發(fā)布時間：2018-05-14 16:05

  本文選題：慢性間歇性缺氧 + 脂聯(lián)素�。� 參考：《南京醫(yī)科大學(xué)》2014年博士論文

【摘要】：目的慢性間歇性缺氧(CIH)是阻塞性睡眠呼吸暫停低通氣綜合征(OSAHS)的重要病理生理學(xué)特點(diǎn),但其對機(jī)體全身系統(tǒng)和局部組織器官的影響機(jī)制尚未完全闡明。本研究課題旨在：1)探索CIH對上氣道擴(kuò)張肌的損傷及其潛在機(jī)制；2)研究CIH對全身炎癥狀況的影響；3)探討脂聯(lián)素對機(jī)體全身和上氣道擴(kuò)張肌的潛在保護(hù)作用及其可能的機(jī)制。從而為闡明OSAHS的發(fā)病機(jī)制及尋找新的治療手段提供線索。方法將45只健康的雄性Sprague-Dawley大鼠(8周齡,體重180---200 g)隨機(jī)分為三組：A組(空白對照,Control)組、B組(慢性間歇性缺氧,CIH)組和C組(慢性間歇性缺氧+脂聯(lián)素,CIH+Ad)組,每組各15只。在完成總共35天、每天8小時的CIH造模后,采集所有大鼠的血液標(biāo)本,以酶聯(lián)免疫吸附法(ELISA)檢測血清脂聯(lián)素、腫瘤壞死因子(TNF)-α、白細(xì)胞介素(IL)-1β、 IL-6和C-反應(yīng)蛋白(CRP)水平,同時檢測代謝功能相關(guān)生化指標(biāo)。采集所有大鼠的頦舌肌組織標(biāo)本,以如下實(shí)驗(yàn)技術(shù)做相關(guān)檢測：1)透射電子顯微鏡觀察超微結(jié)構(gòu)；2)蛋白免疫印跡技術(shù)(Western Blotting)檢測內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)一未折疊蛋白應(yīng)答反應(yīng)(UPR)及其相關(guān)性細(xì)胞凋亡信號通路的標(biāo)志物蛋白分子表達(dá)水平；3)缺口原位標(biāo)記(TUNEL)法檢測組織細(xì)胞凋亡率；4)丙二醛(MDA)法檢測脂質(zhì)過氧化物水平；5)WST(?)法檢測超氧化物歧化酶(SOD)活力；6)探針標(biāo)記技術(shù)熒光顯微鏡觀察檢測活性氧成分(ROS)水平；7)實(shí)時定量一聚合酶鏈?zhǔn)椒磻?yīng)(RT-qPCR)技術(shù)檢測核因子(NF)-κB和脂聯(lián)素受體(AdR)基因的轉(zhuǎn)錄水平；8)ELISA檢測髓過氧化物酶(MPO)水平。數(shù)據(jù)分析以P0.05作為差異具有統(tǒng)計學(xué)意義。結(jié)果B組大鼠的頦舌肌組織中ERS-UPR及其相關(guān)性細(xì)胞凋亡信號傳導(dǎo)通路中主要標(biāo)志物蛋白分子,包括GRP78、p-PERK.p-eIF2α、p-IREla.XBPls.ATF6. CHOP、BAX/Bcl-2比值、caspase-3和caspase-12等的表達(dá)均顯著高于A、C兩組(所有P0.05)；后者兩組之間差異沒有統(tǒng)計學(xué)意義(所有P0.05)。B組大鼠頦舌肌組織細(xì)胞凋亡率((28.2±21.3)%)顯著高于A組的((11.5±3.7)%,PBA=0.002)和C組的((15.9±10.3)%,PBC=0.019),后者兩組之間差異沒有統(tǒng)計學(xué)意義(PAC=0.390)。MDA在B組大鼠頦舌肌組織中的含量((8.05士4.53) nmol/mg prot)分別高于A組((5.18±3.03)nmol/mg prot,PBA=0.001)和C組((5.74±3.06)nmol/mg prot,PBC=0.002),A、C兩組之間差異無統(tǒng)計學(xué)意義(PAC=0.691)。C組大鼠頦舌肌組織的SOD活力水平((42.42±23.17)U/mg prot)低于B組((61.77±36138)U/mg prot,PCB=0.015),但高于A組((18.62±11.67) U/mg prot,PCA=0.009).C組大鼠頦舌肌組織的ROS相對水平(1.94士1.01)低于B組(3.31±1.56,PCB=0.001),但高于A組(1.08±0.38,PCA=0.038)。B組大鼠頦舌肌組織中NF-κB的mRNA相對量高于A組和C組(所有P0.05),后者兩組之間差異沒有統(tǒng)計學(xué)意義(P0.05)。B組和C組大鼠的頦舌肌組織中MPO含量分別為(0.40±O.29)μIU/mg prot和(0.31±O.17)μIU/mg prot,都高于A組((0.17±0.08)μIU/mg prot,PBA=0.002,PCA=0.047),但B、C兩組之間差異無統(tǒng)計學(xué)意義(P=0.240)。B組大鼠頦舌肌組織的AdR1 mRNA和AdR2 mRNA的相對量都分別低于A組和C組(所有P0.01),后者兩組之間的差異都沒有統(tǒng)計學(xué)意義(所有P0.05)。B組大鼠的血清TNF-α水平((70.87±35.16)pg/mL)分別高于A組((26.54±20.32)pg/mL,PBA0.01)和C組((29.50±22.54)pg/mL, PBC0.01),后者兩組之間差異無統(tǒng)計學(xué)意義(PAC=0.777)。B組((30.54±12.25) pg/mL)和C組((23.04±13.85)pg/mL)大鼠的血清IL-6水平分別高于A組((14.10±8.83)pg/mL,.PBA0.01,PCA=0.045),但B、C兩組的差異沒有統(tǒng)計學(xué)意義(PCB=0.090)。B組大鼠的血清脂聯(lián)素水平((4207.9±2238.5)ng/mL)分別低于A組((7051.2±2431.8)ng/mL,PBA=0.002)和C組((6404.5±2383.6)ng/mL,PBC= 0.015),后者兩組之間差異無統(tǒng)計學(xué)意義(PAC=0.468)。結(jié)論CIH引起低脂聯(lián)素血癥的同時,也觸發(fā)頦舌肌組織內(nèi)質(zhì)網(wǎng)應(yīng)激,并激活下游的未折疊蛋白應(yīng)答反應(yīng)信號傳導(dǎo)通路和誘發(fā)相關(guān)的細(xì)胞凋亡。此外,CIH還導(dǎo)致頦舌肌內(nèi)氧化應(yīng)激和炎癥損傷。與此同時,還引起全身炎癥反應(yīng)。補(bǔ)充脂聯(lián)素可改善內(nèi)質(zhì)網(wǎng)應(yīng)激性細(xì)胞凋亡及氧化應(yīng)激和炎癥損傷,同時也部分的緩解全身炎癥。
