【摘要】：目的:1、通過腦室內(nèi)給藥的方法給予食源性肥胖大鼠leptin短期和長期干預,探討中樞給予leptin干預對食源性肥胖大鼠組織自噬的作用及其能量代謝平衡的影響。2、通過體外培養(yǎng)3T3-L1前脂肪細胞后給予leptin和自噬工具藥物干預,觀察leptin對于3T3-L1前脂肪細胞分化及表型的影響;探究leptin對于3T3-L1前脂肪細胞自噬的作用及機制。方法:1、選取SD大鼠通過給予高脂飼料喂養(yǎng)建立食源性肥胖(diet induced obesity,DIO)動物模型。在喂養(yǎng)過程中監(jiān)測大鼠體重增長情況,在高脂飼料喂養(yǎng)16周后,將高脂組(high fat diet,HFD)大鼠根據(jù)體重進一步分為食源性肥胖組和肥胖抵抗組(diet resistant,DR);普通飼料喂養(yǎng)的大鼠作為對照組(chow feed,CF)。建模期間監(jiān)測大鼠體重和進食量;使用EILSA試劑盒檢測不同組別中SD大鼠的血清leptin水平。2、對SD大鼠通過第三腦室內(nèi)注射leptin進行短期干預。分別將DIO、DR和CF組大鼠隨機分為三組,即:注射leptin的大鼠分為高濃度組(High,H)和低濃度組(Low,L),設立對照組(Control,C)給予人工腦脊液注射。腦室內(nèi)注射leptin后24 h監(jiān)測大鼠體重和進食量;使用EILSA試劑盒檢測不同組別SD大鼠的血清leptin水平;通過HE染色觀察干預后肝臟、脂肪組織的形態(tài)變化;通過Western Blot方法檢測大鼠中樞(下丘腦)和外周(肝臟組織、脂肪組織)內(nèi)自噬相關因子LC3B、Beclin-1和P62蛋白水平的表達,觀察中樞給予leptin短期干預后大鼠中樞和外周組織的自噬變化;探討中樞給予leptin短期干預對DIO大鼠中樞及外周組織的自噬及能量代謝的影響。3、對SD大鼠進行側腦室置管后連續(xù)注射leptin一周進行長期干預。分別將CF和DIO和DR組大鼠隨機分為兩組,即:注射高濃度leptin的大鼠為干預組(High,H),設立對照組(Control,C)給予人工腦脊液注射。在置管成功后,給予各組大鼠連續(xù)注射leptin或人工腦脊液干預一周。在干預過程中,每天定時監(jiān)測大鼠體重和進食量;使用EILSA試劑盒檢測不同組別中SD大鼠的血清leptin水平;通過油紅O染色和HE染色觀察干預后大鼠肝臟、脂肪組織的形態(tài)變化;通過Western Blot檢測大鼠中樞(下丘腦)和外周(脂肪組織、肝臟組織)內(nèi)自噬相關因子LC3B、Beclin-1和P62蛋白水平的表達,觀察中樞給予leptin蛋白長期干預后大鼠中樞和外周組織的自噬變化。探討中樞給予leptin長期干預對DIO大鼠中樞及外周組織的自噬及能量代謝的影響。4、體外培養(yǎng)3T3-L1前脂肪細胞并誘導其分化為成熟脂肪細胞,在誘導分化過程中加入leptin干預,通過油紅O染色觀察leptin對于3T3-L1前脂肪細胞分化及細胞表型的影響;通過RT-PCR檢測脂肪細胞分化、甘油三酯合成、分解,脂肪酸β氧化及棕色脂肪特征性基因的mRNA表達水平來探討leptin對脂肪細胞分化及成脂的影響;在細胞誘導分化的不同階段給予leptin及自噬工具藥(Rapamyin和Bafilomycin A1)干預,檢測自噬相關因子LC3B蛋白水平的表達并在免疫熒光顯微鏡下觀察P62的變化,分析leptin干預在3T3-L1前脂肪細胞誘導分化的不同階段對其自噬的作用。檢測leptin干預后3T3-L1前脂肪細胞自噬及mTOR信號通路相關因子LC3B、P62、Beclin-1、4E-BP1、p-4E-BP1的蛋白水平表達。探討leptin對脂肪細胞分化及自噬的影響是否通過mTOR依賴的信號通路實現(xiàn)。結果:1、造模成功后,DIO組大鼠的體重和能量攝入均高于CF和DR組大鼠。DIO大鼠血清leptin水平高于CF和DR組,存在leptin抵抗。2、中樞給予leptin短期干預后,CF組大鼠的體重下降,但能量攝入較干預前增多;DIO組體重下降,leptin高濃度組大鼠能量攝入較干預前減少,leptin低濃度組大鼠能量攝入與干預前比較差異無統(tǒng)計學意義。DR組大鼠體重下降,但能量攝入與干預前比較無明顯變化。Leptin干預后CF、DIO和DR組大鼠血清leptin水平均下降,但差異無統(tǒng)計學意義。高脂飼料喂養(yǎng)的DIO和DR大鼠存在肝脂肪變性,且DIO大鼠的脂肪細胞體積明顯增大,給予leptin干預24h后,肝細胞和脂肪細胞形態(tài)無改變。給予高脂飼料喂養(yǎng)的DIO和DR大鼠與普通飼料喂養(yǎng)的CF組大鼠比較,其肝臟組織自噬下調(diào),脂肪組織和下丘腦組織的自噬上調(diào)。給予leptin干預后,DIO和DR大鼠的肝臟組織自噬上調(diào),脂肪組織和下丘腦組織自噬下調(diào);CF組大鼠的肝臟組織的自噬下調(diào),脂肪組織和下丘腦組織自噬上調(diào)。3、中樞給予leptin長期干預后,CF組大鼠的體重無明顯改變,但能量攝入較干預前增多;DIO組大鼠體重下降,其能量攝入波動下降;DR大鼠體重下降,同時能量攝入也減少。Leptin長期干預后,CF、DIO和DR組大鼠血清leptin水平較干預前有所增加,但差異無統(tǒng)計學意義;給予leptin干預一周后,DIO和DR組大鼠肝臟組織脂肪變性得到一定程度改善,DIO組大鼠脂肪細胞體積減小,但CF和DR組脂肪細胞體積沒有明顯改變。給予leptin干預后,DIO和DR大鼠的肝臟組織自噬上調(diào),脂肪組織和下丘腦組織自噬下調(diào);CF組大鼠的肝臟組織的自噬下調(diào),脂肪組織和下丘腦組織自噬下調(diào)。4、Leptin干預后3T3-L1前脂肪細胞形態(tài)無改變。在誘導分化過程中給予leptin持續(xù)干預,3T3-L1前脂肪細胞成脂率降低。在給予3T3-L1前脂肪細胞誘導分化早期給予leptin干預后PPARγ、HSL mRNA表達量較對照組增高,在細胞分化的晚期干預組的aP2、DGAT1、DGAT2 mRNA表達量降低。給予leptin干預后,UCP1和PGC-1αmRNA增高。5、在3T3-L1前脂肪細胞誘導分化早期給予leptin干預促進自噬上調(diào),在3T3-L1前脂肪細胞誘導分化晚期給予leptin干預后細胞自噬下調(diào)。在3T3-L1前脂肪細胞分化的早期leptin能增加自噬體的合成,促進自噬體降解,加快3T3-L1前脂肪細胞自噬進程,促進自噬;但在3T3-L1前脂肪細胞分化的中、晚期leptin增加自噬體的合成,但抑制自噬體降解,抑制自噬。Leptin對于3T3-L1前脂肪細胞自噬的調(diào)控是通過mTOR依賴的信號通路實現(xiàn)的。結論:1、中樞給予leptin干預后,可以促進DIO、DR大鼠體重下降,能量消耗增加;CF組大鼠能量消耗增加。同時調(diào)節(jié)大鼠肝臟、脂肪和下丘腦組織自噬,維持能量代謝平衡。2、3T3-L1前脂肪細胞誘導分化時給予leptin干預,細胞形態(tài)未改變,但其成脂分化率降低;在細胞誘導分化早期leptin促進3T3-L1前脂肪細胞自噬上調(diào),促進細胞分化;在細胞誘導分化晚期,leptin抑制3T3-L1前脂肪細胞自噬,促進甘油三酯分解,減少脂滴聚集。且leptin對3T3-L1前脂肪細胞自噬的調(diào)節(jié)通過mTOR依賴的信號通路實現(xiàn)。
