慢性萎缩性胃炎模型大鼠定量蛋白组学及中药干预靶点研究

发布时间：2018-01-05 12:04

  本文关键词：慢性萎缩性胃炎模型大鼠定量蛋白组学及中药干预靶点研究　出处：《中国中医科学院》2017年博士论文　论文类型：学位论文

  更多相关文章： 慢性萎缩性胃炎 定量蛋白组学 高通量组学数据分析 半夏泻心汤加减

【摘要】：背景及目的慢性萎缩性胃炎(chronic atrophic gastritis,CAG)是慢性胃炎的亚型之一。胃黏膜萎缩,肠上皮化生(intestinal metaplasia,IM)及伴发的异型增生(Dysplasia,Dys),与胃癌发病密切相关。CAG是最常见的胃癌前疾病。CAG发病率及检出率随年龄增长而增加,已经严重威胁着我国人民的身体健康。目前,对于CAG的发病原因、发病机制尚不明确,亦无有效药物治疗。本病因为病情进展缓慢、诊断及疗效评价需行胃镜及病理活检有创检查,病变常常灶性分布,而且患者多为中老年人,往往合并其他慢性病,病情复杂,使得针对本病的临床试验较难开展。复制本病动物疾病模型以大大缩短病程,去除不必要因素对于疾病影响,尤其规避伦理问题,可有力地推动本病的病因学、发病学以及防治方法的研究。由于CAG病因复杂,西医学对CAG尚缺乏理想的治疗措施,而中医药辨证治疗疗效甚佳,不但能缓解症状,在延缓病理进展甚至逆转病变方面有一定优势。随着对CAG认识的不断深入和现代分子生物技术的发展,中医药治疗CAG的机制研究也得到了深入和发展。CAG动物模型是深入研究发生机制和评价相关药物效果的必不可少的工具。目前CAG大鼠模型造模方法众多,缺乏客观评价。针对以上问题,本文展开如下研究:对比两种不同慢性萎缩性胃炎大鼠复合造模法,通过成模时间、模型成功率、大鼠死亡率、血清胃蛋白酶原检测、病理组织学观察等方面确定稳定造模方法;利用TMT定量蛋白组学技术鉴定慢性萎缩性胃炎模型大鼠与正常大鼠胃黏膜的差异蛋白,采用高通量组学数据分析,阐释本病发病的生物学基本分子机制,结合蛋白富集分析及节点蛋白连接度分析筛选靶点蛋白,利用酶联免疫吸附(ELISA)及实时荧光聚合酶链式反应(RT-PCR)检测靶点蛋白在CAG模型大鼠胃黏膜中异常表达,从而验证蛋白组学结果;选用以半夏泻心汤加减,立法于辛开苦降、益气活血的中药协定方为干预药物,探索中药对CAG的治疗作用,初步筛选中药作用靶点。动物实验分为以下三部分。1.慢性萎缩性胃炎动物模型的探索材料与方法实验动物SPF级4周龄健康雄性SD大鼠60只。按体重随机分为3组。正常组大鼠 8 只;N-甲基-N'-硝基-N-亚硝基胍(N-Methyl-N-nitroso-N'-nitroguanidine,MNNG)复合高盐热淀粉糊造模法组(M1组)26只SD大鼠;MNNG复合酒精综合造模法(M2组)26只SD大鼠。正常组大鼠予SPF级正常水料饲养,每天灌胃1次5ml生理盐水。M1组予0.03%雷尼替丁饲料喂养,自由饮用2%水杨酸钠溶液,每日灌胃120μg/mLMNNG次,每周禁食1次,每次18小时,自第14周起禁食第2天灌胃高盐热淀粉糊,当天不再灌胃MNNG溶液。M2组大鼠予0.03%雷尼替丁饲料喂养,每日自由饮用2%水杨酸钠溶液,每日灌胃120 u g/mL MNNG 1次,每周禁食1次,每次18小时,自第14周起禁食第2天灌胃35%酒精,当天不再灌胃MNNG溶液,连续造模至18周,M1、M2组大鼠每2周随机处死2只,取胃观察胃黏膜病理改变。通过成模时间、模型成功率、大鼠死亡率、血清胃蛋白酶原检测、病理组织学观察等方面确定稳定造模方法。结果MNNG复合酒精灌胃造模方法及MNNG复合高盐热淀粉糊造模方法均可较好复制CAG病变过程;两组大鼠胃黏膜病理积分、血清胃蛋白酶原水平无统计学差异(P0.05);两组大鼠肝细胞HE染色病理切片观察均发现空泡变性,为早期肝脏损伤表现,考虑与MNNG在肝脏中代谢相关;MNNG复合酒精灌胃造模方法动物死亡率较低,并发症较少,可用于后续萎缩性胃炎动物试验的应用研究。2.慢性萎缩性胃炎模型大鼠定量蛋白组学研究材料与方法利用TMT(Tandem Mass Tags)定量蛋白组学技术筛选MNNG复合酒精灌胃CAG模型大鼠胃窦黏膜组织(制备方法同实验1)与正常大鼠胃窦黏膜组织的差异蛋白,采用高通量组学数据分析阐释慢性萎缩性胃炎胃黏膜发生的生物学基本分子机制。并结合蛋白功能富集分析结果、信息通路富集结果、蛋白互作网络分析、节点蛋白连接度分析,筛选关键靶点蛋白。结果(1)通过TMT定量蛋白组学鉴定CAG大鼠胃窦黏膜组织与正常大鼠胃窦黏膜组织的差异表达蛋白共鉴定6937个蛋白,有统计学意义差异蛋白363个,采用GO 分析软件 David 生物信息学软件(http://david.abcc.ncifcrf.gov/summary.jsp)分别从分子功能、亚细胞定位和生物学途径、信号转导通路方面对差异蛋白进行功能注释。