mL MNNG daily gavage 1 times, 1 times a week fasting for 18 hours. Every time, since fourteenth Week second days fasting intragastric administration of 35% alcohol, the day no longer intragastric administration of MNNG solution, continuous modeling to 18 weeks, M1, M2 group rats were sacrificed every 2 weeks 2, the stomach gastric mucosa pathological changes were observed. Through the model, the success rate of the model, rat mortality, serum pepsinogen detection. Histopathological observation on stable modeling method. Results MNNG composite intragastric modeling method and MNNG composite high salt hot starch paste molding method can better replicate CAG disease process; two groups of rat gastric mucosa pathological scores, no significant difference of serum pepsinogen level (P0.05); the two groups of rat liver cells HE stained pathological sections were found in vacuolar degeneration and early liver damage, and consider the MNNG in liver metabolism; MNNG composite alcohol gavage model animal mortality is low, fewer complications, and can be used for atrophic gastritis animal test Study on Application of.2. model in rats with chronic atrophic gastritis quantitative proteomics research materials and methods by using TMT (Tandem Mass Tags) quantitative proteomics screening of MNNG composite intragastric mucosa of gastric CAG rats (preparation method with experiment 1) proteins and gastric mucous membrane tissue of normal rats the high-throughput analysis of biological groups, the molecular mechanisms underlying the interpretation of gastric mucosa of chronic atrophic gastritis. Combined with the data of protein function enrichment analysis results, the information pathway enrichment results, protein interaction network analysis, node protein connectivity analysis, screening key target protein. Results (1) protein identified 6937 protein the expression difference of gastric mucosa and identification of CAG rat and normal rat gastric mucosa by TMT quantitative protein group, there were statistically significant differences in protein 363, using GO analysis software David Bioinformatics Software (http://david.abcc.ncifcrf.gov/summary.jsp) separately from the molecular function, subcellular localization and biological pathways, signal transduction pathway for functional annotation of proteins. The molecular function of binding proteins mainly showed enrichment of protein subcellular localization in cytoplasm, foreign body outside the cell, extracellular space, mitochondria, cytoplasm, focal adhesions, cytoplasmic region the area around the nucleus, key biological pathways in response to drugs, with ethanol, other in response to starvation, hypoxia, protein hydrolysis, classical reaction complement, cell adhesion and other biological processes play a role. Involved in focal adhesion pathway, MAPK signaling pathway, amebiasis, virus carcinogenesis, tight junctions, tumor proteoglycans, complement system protein digestion and absorption of several signal transduction pathways, the STRING network software analysis showed that the difference of protein interaction network (2) and complex. The interaction of network data into Cytoscape3.2 software, according to the node protein connectivity analysis, determine the center node of the network signaling protein. Classification of KEGG database, functional annotation, and screened the key protein selected FLNa, TGFR- beta 1, Bad protein, ELISA, RT-PCR method to verify the proteomics results (see Experiment 3). (3) based on the differential protein analysis showed that with the pro apoptotic effect of Casp3 decreased, while the Bcl-2 family of Pro apoptotic factor Bad upregulation of.Caspase family in apoptosis of Bcl-2 family depends on the downstream.Bad combined with Bcl-2 proteomics results did not show abnormal expression of Bcl-2. In order to explore the expression of apoptosis Bad increase of Bcl-2 family control, using the method of RT-PCR expression of Bcl-2 mRNA identification of CAG rats and normal rats gastric antrum mucosa (see Experiment 3).3. proteins proved slow and traditional Chinese medicine intervention 鎬ц悗緙╂，

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