基于差异蛋白组学对外湿环境下脾阳虚大鼠的发病机制研究

发布时间：2018-08-23 21:08

【摘要】：目的:明确人工模拟外湿环境对正常及脾阳虚大鼠一般情况、脾组织形态学改变、脾组织蛋白质差异表达的影响,探讨湿邪对不同体质(正常与脾阳虚)机体的致病机理。方法:1.选取40只SD大鼠随机分为对照组、脾阳虚组、外湿组、脾阳虚加外湿组,每组10只。脾阳虚组模型制作采用番泻叶灌胃与皮下注射利血平交替进行并强迫大鼠游泳的复合方法。脾阳虚造模后,给予外湿组与脾阳虚加外湿组人工模拟外湿环境一周,于造模前后观察并记录各组大鼠的一般情况包括体质量、进食量、饮水量、精神活动、皮毛光泽度及大便性状并进行宏观症候评分。2.模拟外湿环境造模完成后,无菌摘除脾脏,采用Con A诱导脾淋巴细胞增殖,以此表示淋巴细胞的功能水平。记录并计算脾淋巴细胞增殖能力。3.模拟外湿环境造模完成后,取脾组织进行常规脱水、浸蜡、包埋。每个组织块切取5张切片,60℃恒温箱内过夜干燥切片,5张切片均做常规的HE染色,光镜下观察形态学变化。4.模拟外湿环境造模完成后,取出脾组织10mg/只,进行双向电泳实验以及图象分析,采用肽质量指纹图谱和电喷雾串联质谱的方法鉴定蛋白质。结果:1.外湿对正常及脾阳虚大鼠一般情况的比较。外湿环境下脾阳虚大鼠较正常大鼠体质量明显减轻,脾阳虚加外湿组大鼠体质量(239.30±37.60)较外湿组(321.70±18.74)显著降低,P0.01。脾阳虚加外湿组大鼠在进食、饮水、精神状态、毛色、大便性状等方面较外湿组发生了一定的变化。脾阳虚加外湿组大鼠进食量(19.33±2.88)较外湿组(23.83±2.93)降低,P0.05。外湿组(37.50±1.87)、脾阳虚加外湿组大鼠饮水量(36.67±4.18)较对照组(42.67±2.34)显著降低,P0.01。脾阳虚加外湿组大鼠造模初期出现情绪逐渐改变,呈现易激怒、好争斗状态,放在泳池中游泳时,纷纷跃起。造模后逐渐出现精神萎靡不振,扎堆,嗜睡,反应迟钝,行动迟缓,毛色枯槁,消瘦,粪便软、不成形,甚至稀溏。外湿组大鼠脱毛现象较轻,精神状态较好,偶可见嗜睡,抓取时叫声比较温柔。2.外湿对正常及脾阳虚大鼠脾淋巴细胞增殖力的比较。外湿环境下,脾阳虚大鼠与正常大鼠脾淋巴细胞增殖力较对照组均显著降低。与对照组脾淋巴细胞增殖力(47.94±1.13)相比,外湿组脾淋巴细胞增殖力(15.15±2.18)、脾阳虚加外湿组脾淋巴细胞增殖力(14.42±2.24)均显著降低P0.01。脾阳虚加外湿组脾淋巴细胞增殖力(14.42±2.24)较外湿组脾淋巴细胞增殖力(15.15±2.18)无差别P=0.476,P0.05。与脾阳虚加外湿组脾淋巴细胞增殖力(14.42±2.24)相比,脾阳虚组脾淋巴细胞增殖力(36.07±3.05)显著增强P0.01。3.外湿对正常及脾阳虚大鼠脾组织形态学的改变。外湿组大鼠脾组织白髓边缘带增宽,白髓发生中心增大,脾索内见吞噬浅黄色色素细胞。脾阳虚加外湿组大鼠脾组织白髓边缘带增宽较明显,脾窦内见吞噬浅黄色色素细胞,脾窦扩张、充血,脾索内见吞噬浅黄色色素细胞。4.外湿对正常及脾阳虚大鼠脾组织差异蛋白斑点表达情况的比较。各实验组选三个样本进行差异蛋白组学比较,两两比较差异蛋白图谱,进行蛋白质组学分析,鉴定出40个蛋白斑点,脾阳虚加外湿组与对照组比较表达上调的有13个,表达下调的有20个;外湿组与对照组比较表达上调的有14个,表达下调的有19个;外湿组与脾阳虚加外湿组比较表达上调的有15个,表达下调的有10个。5.外湿对正常及脾阳虚大鼠脾组织差异蛋白质的比较。在实验组2-DE凝胶上从表达差异蛋白质斑点中选取点影像清晰且表达水平改变明显的22个蛋白质斑点作为最终质谱鉴定的对象,利用PMF的方法成功鉴定了20个蛋白质。外湿环境下正常大鼠表达上调的有11个,它们是载脂蛋白A-I、苹果酸脱氢酶、特定短链酰基辅酶A脱氢酶、半乳糖凝集素5、低分子量的磷酸酪氨酸蛋白磷酸酶LMW-PTP、阴离子型胰蛋白酶原-2、胞内氯离子通道蛋白1、f肌动蛋白β亚基、磷酸甘油酸变位酶1、膜突蛋白、肌动蛋白相关蛋白2/3复合物亚基5,表达下调的有4个,它们是3-磷酸甘油醛脱氢酶、哺乳动物过氧化物酶2、Igκ链C区,B等位基、结合珠蛋白。外湿环境下脾阳虚大鼠表达上调的有9个,它们是载脂蛋白A-I、苹果酸脱氢酶、特定短链酰基辅酶A脱氢酶、半乳糖凝集素5、低分子量的磷酸酪氨酸蛋白磷酸酶LMW-PTP、阴离子型胰蛋白酶原-2、胞内氯离子通道蛋白1、磷酸甘油酸变位酶1、膜突蛋白,表达下调的有5个,它们是3-磷酸甘油醛脱氢酶、哺乳动物过氧化物酶2、结蛋白、Igκ链C区,B等位基、肌球蛋白调节轻链9。结论:1.模拟外湿环境因素对正常及脾阳虚组大鼠的一般情况都有影响,包括体质量、进食量、饮水量、精神状态、活动度、皮毛光泽、皮肤黏膜、排泄物等方面,并且对脾阳虚组影响更为明显。2.模拟外湿环境因素显著降低了正常及脾阳虚组大鼠细胞免疫功能。模拟外湿环境因素(外因)相对于脾阳虚因素(内因)对机体免疫抑制作用更强。模拟外湿环境因素(外因)与脾阳虚因素(内因)具有协同致机体免疫机能下降的作用。3.模拟外湿环境因素对正常及脾阳虚大鼠脾组织形态学都有影响,但脾阳虚大鼠脾组织形态改变明显,表现在组织细胞病变加重。4.模拟外湿环境因素对正常及脾阳虚大鼠脾组织蛋白质存在差异表达。模拟外湿环境因素与脾阳虚因素作用于机体差异蛋白质有其特异性,但也存在交集,且交集很大。这些差异蛋白质功能与细胞结构、能量代谢、细胞凋亡、细胞增殖与分化、信号转导、钙稳态调节、免疫应答、免疫保护、肿瘤发生相关。不同因素作用下的特异性差异蛋白质可能是与脾阳虚证及湿邪侵袭相关的疾病特异性蛋白,并有可能成为诊断脾阳虚证的分子标志物及湿邪致病的分子标志物。
[Abstract]:Objective: To investigate the effect of dampness pathogen on normal and spleen yang deficiency rats, spleen histomorphology and protein differential expression, and to explore the pathogenesis of dampness pathogen in different constitutions (normal and Spleen Yang deficiency). Methods: 40 SD rats were randomly divided into control group, spleen yang deficiency group, external dampness group, spleen yang deficiency plus external dampness group. The model of Spleen-Yang deficiency group was made by senna leaf gavage and subcutaneous injection of reserpine alternately and forced to swim in rats. Food intake, water intake, mental activity, fur gloss and stool characteristics and macroscopic symptom score. 