分子功能富集显示差异蛋白主要为binding蛋白,亚细胞定位在细胞质、细胞外的外来体、细胞外空间、线粒体、胞质、粘着斑、细胞质细胞核周围的地区等区域,生物学途径主要参与应对药物、应对乙醇、应对饥饿,其他在蛋白质水解、缺氧、补体经典反应、细胞粘附等生物过程起到作用。涉及粘着斑通路、MAPK信号通路、阿米巴病、病毒致癌、紧密连接、肿瘤的蛋白聚糖、补体系统、蛋白质消化吸收等多条信号转导通路,经STRING网软件分析表明差异蛋白互作网络复杂。(2)并将互作网络数据导出到Cytoscape3.2软件中,根据节点蛋白连接度分析,判断网络中心节点蛋白。综合KEGG数据库的信号通路分类、功能注释等,筛选出关键蛋白选取FLNa、TGFR-β 1、Bad蛋白,以ELISA、RT-PCR方法验证本次蛋白组学实验结果(见实验3)。(3)通过对差异蛋白进行分析发现具有促凋亡作用的Casp3降低,而Bcl-2家族促凋亡因子Bad上调。Caspase家族处于Bcl-2家族下游。Bad的促凋亡作用依赖于与Bcl-2结合,蛋白组学结果并未显示Bcl-2表达异常。为了探究Bad表达的上调对Bcl-2家族调控的凋亡的影响,采用RT-PCR方法鉴定CAG模型大鼠与正常大鼠胃窦黏膜中Bcl-2 mRNA表达(见实验3)。3.差异蛋白的验证及中药干预慢性萎缩性胃炎大鼠作用靶点研究材料与方法100只SPF级SD大鼠根据体重随机分为两组。空白组(N组)10只,给以SPF级动物标准饮食喂养,每日灌胃生理盐水1次,持续至实验结束。造模组90只,按上述方法进行CAG造模,于实验第18,20,22,24,26周末各随机抽检2只处死,取胃黏膜观察病理改变。造模成功后,将剩余大鼠按体重随机分成4组,模型组(M组),模型对照组(C组),慢性萎缩性胃炎协定方高剂量组(G组)、慢性萎缩性胃炎协定方低剂量组(D组)。成模后N组、M组大鼠即刻处死。其余大鼠均给予SPF级动物标准饮食喂养。C组予生理盐水3mL/kg灌胃,每日1次;G组、D组分别予慢性萎缩性胃炎协定方配方颗粒剂制备药液5ml,分别相当于颗粒剂3g/kg/d、1.5g/kg/d(相当于生草药21.9g/kg/d、11g/kg/d)灌胃,每日1次。治疗阶段持续8周,治疗结束后处死大鼠。观察大鼠胃黏膜病理改变,评价中药协定方治疗CAG的疗效。利用ELISA法测定N组、M组胃窦黏膜TGF-β1、FLNa、Bad蛋白表达水平,RT-PCR法测定N组、M组、G组、D组胃窦黏膜 TGF-β1、FLNa、Bad、Bcl-2 的 mRNA 水平。结果(1)中药协定方可显著改善CAG模型大鼠胃窦黏膜萎缩(P0.05)、异型增生(P0.05),肠上皮化生积分较模型组大鼠有所下降,但未见明显统计学差异。(2)ELISA法测定TGF-β1、FLNa、Bad蛋白在M组、C组大鼠胃黏膜表达较N组上调(P0.05);RT-PCR法测定M组、C组大鼠胃黏膜TGF-β 1mRNA、FLNamRNA、BadmRNA表达较N组上调(P0.05)。M组、C组大鼠与N组大鼠Bcl-2的mRNA表达水平无明显差异,与蛋白组学结果一致。考虑CAG模型大鼠胃黏膜中异常上调的Bad蛋白可能无活性,未能引起下游Bcl-2家族的表达改变。(3)RT-PCR测定中药组TGF-β1、FLNa、Bad的mRNA表达量较M组、C组水平下调(P0.05)。结论(1)本研究参照相关文献并加以改进,对比MNNG溶液复合酒精灌胃及MNNG复合高热淀粉糊灌胃,综合对比成模时间、死亡率、病理转化率、肝脏病理改变等确定MNNG溶液复合酒精灌胃模型该模型操作简单易行,且稳定性较好,但仍有耗时较长的不足,有待于今后进一步改进。(2)采用高通量蛋白组学法筛选MNNG溶液复合酒精灌胃模型大鼠与正常大鼠胃窦黏膜间差异蛋白,通过GO分析及Pathway分析可说明,CAG胃黏膜病变涉及多项、复杂生物途径,影响粘着斑、MAPK、紧密连接、蛋白聚糖改变、磷脂酰肌醇信号系统等多条通路。根据节点蛋白连接度分析筛选出FLNa、Bad、及TGF-β1为关键蛋白,并用ELISA、RT-PCR方法验证结果与蛋白组学一致。(3)慢性萎缩性胃炎中药协定方立法辛开苦降、益气活血法,治疗CAG可明显改善CAG大鼠模型胃黏膜萎缩、异型增生病理积分,作用靶点可能与下调CAG胃黏膜异常表达的FLNa、Bad、及TGF-β1等疾病关键蛋白相关,但其治疗CAG的具体分子作用机制,还需进一步研究
[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悗缂╂，

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