本文選題：肥胖癥　切入點(diǎn)：大鼠　出處：《湖北中醫(yī)藥大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：目的通過中醫(yī)藥理論及臨床實(shí)踐研究,探討肥胖癥的中西醫(yī)發(fā)病機(jī)制,為臨床應(yīng)用中醫(yī)理論治療肥胖癥提供理論指導(dǎo),通過高脂飲食誘導(dǎo)肥胖動(dòng)物模型,探討溫腎健脾化痰方對(duì)肥胖癥大鼠的影響,從分子水平及藥理學(xué)方面研究溫腎健脾化痰對(duì)肥胖癥的作用機(jī)制,從而為溫腎健脾化痰法臨床治療肥胖癥提供理論依據(jù)。方法本研究從理論研究和實(shí)驗(yàn)研究兩個(gè)方面進(jìn)行。理論研究:從肥胖癥的診斷、分類和病因病機(jī)以及辨證論治方面進(jìn)行了系統(tǒng)的論述,并從理、法、方、藥四個(gè)方面對(duì)溫腎健脾化痰方治療肥胖癥的機(jī)制進(jìn)行了理論探討。實(shí)驗(yàn)研究:將60只體重100~150g的健康雄性SD大鼠隨機(jī)分為2組,一組為正常對(duì)照組10只,給予普通飼料喂養(yǎng),自由取食飲水;其余50只給予高脂飼料喂養(yǎng),自由取食飲水。每周測(cè)量體重、身長2次,喂養(yǎng)4周后選取體質(zhì)量增加大于正常組平均體質(zhì)量15%且Lee's指數(shù)比正常組平均水平增加大于1.5%的大鼠,作為高脂飲食誘導(dǎo)肥胖造模成功大鼠,選取40只造模成功的大鼠隨機(jī)分為4組,分別為模型組、低劑量組、中劑量組和高劑量組,每組10只。正常組和模型組大鼠給予生理鹽水1 ml/100g·d灌胃;低、中、高劑量組大鼠分別給予低、中、高劑量溫腎健脾化痰方1ml/100g·d灌胃。正常組繼續(xù)喂以普通飼料,其他4組繼續(xù)予高脂飼料喂養(yǎng),實(shí)驗(yàn)過程中每周測(cè)量體重2次,并根據(jù)體重調(diào)整給藥量,連續(xù)治療10周。觀察各組大鼠一般情況和體質(zhì)量的變化,采用全自動(dòng)生化分析儀檢測(cè)各組大鼠血脂及肝功能。運(yùn)用HE染色觀察各組大鼠肝臟組織和脂肪組織的病理形態(tài)學(xué)改變。采用RT-PCR法觀察溫腎健脾化痰方對(duì)肝臟組織SOCS3m RNA表達(dá)的影響,免疫組織化學(xué)法觀察其對(duì)各組大鼠脂肪組織Ghrelin水平的影響。結(jié)果1)各組大鼠體質(zhì)量的變化:溫腎健脾化痰方低、中、高劑量均能明顯控制肥胖癥大鼠體重的增加,使體重增加的速度明顯變緩,與模型組相比具有統(tǒng)計(jì)學(xué)意義(P0.01),且中劑量組和高劑量組效果明顯優(yōu)于低劑量組并具有統(tǒng)計(jì)學(xué)意義(P0.01),而中、高劑量組之間差異無統(tǒng)計(jì)學(xué)意義。2)各組大鼠肝臟組織及脂肪組織病理學(xué)變化:正常組肝細(xì)胞形態(tài)排列正常,肝小葉規(guī)則,細(xì)胞質(zhì)均勻;模型組均出現(xiàn)程度不等的彌漫性肝細(xì)胞脂肪變性,肝細(xì)胞體積增大,胞漿內(nèi)可見數(shù)量不等、大小不一的圓形脂滴或脂肪空泡,胞核被推向周邊;低劑量組脂變肝細(xì)胞與模型組相當(dāng);中、高劑量組脂變肝細(xì)胞較模型組明顯減少,可見少量脂肪空泡。與正常組大鼠相比,模型組大鼠脂肪細(xì)胞明顯增大,單位視野內(nèi)脂肪細(xì)胞數(shù)明顯減少;低劑量組大鼠脂肪細(xì)胞與模型組相當(dāng);中、高劑量組大鼠脂肪細(xì)胞較模型組明顯減小,單位視野內(nèi)脂肪細(xì)胞數(shù)明顯增多,且中劑量組的變化更為顯著。3)各組大鼠肝功能比較:與正常組相比,模型組與低、中、高劑量組的ALT,AST均顯著升高(P0.01,P0.05);與模型組相比,溫腎健脾化痰方低、中、高劑量均可顯著降低ALT,AST水平(P0.01),且中、高劑量組效果顯著優(yōu)于低劑量組(P0.01),高劑量組在降低AST水平方面又顯著優(yōu)于中劑量組(P0.01),但在降低ALT水平方面則無明顯區(qū)別(P0.05)。4)各組大鼠血脂比較:與正常組相比,模型組與低、中、高劑量組的TC,TG,LDL-C均顯著性升高(P0.05),HDL-C顯著性降低(P0.05,提示肥胖癥大鼠模型存在明顯的血脂紊亂。與模型組相比,溫腎健脾化痰方低、中、高劑量組均可顯著性降低TC,TG以及LDL-C(P0.01),升高HDL-C(P0.01),且三組之間比較也有顯著性差異(P0.01)。5)各組大鼠肝臟組織SOCS3m RNA表達(dá):模型組大鼠肝臟組織內(nèi)SOCS3m RNA表達(dá)量顯著升高(P0.01),經(jīng)溫腎健脾化痰方治療后,與模型組比較,藥物治療組SOCS3m RNA表達(dá)均顯著下降(P0.01),其中高劑量組與低、中劑量組相比有顯著性差異(P0.01),與正常組相比無明顯區(qū)別(P0.05)。6)各組大鼠脂肪組織Ghrelin基因表達(dá):模型組大鼠脂肪組織內(nèi)Ghrelin基因表達(dá)量顯著升高(P0.05),經(jīng)溫腎健脾化痰方治療后,與模型組比較,藥物治療組Ghrelin基因表達(dá)均顯著下降(P0.01),其中高、中劑量組相比無明顯區(qū)別(P0.05),但與低劑量組相比有顯著性差異(P0.01)各組大鼠脂肪組織Ghrelin免疫組化檢測(cè)結(jié)果:光鏡下脂肪細(xì)胞的細(xì)胞核呈棕黃色或棕褐色的為Ghrelin陽性細(xì)胞,陰性細(xì)胞核為藍(lán)色或藍(lán)紫色。