【摘要】：目的:明確人工模擬外濕環(huán)境對正常及脾陽虛大鼠一般情況、脾組織形態(tài)學改變、脾組織蛋白質差異表達的影響,探討濕邪對不同體質(正常與脾陽虛)機體的致病機理。方法:1.選取40只SD大鼠隨機分為對照組、脾陽虛組、外濕組、脾陽虛加外濕組,每組10只。脾陽虛組模型制作采用番瀉葉灌胃與皮下注射利血平交替進行并強迫大鼠游泳的復合方法。脾陽虛造模后,給予外濕組與脾陽虛加外濕組人工模擬外濕環(huán)境一周,于造模前后觀察并記錄各組大鼠的一般情況包括體質量、進食量、飲水量、精神活動、皮毛光澤度及大便性狀并進行宏觀癥候評分。2.模擬外濕環(huán)境造模完成后,無菌摘除脾臟,采用Con A誘導脾淋巴細胞增殖,以此表示淋巴細胞的功能水平。記錄并計算脾淋巴細胞增殖能力。3.模擬外濕環(huán)境造模完成后,取脾組織進行常規(guī)脫水、浸蠟、包埋。每個組織塊切取5張切片,60℃恒溫箱內過夜干燥切片,5張切片均做常規(guī)的HE染色,光鏡下觀察形態(tài)學變化。4.模擬外濕環(huán)境造模完成后,取出脾組織10mg/只,進行雙向電泳實驗以及圖象分析,采用肽質量指紋圖譜和電噴霧串聯(lián)質譜的方法鑒定蛋白質。結果:1.外濕對正常及脾陽虛大鼠一般情況的比較。外濕環(huán)境下脾陽虛大鼠較正常大鼠體質量明顯減輕,脾陽虛加外濕組大鼠體質量(239.30±37.60)較外濕組(321.70±18.74)顯著降低,P0.01。脾陽虛加外濕組大鼠在進食、飲水、精神狀態(tài)、毛色、大便性狀等方面較外濕組發(fā)生了一定的變化。脾陽虛加外濕組大鼠進食量(19.33±2.88)較外濕組(23.83±2.93)降低,P0.05。外濕組(37.50±1.87)、脾陽虛加外濕組大鼠飲水量(36.67±4.18)較對照組(42.67±2.34)顯著降低,P0.01。脾陽虛加外濕組大鼠造模初期出現(xiàn)情緒逐漸改變,呈現(xiàn)易激怒、好爭斗狀態(tài),放在泳池中游泳時,紛紛躍起。造模后逐漸出現(xiàn)精神萎靡不振,扎堆,嗜睡,反應遲鈍,行動遲緩,毛色枯槁,消瘦,糞便軟、不成形,甚至稀溏。外濕組大鼠脫毛現(xiàn)象較輕,精神狀態(tài)較好,偶可見嗜睡,抓取時叫聲比較溫柔。2.外濕對正常及脾陽虛大鼠脾淋巴細胞增殖力的比較。外濕環(huán)境下,脾陽虛大鼠與正常大鼠脾淋巴細胞增殖力較對照組均顯著降低。與對照組脾淋巴細胞增殖力(47.94±1.13)相比,外濕組脾淋巴細胞增殖力(15.15±2.18)、脾陽虛加外濕組脾淋巴細胞增殖力(14.42±2.24)均顯著降低P0.01。脾陽虛加外濕組脾淋巴細胞增殖力(14.42±2.24)較外濕組脾淋巴細胞增殖力(15.15±2.18)無差別P=0.476,P0.05。與脾陽虛加外濕組脾淋巴細胞增殖力(14.42±2.24)相比,脾陽虛組脾淋巴細胞增殖力(36.07±3.05)顯著增強P0.01。3.外濕對正常及脾陽虛大鼠脾組織形態(tài)學的改變。外濕組大鼠脾組織白髓邊緣帶增寬,白髓發(fā)生中心增大,脾索內見吞噬淺黃色色素細胞。脾陽虛加外濕組大鼠脾組織白髓邊緣帶增寬較明顯,脾竇內見吞噬淺黃色色素細胞,脾竇擴張、充血,脾索內見吞噬淺黃色色素細胞。4.