【摘要】：目的: 關(guān)于慢性間歇性缺氧（CIH）對頦舌肌功能的影響以及脂聯(lián)素的干預(yù)機(jī)制研究至今尚未完全闡明。本課題通過建立慢性間歇性缺氧大鼠模型，研究（1）CIH對大鼠頦舌肌線粒體結(jié)構(gòu)和功能的影響；（2）脂聯(lián)素對CIH誘導(dǎo)的大鼠頦舌肌線粒體損傷的干預(yù)調(diào)節(jié)作用以及可能的機(jī)制研究，從而為防治阻塞性睡眠呼吸暫停低通氣綜合征提供新的途徑。 方法: 本研究首先建立大鼠間歇性缺氧模型，30只健康雄性Sprague Dawley(SD)大鼠，8周齡，體重180～200g，隨機(jī)分組，分為正常對照組（Control組）、慢性間歇性缺氧組（CIH組）和慢性間歇性缺氧組+脂聯(lián)素組（CIH+AD組），每組10只。造模成功后，首先檢測不同處理組大鼠血清脂聯(lián)素水平的變化。接著通過透射電子顯微鏡、定量real-time PCR、琥珀酸脫氫酶（SDH）染色法、細(xì)胞色素氧化酶（COX）染色法和Western Blot觀察不同處理組之間大鼠頦舌肌線粒體合成、超微結(jié)構(gòu)和氧化能力的改變。在明確CIH對大鼠頦舌肌線粒體的損傷后，我們探討了脂聯(lián)素的干預(yù)作用以及可能的機(jī)制研究。機(jī)制研究主要檢測脂聯(lián)素介導(dǎo)的信號通路對大鼠頦舌肌線粒體的影響以及不同處理組間頦舌肌肌纖維改變，檢測主要依賴于Western blot和real-time PCR技術(shù)。機(jī)制研究重點(diǎn)觀察了脂聯(lián)素是對LKB1-AMPK-PGC1-α通路的激活情況。 結(jié)果: CIH造模成功后，我們檢測了大鼠血清脂聯(lián)素水平。CIH組大鼠血清脂聯(lián)素濃度為(1051.7±201.8)ng/ml，與對照組（濃度（2356±276.7）ng/ml）相比明顯降低；CIH+AD組脂聯(lián)素濃度為（1934.7±253.3）ng/ml，明顯高于CIH組，差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果表明：慢性間歇性缺氧可以引發(fā)大鼠血清脂聯(lián)素水平降低，而當(dāng)給予外源性脂聯(lián)素后，CIH+AD組血清脂聯(lián)素水平顯著升高，但仍略低于對照組。 為了闡明CIH對頦舌肌功能的影響，我們著重研究的了CIH對線粒體合成、超微結(jié)構(gòu)和功能的影響。實(shí)驗(yàn)結(jié)果顯示：（1）間歇性缺氧35天后，大鼠頦舌肌線粒體含量降低，參與線粒體合成重要調(diào)控基因（如PGC1-α、Esrrα、Nrf1、Cycs）的mRNA表達(dá)顯著下降。以上的結(jié)果均提示間歇性缺氧35后大鼠頦舌肌線粒體合成發(fā)生障礙。（2）慢性間歇性缺氧條件下，大鼠頦舌肌肌膜下和肌纖維間線粒體的面積變小，線粒體的內(nèi)外膜結(jié)構(gòu)變得紊亂，基質(zhì)電子密度顯著降低，線粒體嵴排列不規(guī)則，嵴數(shù)量減少，嵴變短，這些說明CIH誘導(dǎo)了頦舌肌線粒體超微結(jié)構(gòu)的損傷。（3）間歇性缺氧條件下，與線粒體功能成正相關(guān)基因的轉(zhuǎn)錄水平顯著降低，SDH染色和COX染色提示CIH組較對照組，SDH和COX陽性細(xì)胞明顯減少。以上結(jié)果均說明CIH環(huán)境下，大鼠頦舌肌線粒體功能顯著受損，肌纖維的氧化能力明顯減弱。 接著，我們研究了脂聯(lián)素對CIH誘導(dǎo)的頦舌肌線粒體損傷的影響。實(shí)驗(yàn)結(jié)果顯示：（1）當(dāng)給予外源性脂聯(lián)素后，CIH+AD組大鼠頦舌肌線粒體的含量顯著高于CIH組；基因水平研究發(fā)現(xiàn)，CIH+AD組與線粒體合成密切相關(guān)基因mRNA表達(dá)水平顯著高于CIH組。以上結(jié)果均提示當(dāng)給予外源性脂聯(lián)素后，可以減輕因慢性間歇性缺氧所致的大鼠頦舌肌線粒體合成障礙。（2）當(dāng)給予外源性脂聯(lián)素后，CIH+AD組大鼠頦舌肌線粒體超微結(jié)構(gòu)損傷較CIIH組相比，顯著改善，主要表現(xiàn)為肌膜下和肌纖維間線粒體的面積較CIH組明顯增加，線粒體內(nèi)外膜結(jié)構(gòu)變得完整，結(jié)構(gòu)清晰，基質(zhì)形態(tài)和結(jié)構(gòu)也較完整，密度適中，線粒體嵴較CIH組排列規(guī)整。（3）定量real time-PCR結(jié)果提示：CIH+AD組與CIH組比，與線粒體功能呈正相關(guān)基因的轉(zhuǎn)錄水平顯著升高，差異具有統(tǒng)計(jì)學(xué)意義。SDH染色和COX染色均提示：CIH+AD組與CIH組相比，SDH和COX陽性細(xì)胞明顯增加。以上結(jié)果均表明：當(dāng)給予外源性脂聯(lián)素后，明顯改善慢性間歇性缺氧所致大鼠頦舌肌線粒體功能損傷，顯著提升肌纖維的氧化能力。 隨后我們檢測不同處理組頦舌肌肌纖維的變化。Western Blot檢測結(jié)果提示慢性間歇性缺氧環(huán)境下，Myosin chain1和Troponin I的蛋白表達(dá)水平明顯低于正常對照組，MHC1基因的mRNA表達(dá)水平亦顯著低于正常對照組；當(dāng)給予外源性脂聯(lián)素干預(yù)后，Myosin chain1、Troponin I的蛋白表達(dá)水平以及MHC1基因的mRNA表達(dá)水平均高于CIH組。以上結(jié)果表明慢性間歇性缺氧環(huán)境下，頦舌肌I型即氧化型纖維的含量較正常對照組顯著降低；當(dāng)給予外源性脂聯(lián)素干預(yù)后，頦舌肌I型纖維的含量明顯增高，即脂聯(lián)素可以減輕慢性間歇性缺氧所致的氧化型纖維的損傷，從而提升頦舌肌的收縮功能。 最后，，我們檢測了脂聯(lián)素介導(dǎo)的信號通路的激活情況。Western Blot檢測結(jié)果提示慢性間歇性缺氧環(huán)境下，LKB1-AMPK-PGC1-α通路的活化被部分抑制；當(dāng)我們給予外源性脂聯(lián)素干預(yù)后，該通路又被重新激活。因此我們推論：脂聯(lián)素可能通過激活LKB1-AMPK-PGC1-α通路從而改善慢性間歇性缺氧所致的大鼠頦舌肌結(jié)構(gòu)和功能的損傷。 結(jié)論: 我們的研究表明，脂聯(lián)素可能通過顯著激活LKB1-AMPK-PGC1a通路改善CIH誘導(dǎo)的頦舌肌線粒體結(jié)構(gòu)和功能的損傷。
[Abstract]:Purpose: The effect of chronic intermittent hypoxia (CIH) on the function of the tongue and the mechanism of the intervention of adiponectin have not been completed yet. The effects of (1) CIH on the structure and function of the mitochondrial structure and function of the rat's tongue muscle were studied by establishing a model of chronic intermittent hypoxia in rats. (2) The effect of adiponectin on the mitochondrial damage of the rat tongue muscle induced by CIH and the possible mechanism In order