[Abstract]:AIM: 1. To investigate the effect of leptin on autophagy and energy metabolism balance in rats with food-borne obesity by intraventricular administration of leptin. 2. To observe the effect of leptin on autophagy and energy metabolism balance in rats with food-borne obesity. To investigate the effect of leptin on the autophagy of 3T3-L1 preadipocytes and its mechanism. Methods: 1. SD rats were fed with high fat diet to establish an animal model of diet-induced obesity (DIO). Sixteen weeks later, the rats in the high fat diet (HFD) group were further divided into the diet resistant (DR) group and the diet resistant (DR) group according to their body weight; the rats fed with ordinary diet were used as the control group (CF). The body weight and food intake of the rats were monitored during the modeling period; the serum leptin levels of SD rats in different groups were detected by EILSA kit. 2. Short-term intervention was carried out by injecting leptin into the third ventricle of SD rats. The rats in DIO, DR and C F groups were randomly divided into three groups. The rats injected with leptin were divided into high concentration group (High, H) and low concentration group (Low, L), and the control group (Control, C) was injected with artificial cerebrospinal fluid. Levels of serum leptin in SD rats of different groups were detected by EILSA kit, morphological changes of liver and adipose tissue were observed by HE staining, and the expression of autophagy-related factors LC3B, Beclin-1 and P62 protein in central (hypothalamus) and peripheral (liver, adipose tissue) were detected by Western Blot method. The changes of autophagy in central and peripheral tissues of SD rats after short-term intervention with leptin were investigated. The effects of short-term intervention with leptin on autophagy and energy metabolism in central and peripheral tissues of DIO rats were investigated. 3. SD rats were injected with leptin for a week after catheterization in lateral ventricle for long-term intervention. The rats in each group were injected with leptin or artificial cerebrospinal fluid continuously for one week after successful catheterization. During the intervention, the rats'body weight and food intake were monitored regularly every day, and different groups were detected by EILSA kit. Levels of serum leptin in SD rats were observed by oil red O staining and HE staining, and the morphological changes of liver and adipose tissue were observed after intervention. To investigate the effects of long-term intervention with leptin on autophagy and energy metabolism in central and peripheral tissues of DIO rats. 4. Preadipocytes 3T3-L1 were cultured in vitro and induced to differentiate into mature adipocytes. Leptin was added into the process of differentiation and observed by oil red O staining. To investigate the effect of leptin on the differentiation and cell phenotype of 3T3-L1 preadipocytes, to investigate the effect of leptin on adipocyte differentiation and adipogenesis by RT-PCR detection of adipocyte differentiation, triglyceride synthesis, decomposition, fatty acid beta oxidation and mRNA expression of brown fat characteristic genes. The expression of autophagy-related factor LC3B protein was detected by immunofluorescence microscopy, and the effect of leptin on autophagy in different stages of 3T3-L1 preadipocyte differentiation was analyzed. The protein levels of LC3B, P62, Beclin-1, 4E-BP1 and p-4E-BP1 were detected. The effects of leptin on adipocyte differentiation and autophagy were investigated by mTOR-dependent signaling pathway. Results: 1. After successful modeling, the body weight and energy intake of DIO rats were higher than those of CF and DR rats. The serum leptin levels of DIO rats were higher than those of CF and DR rats. The body weight of CF rats