外濕對正常及脾陽虛大鼠脾組織差異蛋白斑點表達情況的比較。各實驗組選三個樣本進行差異蛋白組學比較,兩兩比較差異蛋白圖譜,進行蛋白質組學分析,鑒定出40個蛋白斑點,脾陽虛加外濕組與對照組比較表達上調的有13個,表達下調的有20個;外濕組與對照組比較表達上調的有14個,表達下調的有19個;外濕組與脾陽虛加外濕組比較表達上調的有15個,表達下調的有10個。5.外濕對正常及脾陽虛大鼠脾組織差異蛋白質的比較。在實驗組2-DE凝膠上從表達差異蛋白質斑點中選取點影像清晰且表達水平改變明顯的22個蛋白質斑點作為最終質譜鑒定的對象,利用PMF的方法成功鑒定了20個蛋白質。外濕環(huán)境下正常大鼠表達上調的有11個,它們是載脂蛋白A-I、蘋果酸脫氫酶、特定短鏈�；o酶A脫氫酶、半乳糖凝集素5、低分子量的磷酸酪氨酸蛋白磷酸酶LMW-PTP、陰離子型胰蛋白酶原-2、胞內氯離子通道蛋白1、f肌動蛋白β亞基、磷酸甘油酸變位酶1、膜突蛋白、肌動蛋白相關蛋白2/3復合物亞基5,表達下調的有4個,它們是3-磷酸甘油醛脫氫酶、哺乳動物過氧化物酶2、Igκ鏈C區(qū),B等位基、結合珠蛋白。外濕環(huán)境下脾陽虛大鼠表達上調的有9個,它們是載脂蛋白A-I、蘋果酸脫氫酶、特定短鏈�；o酶A脫氫酶、半乳糖凝集素5、低分子量的磷酸酪氨酸蛋白磷酸酶LMW-PTP、陰離子型胰蛋白酶原-2、胞內氯離子通道蛋白1、磷酸甘油酸變位酶1、膜突蛋白,表達下調的有5個,它們是3-磷酸甘油醛脫氫酶、哺乳動物過氧化物酶2、結蛋白、Igκ鏈C區(qū),B等位基、肌球蛋白調節(jié)輕鏈9。結論:1.模擬外濕環(huán)境因素對正常及脾陽虛組大鼠的一般情況都有影響,包括體質量、進食量、飲水量、精神狀態(tài)、活動度、皮毛光澤、皮膚黏膜、排泄物等方面,并且對脾陽虛組影響更為明顯。2.模擬外濕環(huán)境因素顯著降低了正常及脾陽虛組大鼠細胞免疫功能。模擬外濕環(huán)境因素(外因)相對于脾陽虛因素(內因)對機體免疫抑制作用更強。模擬外濕環(huán)境因素(外因)與脾陽虛因素(內因)具有協(xié)同致機體免疫機能下降的作用。3.模擬外濕環(huán)境因素對正常及脾陽虛大鼠脾組織形態(tài)學都有影響,但脾陽虛大鼠脾組織形態(tài)改變明顯,表現(xiàn)在組織細胞病變加重。4.模擬外濕環(huán)境因素對正常及脾陽虛大鼠脾組織蛋白質存在差異表達。模擬外濕環(huán)境因素與脾陽虛因素作用于機體差異蛋白質有其特異性,但也存在交集,且交集很大。這些差異蛋白質功能與細胞結構、能量代謝、細胞凋亡、細胞增殖與分化、信號轉導、鈣穩(wěn)態(tài)調節(jié)、免疫應答、免疫保護、腫瘤發(fā)生相關。不同因素作用下的特異性差異蛋白質可能是與脾陽虛證及濕邪侵襲相關的疾病特異性蛋白,并有可能成為診斷脾陽虛證的分子標志物及濕邪致病的分子標志物。
[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.
【學位授予單位】：湖北中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R228

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