to provide a new method for the prevention and treatment of obstructive sleep apnea-hypopnea syndrome The way. Methods: In this study, an intermittent hypoxia model was established in rats,30 healthy male Sprague Dawley (SD) rats,8 weeks of age,180 to 200 g of body weight, and randomly divided into normal control group (control group), chronic intermittent hypoxia group (CIH group) and chronic intermittent hypoxia group + adiponectin group (control group). CIH + AD Group)10 rats in each group. After the model was successful, the serum of rats in different treatment groups was first tested. The changes of the level of adiponectin were observed by transmission electron microscopy, quantitative real-time PCR, succinate dehydrogenase (SDH) staining, cytochrome oxidase (COX) staining and Western Blot. And the effect of the intervention of adiponectin was discussed. The mechanism study mainly detected the effect of adiponectin-mediated signaling pathway on the mitochondria of the rat's tongue and the muscle fiber of the tongue in different treatment groups, and the detection mainly depended on Western blot and real-t. The mechanism study focused on the effect of adiponectin on LKB1-AMPK-PGC1- path of passage Results: The CIH model was successful. The level of serum adiponectin in the rat was detected. The serum adiponectin concentration in the CIH group was (1051.7-201.8) ng/ ml, and the concentration of adiponectin in the CIH + AD group was significantly lower than that of the control group (2356-26.7) ng/ ml. The concentration of adiponectin in the CIH + AD group was (1934.7-253.3) ng/ ml, which was significantly higher than that of the control group. The results showed that the level of adiponectin in the serum of the rats can be reduced by chronic intermittent hypoxia, and the serum adiponectin in the CIH + AD group after administration of the exogenous adiponectin In order to clarify the effect of the CIH on the function of the tongue, we focus on the CIH. The effects of mitochondrial synthesis, ultrastructure and function were studied. The results showed that: (1) After 35 days of intermittent hypoxia, the content of the mitochondria in the rat's tongue was decreased, and the important regulatory genes involved in the synthesis of mitochondria (such as PGC1-1, Esrr, Nrf1) were involved. And Cycs) showed a significant decrease in the expression of mRNA. (2) Under the condition of chronic intermittent hypoxia, the area of the mitochondria between the muscle and the muscle fibers of the rat tongue is smaller, the inner and outer membrane structures of the mitochondria become disordered, the electron density of the matrix is obviously reduced, and the line particles the volume of the body is irregular, the number of the elements is reduced, the volume of the body is shortened, H. The ultrastructure of the mitochondria in the tongue of the tongue was induced. (3) Under the condition of intermittent hypoxia, the level of transcription of the gene related to the function of the mitochondria was significantly lower, and the staining of SDH and the staining of the COX-2 in the CIH group were significantly lower than that in the control group. In group, SDH and COX-positive cells were significantly reduced. The results indicated that the function of the mitochondrial function of the rat's tongue in the presence of CIH Significantly, the oxidation ability of the