2. After modeling in simulated wet environment, spleen was removed aseptically and splenic lymphocyte proliferation was induced by Con A to express the level of lymphocyte function. 3. Splenic lymphocyte proliferation was recorded and calculated after modeling in simulated wet environment. The spleen tissues were taken out for routine dehydration, paraffin immersion and embedding. Each tissue block was cut into 5 slices, dried overnight in a 60 C thermostat, and 5 slices were stained with routine HE. Morphological changes were observed under light microscope. 4. After modeling in simulated wet environment, spleen tissues were taken out 10 mg / mouse for two-dimensional electrophoresis and image analysis. Mass fingerprint and electrospray ionization tandem mass spectrometry were used to identify proteins. Results: 1. Comparing the effects of external dampness on normal and Spleen-Yang deficiency rats, the body weight of Spleen-Yang deficiency rats in external dampness environment was significantly lighter than that of normal rats, and the body weight of Spleen-Yang deficiency plus external dampness group (239.30 + 37.60) was significantly lower than that of external dampness group (321.70 + 18.74), P 0.01. The rats in the Yang deficiency plus external dampness group had some changes in food intake, drinking water, mental state, hair color and stool properties compared with those in the external dampness group. 34) significantly lower, P 0.01. Spleen-yang deficiency plus external humidity group rats in the early modeling mood gradually changed, showing irritable, aggressive state, swimming in the pool, have jumped up. Compared with normal and spleen-yang-deficiency rats, the proliferation of spleen lymphocytes in spleen-yang-deficiency rats and normal rats was significantly decreased under the environment of external humidity. 1.13 Compared with the control group, the proliferative capacity of splenic lymphocyte (15.15+2.18) and the proliferative capacity of splenic lymphocyte (14.42+2.24) of splenic Yang deficiency plus external dampness group decreased significantly (P 0.01). The proliferative capacity of splenic lymphocyte (14.42+2.24) of splenic Yang deficiency plus external dampness group was higher (15.15+2.18) than that of external dampness group (P = 0.476, P 0.05). The proliferative ability of splenic lymphocytes in Spleen-Yang deficiency group (36.07 65507 The marginal band of white pulp in spleen tissue of rats in group A was widened obviously, and light yellow pigment cells were phagocytized in splenic sinus, splenic sinus dilated and congested, and light yellow pigment cells were phagocytized in splenic cord. 4. The expression of differential protein spots in spleen tissue of normal and splenic Yang deficiency rats was compared with that of normal rats. Compared with the control group, 13 spots were up-regulated and 20 were down-regulated, 14 were up-regulated and 19 were down-regulated in the external dampness group, and 19 were up-regulated in the external dampness group and the Spleen-Yang deficiency plus external dampness group. Fifteen of them were down-regulated, and 10.5% of them were down-regulated. The difference proteins in spleen tissues between normal and spleen-yang-deficiency rats were compared. Twenty-two protein spots