[Abstract]:Objective chronic intermittent hypoxia (CIH) is an important pathophysiological feature of obstructive sleep apnea hypopnea syndrome (OSAHS), but the mechanism of its influence on systemic systemic and local tissues has not been fully elucidated. 1) the aim of this study is to explore the damage of CIH to the upper airway dilator and its potential mechanism; 2) to study CIH The effect on the systemic inflammatory condition; 3) to explore the potential protective effect of adiponectin on the body and the upper airway dilator and its possible mechanism, thus providing clues for clarifying the pathogenesis of OSAHS and finding new treatment methods. Methods 45 healthy male Sprague-Dawley rats (8 weeks of age, and weight 180---200 g) were randomly divided into three groups Group A (blank control, Control), group B (chronic intermittent hypoxia, CIH) and C group (chronic intermittent hypoxia + adiponectin, CIH+Ad) group, 15 rats in each group. After completing a total of 35 days and 8 hours of CIH model each day, the blood samples of all rats were collected and serum adiponectin and tumor necrosis factor (TNF) - alpha were detected by enzyme linked immunosorbent assay (ELISA). The levels of interleukin (IL) -1 beta, IL-6 and C- reactive protein (CRP) were measured, and the biochemical indexes of metabolic function were detected. The genial tongue muscle specimens of all rats were collected, and the related tests were performed as follows: 1) transmission electron microscopy was used to observe the ultrastructure; 2) protein immunoblotting (Western Blotting) was used to detect endoplasmic reticulum stress (ERS). An unfolded protein response response (UPR) and its associated apoptotic signaling pathway marker protein molecular expression level; 3) the apoptosis rate of tissue cells was detected by the nick in situ labeling (TUNEL) method; 4) malondialdehyde (MDA) assay was used to detect the lipid peroxide level; 5) WST (?) method was used to detect the activity of superoxide dismutase (SOD); 6) probe labeling technique The level of reactive oxygen species (ROS) was detected by fluorescence microscopy; 7) real-time quantitative polymerase chain reaction (RT-qPCR) technique was used to detect the transcription level of nuclear factor (NF) - kappa B and adiponectin receptor (AdR) gene; 8) ELISA was used to detect myeloperoxidase (MPO) level. Data analysis was statistically significant in P0.05 as a difference. The main marker protein molecules in ERS-UPR and its associated apoptotic signal transduction pathway in the tongue muscle tissue, including GRP78, p-PERK.p-eIF2 alpha, p-IREla.XBPls.ATF6. CHOP, BAX/Bcl-2 