觀察各組大鼠的脂肪細(xì)胞:模型組大鼠脂肪細(xì)胞內(nèi)Ghrelin陽性細(xì)胞明顯較正常組增多,經(jīng)溫腎健脾化痰方藥物治療的大鼠脂肪細(xì)胞內(nèi)Ghrelin陽性細(xì)胞較模型組明顯減少,以中、高劑量組較明顯。IPP分析結(jié)果也顯示:與正常組相比,模型組、低、中、高劑量組Ghrelin陽性表達(dá)率明顯升高(P0.05),經(jīng)治療后,低、中、高劑量組大鼠脂肪Ghrelin陽性表達(dá)率較模型組顯著下降(P0.01)。結(jié)論理論研究:肥胖癥的病機(jī)為本虛標(biāo)實(shí),表現(xiàn)以脾腎虧虛為本,以痰濕、血瘀等為標(biāo)。溫腎健脾化痰方的組方體現(xiàn)了肥胖癥的辨證論治,為運(yùn)用其治療肥胖癥提供了理論依據(jù)。實(shí)驗(yàn)研究:1)溫腎健脾化痰方在控制肥胖模型大鼠體重增加,減輕肝功能損害,減少腹內(nèi)脂肪,降低血清膽固醇及甘油三酯減輕肝臟脂肪變性方面有良好作用。2)溫腎健脾化痰方可調(diào)控食欲,增加能量代謝,抑制脂肪合成,改善糖、脂代謝,減輕肥胖,可能與其通過調(diào)節(jié)肥胖癥模型大鼠的SOCS3m RNA水平來實(shí)現(xiàn)的。3)溫腎健脾化痰方可調(diào)節(jié)肥胖癥模型大鼠Ghrelin基因的表達(dá),而調(diào)節(jié)這些基因表達(dá)不僅可直接影響食欲和飲食,提高能量代謝效率,還能通過調(diào)節(jié)瘦素等脂肪因子,達(dá)到治療肥胖癥的目的。
[Abstract]:Objective to study the TCM theory and clinical practice of traditional Chinese medicine and Western medicine, to explore the pathogenesis of obesity, and provide theoretical guidance for the clinical application of the theory of traditional Chinese medicine for the treatment of obesity, the high fat diet induced obesity animal model to investigate the effects of Wenshen Jianpi Huatan Decoction on obese rats, at the molecular level and pharmacology research on obesity and phlegm of warming kidney and invigorating spleen the mechanism of action, so as to provide a theoretical basis for the clinical treatment of warming kidney and invigorating spleen and resolving phlegm obesity. Methods this study from two aspects of theoretical research and experimental research. The theoretical research: from obesity diagnosis, classification and pathogenesis and syndrome differentiation were discussed, and the principle, method, party. The mechanism of the four aspects of medicine Wenshen Jianpi Huatan Decoction in the treatment of obesity are discussed. Experimental study: 60 healthy male SD rats weighing 100~150g random divided into 2 groups As a group, 10 rats in normal control group, given normal diet, free feeding, drinking water; the remaining 50 rats were given high fat diet, feeding free drinking water. Weekly measurement of body weight, body length 2 times, after 4 weeks of feeding selection of body weight increased more than the normal group average body mass index increased more than 1.5% and 15% Lee's in rats compared with normal group average, as the high fat diet induced obese rats, 40 rats were randomly divided into 4 groups of successful modeling, respectively, model group, low dose group, middle dose group and high dose