with distinct image and distinct change in expression level were selected from the 2-DE gel of the experimental group as the final target of mass spectrometry identification. Eleven proteins were up-regulated in normal rats under wet conditions. They were apolipoprotein A-I, malic dehydrogenase, specific short-chain acyl coenzyme A dehydrogenase, galactose lectin 5, low-molecular-weight phosphotyrosine protein phosphatase LMW-PTP, anionic trypsinogen-2, intracellular chloride channel protein 1, F-actin beta subunit. Phosphoglycerate mutase 1, membrane process protein, actin-associated protein 2/3 complex subunit 5, down-regulated by 4, they are 3-phosphate glyceraldehyde dehydrogenase, mammalian peroxidase 2, Ig-kappa chain C region, B allele, binding globin. Spleen-yang deficiency rats in wet environment up-regulated by 9, they are apolipoprotein A-I, malic acid Dehydrogenase, specific short-chain acyl coenzyme A dehydrogenase, galactose lectin 5, low-molecular-weight phosphotyrosine protein phosphatase LMW-PTP, anionic trypsinogen 2, intracellular chloride channel protein 1, phosphoglyceric acid mutase 1, membrane process protein, 5 down-regulated, they are 3-phosphoglyceraldehyde dehydrogenase, mammalian peroxide Enzyme 2, desmin, Ig-kappa chain C region, B allele, myosin modulation light chain 9. Conclusion: 1. Simulated external wet environment factors have an impact on normal and Spleen-Yang deficiency rats in general, including body weight, food intake, water intake, mental state, activity, fur gloss, skin mucosa, excreta and so on, and the Spleen-Yang deficiency group is more obvious. 2. Simulated external wet environment factors significantly reduced the cellular immune function of normal and Spleen-Yang deficiency rats. Simulated external wet environment factors (external factors) had stronger immunosuppressive effect than Spleen-Yang deficiency factors (internal factors). Simulated external wet environment factors (external factors) and Spleen-Yang deficiency factors (internal factors) had synergistic effect on the decline of immune function. Simulated external wet environment factors have effects on the spleen histomorphology of normal and Spleen-Yang deficiency rats, but the spleen tissue morphology of Spleen-Yang deficiency rats has obvious changes, showing in the aggravation of histocytopathy. 4. Simulated external wet environment factors on normal and Spleen-Yang deficiency rats spleen tissue protein differential expression. Simulated external wet environment factors and Spleen-Yang deficiency factors Different proteins in the body have their own specificity, but there are also intersections, and the intersection is very large. These differential proteins may be related to cell structure, energy metabolism, cell apoptosis, cell proliferation and differentiation, signal transduction, calcium homeostasis regulation, immune response, immune protection, and tumorigenesis. Disease-specific proteins associated with Spleen-Yang deficiency syndrome and damp pathogen invasion may be used as molecular markers for the diagnosis of Spleen-Yang deficiency syndrome and damp pathogenic molecular markers.
【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R228

【参考文献】