【摘要】：慢性炎癥反應(yīng)介導(dǎo)的胰島素抵抗是肥胖發(fā)展為二型糖尿病(type 2 diabetes,T2DM)的主要原因,因此,肥胖被認(rèn)為是誘發(fā)T2DM的重要風(fēng)險因子之一。大量臨床資料顯示,當(dāng)人體攝入的能量遠(yuǎn)遠(yuǎn)超過新陳代謝所需要的能量時,會造成機(jī)體脂肪堆積,使機(jī)體內(nèi)能量代謝平衡失調(diào),導(dǎo)致肥胖。已有研究表明,肥胖組織含有大量的脂肪細(xì)胞,這些細(xì)胞含有的游離脂肪酸(free fatty acids,FFA)會促進(jìn)大量炎性因子產(chǎn)生和釋放,長期作用就形成了機(jī)體的炎性環(huán)境,下丘腦(hypothalamus)是機(jī)體的飲食調(diào)節(jié)中樞,也是代謝調(diào)控中心,研究表明,下丘腦炎癥反應(yīng)可以降低機(jī)體對胰島素和瘦素的敏感性,導(dǎo)致胰島素和瘦素抵抗,打破機(jī)體對能量攝入的調(diào)控平衡,最終誘發(fā)T2DM。因此,炎癥反應(yīng)在下丘腦胰島素抵抗(Insulin resistance,IR)和瘦素抵抗(Leptin resistance,LR)中發(fā)揮重要作用。趨化因子(Chemokines)是一類能夠促進(jìn)白細(xì)胞遷移到感染部位的小分子量蛋白質(zhì),它通過與其受體的特異性結(jié)合,促進(jìn)各類炎癥因子的釋放,在機(jī)體炎癥反應(yīng)中發(fā)揮重要作用。我們的前期研究表明,趨化因子受體CCR7基因在下丘腦中高表達(dá),這一結(jié)果提示CCR7可能參與下丘腦IR的病理生理學(xué)發(fā)生過程,因此,本研究著重探索CCR7在高脂飲食誘導(dǎo)的T2DM小鼠下丘腦IR的分子機(jī)制。實(shí)驗(yàn)選取6周齡雄性ICR小鼠,隨機(jī)分為兩組,一組飼喂含11.4%脂肪的普通飼料,設(shè)為對照組(Ctrl組);另一組飼喂含60%脂肪的高脂飼料(HFD組),設(shè)為模型組;兩組小鼠分別處理16周,期間每周固定時間測量并記錄各組小鼠的體重、血糖及攝食量,第16周對兩組小鼠進(jìn)行葡萄糖耐受(OGTT)與胰島素耐受(ITT)評價,結(jié)果顯示HFD組小鼠的c均顯著高于Ctrl組(P0.001),且HFD組小鼠產(chǎn)生了葡萄糖不耐受和胰島素抵抗。綜合分析實(shí)驗(yàn)數(shù)據(jù)后,評估T2DM模型小鼠的構(gòu)建,于第17周,將上述Ctrl組和HFD組兩組小鼠進(jìn)一步分為四組,總體分組如下:Ctrl Scramble sgRNA組、HFD Scramble sgRNA組、HFD CCR7sgRNA組、Ctrl CCR7 sgRNA組,利用CRISPR-Cas9基因編輯技術(shù)敲除小鼠下丘腦CCR7基因(利用腦立體定位儀分別向下丘腦雙側(cè)基底部VMH區(qū)域,前囟點(diǎn)后1.58mm,中間線外側(cè)0.3mm,顱骨表面下方5.68mm處注射慢病毒包裝的CCR7sgRNA顆粒(病毒滴度為1x106TU/ml),Scramble組注射空載慢病毒顆粒);術(shù)后動物處理與術(shù)前相同,繼續(xù)每周測量并記錄各組小鼠的體重、血糖及攝食量,待各項(xiàng)生理指標(biāo)穩(wěn)定后,即腦立體定位注射手術(shù)5周左右,利用蛋白免疫印跡技術(shù)(western blotting technology,WB)和免疫熒光組織化學(xué)技術(shù)(immunofluorescence histochemistry technology,IF)對四組小鼠下丘腦中CCR7蛋白表達(dá)量進(jìn)行測定,評價CCR7基因在相應(yīng)處理組小鼠下丘腦中的敲除情況。各組小鼠生理生化指標(biāo)檢測結(jié)果顯示:與Ctrl Scramble sgRNA組相比,HFD Scramble sgRNA組小鼠的體重和血糖水平顯著提高(P0.001);與HFD Scramble sgRNA組相比,HFD CCR7 sgRNA組小鼠體重和血糖水平明顯下降,并存在顯著性差異(P0.001),而與Ctrl Scramble sgRNA組相比,Ctrl CCR7 sgRNA組小鼠的體重和血糖水平無顯著性差異;這表明HFD CCR7 sgRNA組小鼠的葡萄糖不耐受與胰島素抵抗得到改善。此外,各組小鼠在實(shí)驗(yàn)各階段攝食量穩(wěn)定且無顯著性組別差異。進(jìn)一步利用WB和IF的方法檢測不同處理組小鼠下丘腦中NLRP3炎癥小體介導(dǎo)的炎癥信號通路關(guān)鍵蛋白,如NLRP3,ASC,Caspase 1,IL-1β等,及胰島素信號通路相關(guān)蛋白IRS-1和Akt的表達(dá)變化情況,結(jié)果顯示:HFD組小鼠下丘腦中CCR7蛋白表達(dá)顯著高于Ctrl組,同時NLRP3,ASC,Caspase-1,IL-1β,等蛋白表達(dá)量也顯著增多,胰島素受體底物1(IRS-1)磷酸化增強(qiáng);特異性敲除CCR7基因后,HFD CCR7 sgRNA組與HFD Scramble sgRNA組相比,以上炎癥通路蛋白表達(dá)量均顯著下降(P0.001),IRS-1蛋白磷酸化程度降低;而Ctrl CCR7sgRNA組與Ctrl Scramble sgRNA組相比,炎癥通路相關(guān)蛋白表達(dá)量及IRS-1蛋白酸化情況均沒有明顯變化,IF檢測結(jié)果與WB檢測結(jié)果一致。綜上所述,CCR7介導(dǎo)高脂飲食小鼠下丘腦中NLRP3炎癥小體的活化,激活Caspase-1蛋白,促進(jìn)IL-1β和TNF-α等炎性因子的成熟和釋放,放大下丘腦炎癥反應(yīng),負(fù)調(diào)節(jié)下丘腦胰島素信號和瘦素信號,最終導(dǎo)致IR和LR,這些結(jié)果提示CCR7是治療T2DM下丘腦IR的關(guān)鍵候選靶點(diǎn)。
[Abstract]:Chronic inflammatory response-mediated insulin resistance is a major cause of obesity development for type 2 diabetes mellitus (T2DM), and therefore, obesity is considered one of the key risk factors for inducing T2DM. A lot of clinical data show that when the energy consumed by the human body far exceeds the energy required by the metabolism, the body fat accumulation can be caused, the energy metabolism in the body is balanced, and the obesity is