muscle fibers was significantly reduced. Then, we studied the fat The effects of adiponectin on the mitochondrial injury induced by CIH were studied. The results showed that: (1) When the exogenous adiponectin was given, the content of the mitochondria in the papillary muscle of the CIH + AD group was significantly higher than that of the CIH group, and the level of the gene found that the CIH + AD group and the mitochondria were closely combined. The expression level of the related gene was significantly higher than that of the CIH group. (2) When the exogenous adiponectin was given, the ultrastructure of the mitochondria in the papillary muscle of the CIH + AD group was significantly improved compared with that of the CIIH group, and the main performance was that the area of the mitochondria in the muscle membrane and the muscle fiber was higher than that of the CIH group. in addition, that internal and external membrane structure of the mitochondria become complete, the structure is clear, the shape and the structure of the matrix are also complete, The results of quantitative real time-PCR showed that the CIH + AD group and the CIH group were in positive correlation with the function of the mitochondria. There was a significant increase in the level of transcription, the difference was of statistical significance, and both the SDH staining and the COX staining showed: CIH + AD group and CI Compared with group H, the positive cells of SDH and COX-positive cells increased significantly. The mitochondrial function is damaged, and the oxidation capacity of the muscle fiber is obviously improved. The results of the Western Blot test indicated that the level of protein expression of Myosin chain1 and Troponin I was significantly lower than that in the control group. The expression of MHC1 gene was also significantly lower than that in the control group. After the intervention of exogenous adiponectin, the level of protein expression of Myosin chain1, Troponin I was The results showed that the content of the oxidized fibers was significantly lower in the control group than in the normal control group under the condition of chronic intermittent hypoxia, and the expression of the MHC1 gene was significantly lower in the control group than in the normal control group. After the intervention of the source of adiponectin, the content of the I-shaped fibers in the tongue of the tongue of the tongue is obviously increased, that is, the adiponectin can reduce the chronic intermittent hypoxia. The resulting oxidized fiber is damaged, thus promoting the contraction function of the papillary muscle. Finally, we examined the activation of the signal pathway mediated by adiponectin. The results of the Western Blot test suggest that the activation of LKB1-AMPK-PGC1-1 pathway is partially inhibited in the chronic intermittent hypoxia environment. System; this pathway was re-activated when we were given an exogenous adiponectin intervention. So we concluded that adiponectin may be derived from the activation of LKB1-AMPK-PGC1-1 pathway. and improve Chronic intermittent hypoxia-induced damage to the structure and function of the tongue of the rat's tongue. Conclusion: We have shown that adiponectin may activate LKB1-AM significantly by significantly activating LKB1-AM.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R766

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