本文關(guān)鍵詞： 脊髓損傷模型 A型肉毒桿菌神經(jīng)毒素重鏈 神經(jīng)損傷與再生 髓磷脂相關(guān)糖蛋白 Nogo-receptor　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:1.在大鼠腰段脊髓損傷模型基礎(chǔ)上局部給予小劑量A型肉毒桿菌神經(jīng)毒素重鏈(botulinum neurotoxin serotype A heavy chain,BoNT/A HC),觀察其對脊髓損傷后局部總體蛋白表達(dá)譜的影響;2.觀察BoNT/A HC對脊髓損傷局部髓磷脂相關(guān)糖蛋白及其受體表達(dá)的影響,為探討B(tài)oNT/A重鏈促進(jìn)神經(jīng)突起再生的機(jī)制提供實驗依據(jù)。方法:1.采用1ml注射器針頭改制的脊髓損傷器械復(fù)制大鼠腰段脊髓半離斷性損傷模型;在模型復(fù)制同時由針頭所連接微量注射器向髓腔內(nèi)注射不同劑量BoNT/A重鏈(2μg、4μg、6μg和8μg);2.于大鼠脊髓損傷后2天和20天提取損傷局部及損傷近端2~3cm組織蛋白進(jìn)行SDS-PAGE+考馬氏亮藍(lán)染色確定蛋白表達(dá)有無差異,同時采用BoNT/A重鏈6mg劑量組進(jìn)行雙向電泳+硝酸銀染色進(jìn)一步確定蛋白表達(dá)差異的點數(shù)或點群分布;3.根據(jù)雙向電泳所出現(xiàn)的點群變化的分子量水平,采用western blot主要檢測BoNT/A重鏈對脊髓組織髓磷脂相關(guān)抑制因子—髓鞘相關(guān)糖蛋白(myelin associated glycoprotein,MAG)及其受體(nogo-receptor,N gR)的表達(dá)的干預(yù)作用。結(jié)果:1.單側(cè)腰段脊髓損傷模型顯微鏡下呈現(xiàn)明顯的一側(cè)脊髓結(jié)構(gòu)破壞,局部神經(jīng)細(xì)胞壞死、白質(zhì)神經(jīng)傳導(dǎo)束崩解不可辨認(rèn);2.SDS-PAGE+考馬斯亮藍(lán)染色顯示,單純脊髓損傷后,損傷局部蛋白表達(dá)與正常相比有明顯不同,有些分子量水平的蛋白表達(dá)明顯增多;BoNT/A重鏈用藥后2天及20天時,局部蛋白的表達(dá)與單純損傷組相比明顯不同,但與正常對照組類同、趨向正�；�;3.雙向電泳+硝酸銀染色的結(jié)果進(jìn)一步確定了這些蛋白表達(dá)變異的現(xiàn)象出現(xiàn)在多個分子量水平,呈現(xiàn)點或點群的表達(dá)變異。有10余種蛋白表達(dá)與單純損傷組明顯不同,呈向正常轉(zhuǎn)化的趨勢;主要的特點是:單純損傷時,某些蛋白點或點群表達(dá)增加,而BoNT/A重鏈干預(yù)后,這些變異又趨向于與正常對照組相似;4.根據(jù)蛋白表達(dá)變異的分子量特點,選取MAG和NgR作為目標(biāo)蛋白的wesetrb-blot結(jié)果顯示:單純脊髓損傷后MAG及NgR表達(dá)皆呈現(xiàn)增多,而髓腔內(nèi)注射BoNT/A重鏈后這兩種蛋白的表達(dá)均減少,差異具有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:1.BoNT/A重鏈可干預(yù)大鼠脊髓損傷后局部蛋白表達(dá)譜變化;2.BoNT/A重鏈可逆轉(zhuǎn)脊髓損傷后局部大量髓磷脂抑制因子的產(chǎn)生及其受體的表達(dá)。
[Abstract]:Objective 1. To give a small dose of botulinum A neurotoxin heavy chain on the basis of lumbar spinal cord injury model in rats. Botulinum neurotoxin serotype A heavy chain. To observe the effect of BoNT/A on the expression profile of local total protein after spinal cord injury. 2. To observe the effect of BoNT/A HC on the expression of myelin associated glycoprotein and its receptor in spinal cord injury. To explore the mechanism of BoNT/A heavy chain promoting neurite regeneration. Methods 1: 1. Spinal cord injury model was established by using 1 ml syringe needle modified spinal cord injury instrument in rats. At the same time, the microinjector connected with the needle was injected into the intramedullary cavity with different doses of BoNT/A 2 渭 g, 4 渭 g, 6 渭 g and 8 渭 g / g respectively. 2. Two and twenty days after spinal cord injury, 3 cm tissue proteins were extracted from the injured area and the proximal end of the injury to determine whether there was a difference in the expression of the protein by SDS-PAGE Coombe bright blue staining. At the same time, the BoNT/A heavy chain 6mg dose group was used to determine the point or point group distribution of protein expression difference by two dimensional electrophoresis silver nitrate staining. 3.According to the molecular weight level of point group changes in two-dimensional electrophoresis. Western blot was used to detect myelin associated inhibitor (myelin sheath associated glycoprotein) of BoNT/A heavy chain in spinal cord tissue. Myelin associated glycoprotein. Results: 1. Unilateral spinal cord injury model showed obvious unilateral spinal cord structure damage under microscope. Local nerve cell necrosis, white matter nerve conduction bundle disintegration unrecognizable; 2. SDS-PAGE Coomassie brilliant blue staining showed that after spinal cord injury alone, the expression of local protein was significantly different from that of normal, and the expression of some proteins with molecular weight increased obviously. On the 2nd and 20th day after BoNT/A heavy chain administration, the expression of local protein was significantly different from that of the simple injury group, but similar to that of the normal control group, and tended to normalize. 3. The results of silver nitrate staining further confirmed that the variation of these proteins appeared at multiple molecular weight levels. The expression of more than 10 proteins was significantly different from that of the simple injury group and showed a trend of normal transformation. The main characteristics were as follows: the expression of some protein points or groups was increased in simple injury, but after the intervention of BoNT/A heavy chain, these variations tended to be similar to those of the normal control group. 4.According to the molecular weight characteristics of protein expression variation. The results of wesetrb-blot with MAG and NgR as target protein showed that the expression of MAG and NgR increased after spinal cord injury alone. However, the expression of these two proteins decreased after intramedullary injection of BoNT/A heavy chain. Conclusion: 1. BoNT / A heavy chain can interfere with the changes of local protein expression profile after spinal cord injury in rats. 2. BoNT-A heavy chain could reverse the production of a large amount of myelin inhibitor and the expression of its receptor after spinal cord injury.
【學(xué)位授予單位】：山西醫(yī)科大學(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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