caused. The research has shown that the obesity tissue contains a large amount of fat cells, free fatty acids (FFA) contained in these cells can promote the production and release of a large number of inflammatory factors, and the long-term effect forms an inflammatory environment of the body, and the hypothalamus (hythaloamus) is the food regulation center of the body. It is also the metabolic control center. The research shows that the hypothalamic inflammation reaction can reduce the sensitivity of the body to the insulin and the leptin, lead to the resistance of the insulin and the leptin, break the regulation and balance of the body to the energy intake, and finally induce the T2DM. Thus, the inflammatory response plays an important role in the hypothalamic insulin resistance (IR) and leptin resistance (LR). Chemokines are a class of small-molecular-weight proteins that can promote the migration of leukocytes to the site of infection, which can promote the release of various inflammatory factors through the specific binding with their receptors, and play an important role in the inflammation reaction of the body. Our early studies have shown that the chemokine receptor CCR7 gene is highly expressed in the hypothalamus, which suggests that CCR7 may be involved in the pathophysiology of the hypothalamic IR. Therefore, this study focuses on the molecular mechanism of the hypothalamic IR in the T2DM mouse induced by high-fat diet. The 6-week-old male ICR mice were randomly divided into two groups. One group was fed with a common feed containing 11.4% fat, which was set as the control group (Ctrl group), and the other group was fed with a high-fat diet (HFD group) containing 60% of fat, which was set as the model group, and the two groups were treated for 16 weeks, respectively. The body weight, blood glucose and food consumption of each group of mice were measured and recorded during the weekly fixed time, and the glucose tolerance (OGTT) and the insulin resistance (ITT) evaluation were performed on the two groups at Week 16, and the results showed that the c of the mice in the HFD group was significantly higher than that in the control group (P 0.001). And the HFD group mice produced glucose intolerance and insulin resistance. After comprehensive analysis of the experimental data, the construction of the model mice with T2DM was evaluated. In the first week, two groups of mice in the control group and the HFD group were further divided into four groups. The overall group was as follows: Ctrl Sramble sgRNA group, HFD Sramble sgRNA group, HFD CCR7sgRNA group, and Ctrl CCR7 sgRNA group. The CCR7 gene (virus titer of 1 x 106TU/ ml) was injected at 5.68 mm below the skull surface by using the CRISPR-Cas9 gene editing technique to knock out the CCR7 gene of the hypothalamus of the mouse (using the brain stereolocator, respectively, to the VMH region at the bottom of the bilateral basal part of the hypothalamus, 1.58 mm after the anterior and the outer side of the middle line, and 5.68 mm below the surface of the skull. The weight, blood sugar and