發(fā)布時(shí)間：2018-05-20 02:09

  本文選題：自噬 + 急性期脊髓損傷　； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：研究背景脊髓損傷(Spinal cord injury,SCI)是一種可造成中樞神經(jīng)系統(tǒng)的重度創(chuàng)傷并可引起多種并發(fā)癥的疾病,脊髓損傷后其中常見(jiàn)的并發(fā)癥之一即是神經(jīng)源性膀胱。因?yàn)榈图?jí)的排尿中樞存在于脊髓腰骶段,所以當(dāng)損傷腰骶或以上時(shí)就會(huì)出現(xiàn)排尿障礙,表現(xiàn)為膀胱逼尿肌過(guò)度活動(dòng)癥、膀胱逼尿肌收縮無(wú)力或者逼尿肌與尿道括約肌協(xié)同失調(diào)等,嚴(yán)重影響患者生活質(zhì)量。已有研究證實(shí)自噬在大鼠脊髓損傷模型中脊髓損傷處活躍,其參與神經(jīng)損傷修復(fù),促進(jìn)自噬表達(dá)也有助于恢復(fù)小鼠后肢的運(yùn)動(dòng)功能,此外,在氣管平滑肌、心肌及血管平滑肌中已證實(shí)自噬現(xiàn)象存在,其異常表達(dá)的水平,在慢性阻塞性肺部疾病、心臟疾病及動(dòng)脈粥樣硬化形成過(guò)程中發(fā)揮作用。自噬是一種選擇性降解胞質(zhì)蛋白、蛋白堆積物及細(xì)胞器的胞內(nèi)代謝機(jī)制,首先形成自噬小體,然后運(yùn)至溶酶體并與其融合形成自噬溶酶體,最終由自噬溶酶體降解胞質(zhì)成分。目的觀察自噬相關(guān)基因、蛋白在急性期脊髓損傷后大鼠膀胱平滑肌組織的表達(dá),以探討其在神經(jīng)源性膀胱發(fā)病機(jī)制可能存在作用。方法24只雄性成年Wistar大鼠隨機(jī)分為兩組即模型組(12只)和對(duì)照組(12只)。模型組應(yīng)用改良Allens'法構(gòu)造急性期脊髓損傷模型,對(duì)照組僅予以椎板切除術(shù)。術(shù)后6h對(duì)大鼠下肢運(yùn)動(dòng)功能進(jìn)行BBB(Basso Beattle Bresnahan)運(yùn)動(dòng)評(píng)分,尼氏染色觀察兩組大鼠脊髓橫斷面形態(tài)學(xué)變化,Western blotting檢測(cè)膀胱平滑肌細(xì)胞微管相關(guān)蛋白 1 輕鏈 3(microtubule associated protein 1 light chain 3,LC3)和 P62 蛋白相對(duì)表達(dá)量,免疫熒光染色法檢測(cè)LC3和P62蛋白在平滑肌細(xì)胞位置及陽(yáng)性細(xì)胞率,RT-PCR檢測(cè)膀胱平滑肌細(xì)胞中基因LC3、P62、Beclin1mRNA相對(duì)水平。結(jié)果1.術(shù)后6h模型組大鼠下肢運(yùn)動(dòng)功能BBB評(píng)分較對(duì)照組低(P0.01)2.尼氏染色觀察鏡下觀察對(duì)照組脊髓灰質(zhì)神經(jīng)元細(xì)胞尼氏小體結(jié)構(gòu)完整,神經(jīng)元胞體呈多角形,模型組可觀察到損傷處脊髓橫斷面神經(jīng)元數(shù)量減少,胞體腫脹變?yōu)閳A形,尼氏小體減少。3.Western blotting提示模型組膀胱平滑肌細(xì)胞蛋白LC3-II表達(dá)比對(duì)照組平滑肌細(xì)胞蛋白高(P0.05),模型組膀胱平滑肌細(xì)胞蛋白P62表達(dá)比對(duì)照組膀胱平滑肌細(xì)胞蛋白降低(P0.05)。4.免疫熒光染色法提示LC3蛋白位于膀胱平滑肌細(xì)胞質(zhì)內(nèi),陽(yáng)性細(xì)胞率比對(duì)照組高(P0.05),P62蛋白亦位于膀胱平滑肌細(xì)胞質(zhì)內(nèi),陽(yáng)性細(xì)胞率比對(duì)照組低(P0.05)。5.RT-PCR檢測(cè)模型組LC3、P62、Beclin1 mRNA水平比對(duì)照組升高(P0.05)。結(jié)論在脊髓損傷急性期大鼠膀胱平滑肌中自噬表達(dá)活躍,可能在脊髓損傷所致神經(jīng)源性膀胱發(fā)病機(jī)制中起作用。
[Abstract]:Background Spinal cord injury (sci) is a kind of disease which can cause severe injury to the central nervous system and cause many complications. One of the common complications after spinal cord injury is neurogenic bladder. Because the lower voiding center is present in the lumbosacral segment of the spinal cord, dysuria occurs when the lumbosacral area is injured or above, which is characterized by bladder detrusor hyperactivity. Bladder detrusor contraction weakness or detrusor and urethral sphincter coordination, seriously affect the quality of life of patients. It has been proved that autophagy is active in the spinal cord injury model of rats, which is involved in the repair of nerve injury, promotes autophagy expression and helps to restore the motor function of the hind limbs of mice, in addition, in the tracheal smooth muscle, Autophagy has been confirmed in myocardium and vascular smooth muscle. The abnormal expression of autophagy plays an important role in the development of chronic obstructive pulmonary disease, heart