group, 10 rats in each group. The normal group and model group were given saline 1 ml/100g D Ig; low, high dose group rats were given low, high dose of Wenshen Jianpi Huatan Decoction 1ml/100g d by gavage. The normal group fed with normal diet, the other 4 groups continue to be fed with high-fat diet during the experiment, body weight was measured weekly for 2 times, and according to the weight adjustment The whole dosage, continuous treatment for 10 weeks. To observe the changes of general condition and body weight of the rats were detected by automatic biochemistry analyzer rats blood lipid and liver function. Using HE staining to observe the pathologic liver tissue of rats and adipose tissue changes. Observation of Wenshen Jianpi Huatan Decoction on expression of SOCS3m RNA in liver organized by RT-PCR method, its impact on the level of Ghrelin in adipose tissue of rats were observed by immunohistochemistry. Results: 1) the body weight of the rats in each group: Wenshen Jianpi Huatan Decoction in low, high dose could significantly increase the weight control in obese rats, the body weight increased significantly slow speed compared with the model group, with statistical significance (P0.01), and the effect of middle dose group and high dose group was significantly better than the low dose group and had statistical significance (P0.01), and between the high dose group, the difference was statistically significant. 2) liver histology of rats and adipose tissue pathology: normal hepatic cell morphology were normal, uniform rules, lobular cytoplasm; model group showed different degrees of diffuse fatty degeneration of liver cells, liver cell volume increases, cytoplasm number ranging from the size of the circular lipid or fat vacuoles, nucleus is pushed to the periphery; low dose group of fat liver cell and model group; high dose group, fatty degeneration of liver cells was significantly reduced compared with the model, a small amount of fat vacuoles. Compared with normal rats, fat cells of rats in the model group increased obviously, number of fat cells in the field units significantly reduced; low dose group rat adipocytes with the model group, high dose group; rat fat cells decreased significantly compared with the model group, the number of fat cells in the field of units increased significantly, and the change in dose group is more significant.3) liver function of rats 鑳芥瘮杈，

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