decreased, but the energy intake increased after short-term intervention with leptin. The body weight of DIO rats decreased, the energy intake of high leptin concentration group decreased, and the energy intake of low leptin concentration group was not significantly different from that before intervention. The serum leptin levels of CF, DIO and DR rats decreased after Leptin intervention, but there was no significant difference between the two groups. Compared with CF group, DIO and DR rats fed with high fat diet had lower autophagy in liver tissue and higher autophagy in adipose tissue and hypothalamus tissue. Tissue autophagy was down-regulated, adipose tissue and hypothalamic tissue autophagy was up-regulated. 3. After long-term intervention with leptin, the body weight of CF rats did not change significantly, but energy intake increased. The body weight of DIO rats decreased and energy intake fluctuated. The body weight of DR rats decreased and energy intake decreased. After long-term intervention with Leptin, CF and DIO rats lost weight and energy intake. The serum leptin level of rats in DIO and DR groups was higher than that before the intervention, but there was no significant difference between the two groups.One week after the intervention with leptin, the fatty degeneration of liver tissue in DIO and DR groups was improved to a certain extent, the volume of adipocytes in DIO group decreased, but the volume of adipocytes in CF and DR groups did not change significantly.After the intervention with leptin, DIO and DR increased. Autophagy of liver tissue was up-regulated, autophagy of adipose tissue and hypothalamus tissue was down-regulated; autophagy of liver tissue was down-regulated, autophagy of adipose tissue and hypothalamus tissue was down-regulated in CF group. The morphology of 3T3-L1 preadipocytes did not change after Leptin intervention. The expression of PPAR-gamma and HSL-mRNA increased after leptin intervention in the early stage of 3T3-L1 preadipocyte differentiation, but decreased in the late stage of cell differentiation. After leptin intervention, the expression of UCP1 and PCG-1alpha mRNA increased. 5. Leptin intervention was given in the early stage of 3T3-L1 preadipocyte differentiation. In the early stage of 3T3-L1 preadipocyte differentiation, leptin can increase autophagy synthesis, promote autophagy degradation, accelerate the autophagy process of 3T3-L1 preadipocyte, and promote autophagy; but in the late stage of 3T3-L1 preadipocyte differentiation, leptin increases. Leptin regulates autophagy of 3T3-L1 preadipocytes through a mTOR-dependent signaling pathway. Conclusion: 1. Leptin can promote the weight loss and energy consumption of DIO, DR rats, and CF rats. 2,3T3-L1 preadipocytes were treated with leptin when they were induced to differentiate, but the adipogenic differentiation rate was decreased; leptin promoted autophagy of 3T3-L1 preadipocytes in the early stage of cell differentiation, and promoted cell differentiation; leptin inhibited 3T3-L1 preadipocytes in the late stage of cell differentiation. L1 preadipocyte autophagy promotes triglyceride decomposition and decreases lipid droplet aggregation, and leptin modulates 3T3-L1 preadipocyte autophagy via a mTOR-dependent signaling pathway.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R589.2

【參考文獻】

相關期刊論文 前1條

1 楊曉寧;張辰雨;王炳蔚;祝世功;鄭瑞茂;;瘦素信號與瘦素抵抗機制研究進展[J];生理科學進展;2015年05期

相關博士學位論文 前1條

1 韓潔;LKB1抑制脂肪生成和分化的機制研究[D];天津醫(yī)科大學;2016年

，

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