disease and atherosclerosis. Autophagy is a mechanism of selective degradation of cytoplasmic proteins, protein accumulations and organelles. Firstly, autophagy bodies are formed, then transported to lysosomes and fused to form autophagy lysosomes, and the cytoplasmic components are degraded by autophagy lysosomes. Objective to investigate the expression of autophagy related genes and proteins in the smooth muscle of bladder after acute spinal cord injury (sci) in order to explore the possible role of autophagy gene and protein in the pathogenesis of neurogenic bladder. Methods Twenty-four adult male Wistar rats were randomly divided into two groups: model group (n = 12) and control group (n = 12). The model group used improved Allens' method to construct acute spinal cord injury model, while the control group was treated with laminectomy only. The lower extremity motor function of rats was evaluated with BBB(Basso Beattle Bresnahan motor score at 6 hours after operation. Morphological changes of spinal cord in the two groups were observed by Nissl staining. The relative expression of microtubule-associated protein 1 (3(microtubule associated protein 1 light chain 3) and P62 protein in bladder smooth muscle cells was detected by Western blotting. The location and positive rate of LC3 and P62 protein in smooth muscle cells were detected by immunofluorescence staining. RT-PCR was used to detect the relative level of gene LC3P62 / Beclin1 mRNA in bladder smooth muscle cells. Result 1. The BBB score of lower extremity motor function in the model group was lower than that in the control group at 6 hours postoperatively (P < 0.01). The structure of the neurons in the control group was intact and the neuronal bodies were polygonal. In the model group, the number of neurons in the cross section of the spinal cord was decreased, and the swelling of the neurons became round. The expression of bladder smooth muscle cell protein (LC3-II) in the model group was higher than that in the control group (P 0.05), and the expression of P62 in the model group was lower than that in the control group (P 0.05 路4). Immunofluorescence staining showed that the LC3 protein was located in the cytoplasm of the bladder smooth muscle, and the positive cell rate was higher than that in the control group. The positive cell rate was lower than that in the control group, and the positive cell rate was lower than that in the control group (P 0.05). 5. The level of LC3 protein in the model group was higher than that in the control group (P 0.05). Conclusion the active expression of autophagy in the bladder smooth muscle of rats with acute spinal cord injury may play an important role in the pathogenesis of neurogenic bladder induced by spinal cord injury.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R651.2

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本文編號(hào)：1912714