food consumption of each group of mice were measured and recorded on a weekly basis, and the weight, blood sugar and food consumption of each group of mice were measured and recorded on a weekly basis. After the physiological indexes were stable, the brain three-dimensional positioning and injection operation was performed for about 5 weeks, and western blotting technology was used. WB and IF were used to measure the expression of CCR7 in the hypothalamus of four groups of mice, and the knockout of CCR7 gene in the hypothalamus of the corresponding treatment group was evaluated. The results showed that the body weight and blood glucose level of the mice in the HFD Sramble sgRNA group were significantly higher than that of the control Sramble sgRNA group (P 0.001), and the body weight and blood glucose level of the HFD CCR7 sgRNA group decreased significantly compared with that of the HFD Sramble sgRNA group (P 0.001). There was no significant difference in body weight and blood glucose levels in the control CCR7 sgRNA group compared to the Ctrl Sramble sgRNA group; this indicated that the glucose intolerance and insulin resistance of the mice in the HFD CCR7 sgRNA group were improved. In addition, that food consumption in each group was stable and there was no significant group difference in each stage of the experiment. The expression of NLRP3, ASC, Caspase-1, IL-1 and the expression of IRS-1 and Akt in the hypothalamus of mice with different treatment groups, such as NLRP3, ASC, Caspase-1, IL-1, and the expression of IRS-1 and Akt, were detected by WB and IF. The results showed that: The expression of CCR7 protein in the hypothalamus of the HFD group was significantly higher than that of the control group, while the expression of the protein, such as NLRP3, ASC, Caspase-1, IL-1, and the like, increased significantly, and the phosphorylation of the insulin receptor substrate 1 (IRS-1) was enhanced. After the specific knockout of the CCR7 gene, the HFD CCR7 sgRNA group compared with the HFD Sramble sgRNA group. The expression of the above-mentioned inflammatory pathway protein decreased significantly (P0.01), and the phosphorylation of the IRS-1 protein was decreased; while the expression of the inflammation pathway-related protein and the expression of the IRS-1 protein did not change significantly compared with the control Sramble sgRNA group, and the results of the IF detection were consistent with the WB detection results. In conclusion, CCR7 mediates the activation of an NLRP3 inflammatory body in the hypothalamus of a high-fat diet mouse, activates the Caspase-1 protein, promotes the maturation and release of the inflammatory factors such as IL-1 and TNF-1, amplifies the hypothalamic inflammation reaction, negatively regulates the hypothalamic insulin signal and the leptin signal, and finally leads to IR and LR, These results suggest that CCR7 is a key candidate for the treatment of the hypothalamic IR of T2DM.
【學(xué)位授予單位】：江蘇師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1

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