本文關(guān)鍵詞： 脊髓損傷 褪黑素 自噬 LC3 Beclin-1　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景及目的作為脊柱損傷最嚴(yán)重的并發(fā)癥,急性脊髓損傷(acute spinal cord injury,ASCI)常引起軀體感覺與功能障礙的嚴(yán)重?fù)p害。對患者本人而言,其帶來了嚴(yán)重的身體和心理傷害,對整個社會而言,其造成了巨大的經(jīng)濟負(fù)擔(dān)。鑒于此,當(dāng)今醫(yī)學(xué)界對急性脊髓損傷的預(yù)防、治療和康復(fù)給予了越來越多的重視和關(guān)注。隨著病理生理學(xué)的發(fā)展,對于急性脊髓損傷的認(rèn)識也逐漸加深,將其分為原發(fā)性和繼發(fā)性脊髓損傷。由于目前臨床無法干預(yù)原發(fā)性脊髓損傷,繼發(fā)性脊髓損傷在ASCI中起著重要的作用,而神經(jīng)細(xì)胞的凋亡與繼發(fā)性脊髓損傷的發(fā)生密切相關(guān)。因此抑制神經(jīng)元細(xì)胞的凋亡成為ASCI早期治療的關(guān)鍵。細(xì)胞自噬是一種高度保守的生物自我保護機制,可以使變性的蛋白及喪失功能的細(xì)胞器降解并重新利用。近年來,研究證實細(xì)胞自噬與凋亡有著相互影響。褪黑素(MT)是由松果腺產(chǎn)生和分泌的兩性分子,廣泛存在于機體內(nèi),目前學(xué)者普遍認(rèn)為,褪黑素通過減輕水腫、抑制凋亡、減少氧化應(yīng)激等機制發(fā)揮其保護神經(jīng)的作用。然而,褪黑素治療脊髓損傷在抗凋亡方面的機制尚無統(tǒng)一結(jié)論。因此,在本研究中,我們通過在ASCI動物模型中予以褪黑素干預(yù),觀察急性脊髓損傷后自噬的發(fā)生并探討褪黑素對自噬的影響,研究褪黑素的作用機制,為臨床應(yīng)用提供參考。材料與方法108只成年SD大鼠,按隨機數(shù)字表法隨機分為三組:假手術(shù)組(A組)、脊髓損傷組(B組)、褪黑素治療組(C組)。A組僅行椎板切除,暴露T11-T12節(jié)段,不損傷脊髓神經(jīng)。B組和C組用改良的Allen's法建立T11-T12脊髓損傷模型,并分別在脊髓損傷后10 min腹腔注射等體積的無水乙醇和100mg/kg的MT制劑。各組分別于術(shù)后6 h、12 h、24 h隨機選取12只大鼠,取T11-T12節(jié)段脊髓標(biāo)本,6個標(biāo)本檢測自噬相關(guān)蛋白的表達(dá)水平,6個標(biāo)本HE染色和免疫熒光切片,觀察脊髓損傷及修復(fù)情況。術(shù)后72h對3組大鼠進行Basso Beattie Bresnahan(BBB)運動功能評分。結(jié)果脊髓損傷后,B、C兩組均出現(xiàn)了嚴(yán)重的下肢神經(jīng)功能障礙,在術(shù)后72h,C組的BBB運動功能評分較B組明顯上升(p㩳0.05);術(shù)后各時間點大鼠脊髓的微管相關(guān)蛋白1輕鏈3(LC3)表達(dá)水平比較:C組最高、B組次之、A組最低。在6h、12 h和24 h時間點上各組之間差異均有統(tǒng)計學(xué)意義(p㩳0.05);術(shù)后各時間點大鼠脊髓的Beclin-1表達(dá)水平比較:C組最高、B組次之、A組最低。在6h、12 h和24 h時間點上各組之間差異均有統(tǒng)計學(xué)意義(p㩳0.05);HE染色切片觀察脊髓損傷程度:A組最輕、C組次之、B組最重,并且各組損傷時間越長,損傷程度越重;免疫熒光顯示:C組LC3陽性神經(jīng)元細(xì)胞最多、B組次之、A組最少。結(jié)論大鼠急性脊髓損傷后早期神經(jīng)組織發(fā)生了自噬,應(yīng)用褪黑素能增強自噬并在一定程度上起到保護神經(jīng)功能的作用。
[Abstract]:Background and objective as the most serious complication of spinal injury, acute spinal cord injuryASCI often causes severe damage to somatosensory and functional disorders. In view of this, the medical profession has paid more and more attention to the prevention, treatment and rehabilitation of acute spinal cord injury. With the development of pathophysiology, The recognition of acute spinal cord injury is gradually deepened, which is divided into primary spinal cord injury and secondary spinal cord injury. Because it is difficult to intervene in primary spinal cord injury, secondary spinal cord injury plays an important role in ASCI. The apoptosis of neurons is closely related to the occurrence of secondary spinal cord injury. Therefore, inhibition of neuronal apoptosis is the key to early treatment of ASCI. Autophagy is a highly conservative biological self-protection mechanism. In recent years, studies have shown that autophagy and apoptosis interact with each other. Melatonin (MTT) is a amphoteric molecule produced and secreted by the pineal gland and is widely present in the body. At present, it is widely believed that melatonin protects nerves by reducing edema, inhibiting apoptosis and reducing oxidative stress. However, there is no uniform conclusion on the mechanism of anti-apoptosis of melatonin in spinal cord injury. In this study, we observed the occurrence of autophagy after acute spinal cord injury and studied the effect of melatonin on autophagy through the intervention of melatonin in ASCI animal model. Materials and methods 108 adult Sprague-Dawley rats were randomly divided into three groups: sham operation group (group A), spinal cord injury group (group B) and melatonin treatment group (group C). T11-T12 spinal cord injury model was established by modified Allen's method in group B and C without spinal cord injury. Anhydrous ethanol and 100 mg / kg MT were injected intraperitoneally 10 min after spinal cord injury. The expression level of autophagy related protein was detected in 6 spinal cord specimens from T11-T12 segment and HE staining and immunofluorescence sections were obtained from 6 specimens. The motor function score of Basso Beattie Bresnahanna BBB was evaluated 72 hours after operation. Results severe lower extremity neurological dysfunction appeared in group C after spinal cord injury. The BBB motor function score of group C was significantly higher than that of group B at 72 hours after operation. The expression level of microtubule associated protein 1 light chain 3 (LC3) in the spinal cord of rats at different time points after operation was lower than that in group B, the highest in group C, and the lowest in group A. there were significant differences in the expression of microtubule associated protein 1 between each group at 12 h and 24 h after operation. The expression level of Beclin-1 in spinal cord of rats at different time points after operation was lower than that in group B, the highest in group C, and the lowest in group A. the differences were statistically significant at the time points of 12 h and 24 h after operation. The degree of spinal cord injury in group A was the lighter than that in group B, and the longer the injury time was, the more serious the injury was. Immunofluorescence showed that the number of LC3 positive neurons was the most in group B and the lowest in group A. conclusion autophagy occurred in the early stage of spinal cord injury in rats. Melatonin can enhance autophagy and protect neural function to some extent.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R651.2

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本文編號：1500070