ratio, caspase-3 and caspase-12, were significantly higher than A, C two group (all P0.05), and the difference between the two groups was not statistically significant (all P0.05). The apoptosis rate ((28.2 + 21.3)%) of the genial myocytes of group.B was significantly higher than that in group A ((11.5 + 3.7)%, PBA=0.002) and C group (15.9 + 10.3)%, PBC=0.019). The difference between the two groups was not statistically significant (PAC=0.390) in the group of genocutaneous muscles of the B group (8.05. 4.53) nmol/mg prot) higher than that of A group (5.18 + 3.03) nmol/mg. Prot, PBA=0.001) and group C ((5.74 + 3.06) nmol/mg prot, PBC=0.002), A, C two groups had no statistically significant difference (PAC=0.691).C group genocutaneous tissue SOD activity level ((42.42 + 23.17) U/mg) lower than that of the group (61.77 + 36138), 0.015), but higher than (18.62 + 11.67) group (18.62 + 11.67) genocutaneous group The relative level of ROS (1.94 se 1.01) was lower than that of group B (3.31 + 1.56, PCB=0.001), but the mRNA relative amount of NF- kappa B in the gennial muscle tissue of group A (1.08 + 0.38, PCA=0.038) was higher than that of A and C groups (all P0.05), the difference between the two groups was (0.40). IU/mg prot and (0.31 + O.17) mu IU/mg prot were all higher than those of A group ((0.17 + 0.08) mu IU/mg prot, PBA=0.002, PCA=0.047), but there was no significant difference between the two groups. The level of serum TNF- alpha ((70.87 + 35.16) pg/mL) in group.B rats ((70.87 + 20.32) pg/mL, PBA0.01) and C group (29.50 + 22.54) pg/mL, PBC0.01), respectively, the difference between the two groups was not statistically significant ((PAC=0.777) in.B group (30.54 + 12.25) and (23.04 + 13.85) (23.04 + 13.85) No higher than group A ((14.10 + 8.83) pg/mL,.PBA0.01, PCA=0.045), but the difference in B, C two groups was not statistically significant ((4207.9 + 2238.5) ng/mL) in group.B rats ((7051.2 + 2431.8) ng/mL, PBA=0.002) and (6404.5 + 2383.6) 0.015), and there was no statistical difference between the latter group (6404.5 + 2383.6). AC=0.468). Conclusion CIH causes hypo hyponatremia, triggers the stress of the genocolx muscle tissue endoplasmic reticulum, activates the downstream unfolded protein response signal transduction pathway and induces apoptosis related. In addition, CIH also causes internal oxidative stress and inflammatory damage in the genial tongue muscle. At the same time, it also causes systemic inflammatory response. It can improve endoplasmic reticulum stress cell apoptosis, oxidative stress and inflammatory injury, and partly alleviate systemic inflammation.

【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】：2014
【分類號】：R766

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