本文關(guān)鍵詞： 鼠神經(jīng)生長因子(mNGF) 水通道蛋白-2(AQP-2) 脊神經(jīng)節(jié) 免疫組化　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景和目的:鼠神經(jīng)生長因子(mNGF)分子量約為26.5×103,它是一種大分子蛋白質(zhì),具有生物活性,mNGF最初是在小鼠頜下腺中提取而獲得的,它具有營養(yǎng)人類正常細(xì)胞的作用,在神經(jīng)損傷后,尤其是對外周神經(jīng)具有促進(jìn)恢復(fù)和再生的作用,目前在臨床上已被廣泛用于治療中樞及外周神經(jīng)損傷,并且獲得了比較理想的的臨床療效。腰脊神經(jīng)節(jié)與神經(jīng)根相鄰,該部位是感覺神經(jīng)的胞體集中區(qū)域,當(dāng)炎癥或者外來暴露對其造成損傷后,患側(cè)肢體會出現(xiàn)感覺功能的異常。隨著人類年齡增長,腰椎會逐漸發(fā)生退行性的改變,如椎間盤突出,椎管狹窄等,這些退變�？蓪�(dǎo)致腰脊神經(jīng)節(jié)(Dorsal root ganglion,DRG)的損傷,因此腰脊神經(jīng)節(jié)損傷后的修復(fù)對臨床具有重要的意義。本研究中利用結(jié)扎實(shí)驗大鼠的坐骨神經(jīng)來模擬建立腰脊神經(jīng)節(jié)損傷的動物模型,通過鼠神經(jīng)生長因子(mNGF)的治療來研究其在腰脊神經(jīng)節(jié)慢性損傷后促進(jìn)神經(jīng)功能恢復(fù)的作用,并研究其對脊神經(jīng)節(jié)內(nèi)水通道蛋白-2(AQP-2)含量變化的影響,了解臨床治療過程中脊神經(jīng)節(jié)內(nèi)分子水平的變化情況,為治療提供理論依據(jù)。方法:選取健康雄性成年SD大鼠90只,體重200-250g,將這90只實(shí)驗鼠隨機(jī)分為三組,假手術(shù)組,手術(shù)組,mNGF治療組,每組平均30只,在假手術(shù)組中將大鼠左側(cè)坐骨神經(jīng)給予暴露,不給予結(jié)扎;手術(shù)組暴露左側(cè)坐骨神經(jīng),在距神經(jīng)起始處附近用4.0含鉻羊腸線結(jié)扎坐骨神經(jīng)4道,每道間隔約1-2 mm,被結(jié)扎的神經(jīng)長約4.5 mm,然后逐層縫合,mNGF組處理同手術(shù)組,三組分別于手術(shù)前,術(shù)后第3天,7天,14天行熱縮足潛伏期、及機(jī)械縮足反應(yīng)閾測試。并與術(shù)后第3天,第7天,和第14天對三個實(shí)驗組中的部分大鼠進(jìn)行處死并留取脊神經(jīng)節(jié)標(biāo)本,然后對標(biāo)本進(jìn)行免疫組織化學(xué)染色觀察脊神經(jīng)節(jié)內(nèi)神經(jīng)元染色情況,Western blot分析來定量分析AQP-2量。結(jié)果:1行為學(xué)測試術(shù)后3天三組間熱縮足潛伏期分別為13.5±0.6s,5.4±0.3s,6.4±0.4s,第7天分別為13.5±0.6s,5.8±0.3s,8.3±0.7s,第14天分別為13.3±0.6s,5.8±0.3s,10.7±0.8s;術(shù)后第3天、7天、14天三組間差異均有統(tǒng)計學(xué)意義(P0.05)。術(shù)后3天三組間機(jī)械縮足反應(yīng)閾分別為34.3±1.3s,15.9±0.8s,22.5±0.8s,第7天分別為34.3±0.9s,15.9±1.0s,24.8±0.8s,第14天分別為34.3±0.5s,15.6±0.6s,28.6±1.3s;術(shù)后第3天、7天、14天三組間差異均有統(tǒng)計學(xué)意義(P0.05)。2免疫組化染色假手術(shù)組的脊神經(jīng)節(jié)免疫組化染色術(shù)后第3天,第7天,第14天染色較淺,隨時間增長染色變化不大;手術(shù)組于術(shù)后第3天進(jìn)行免疫組織化學(xué)染色可見大量點(diǎn)狀的DRG細(xì)胞被染成棕黃色,隨著時間增長,在第7天,第14天,陽性細(xì)胞染色變化不大;mNGF組于術(shù)后3天和第7天染色程度與手術(shù)組相差不大,第14天可見染色細(xì)胞數(shù)明顯減少,染色變淺。3 Western blot分析分別與術(shù)后3天,7天,14天對假手術(shù)組,手術(shù)組,以及mNGF組脊神經(jīng)節(jié)內(nèi)AQP-2表達(dá)量行定量分析,結(jié)果以積分光密度(IOD)值的平均值±標(biāo)準(zhǔn)差表示,術(shù)后3天假手術(shù)組與手術(shù)組和mNGF組分別為328±20,2744±77,2173±110,三組間有統(tǒng)計學(xué)差異(P0.05),術(shù)后第7天三組IOD值分別為323±16,2299±93,1664±54、三組間有統(tǒng)計學(xué)差異(P0.05),術(shù)后第14天三組IOD值分別為323±16,1948±40,841±46,三組間有統(tǒng)計學(xué)差異(P0.05)。結(jié)論:腰脊神經(jīng)節(jié)損傷后給予應(yīng)用mNGF進(jìn)行治療,可明顯增長大鼠熱縮足潛伏期,提高機(jī)械縮足反應(yīng)閾,減輕感覺過敏癥狀,同時腰脊神經(jīng)節(jié)慢性擠壓傷可造成腰脊神經(jīng)節(jié)內(nèi)AQP-2表達(dá)增多,mNGF可降低AQP-2的表達(dá)量。
[Abstract]:Background and objective: mouse nerve growth factor (mNGF) molecular weight is about 26.5 * 103, it is a kind of macromolecular protein with biological activity, mNGF was extracted and obtained in the mouse submandibular gland, it has the nutrition of human normal cells, after nerve injury, especially with peripheral nerve promote the recovery and regeneration, is currently in clinical practice has been widely used in the treatment of central and peripheral nerve injury, and has obtained the ideal clinical curative effect. Spinal ganglion and nerve root adjacent to the site of somatic sensory nerve area when inflammation or damage to the external exposure after suffering limb abnormalities occur in sensory function. With the growth of age, will gradually lumbar degenerative changes, such as disc herniation, spinal stenosis, which often leads to degeneration of lumbar spinal nerve section (Dorsal root ganglion, DRG) damage, so the spinal ganglion injury repair has important significance for clinical. To simulate the animal model of spinal ganglion injury in rats by ligation of the sciatic nerve in this study, the rat nerve growth factor (mNGF) treatment to study the recovery of neurological function in patients with chronic injury spinal ganglion, and to study the spinal ganglion of aquaporin -2 (AQP-2) content changes, understand the changes in the molecular level in the spinal ganglia in the course of clinical treatment, and provide theoretical basis for treatment. Methods: healthy adult male SD rats 90, weight 200-250g, the 90 experimental rats were randomly divided into three groups, sham operation group, operation group, mNGF treatment group, each group had an average of 30, exposed in the sham group, rats with left sciatic nerve ligation surgery group, not given; the left sciatic nerve was exposed. 璺濈緇忚搗濮嬪闄勮繎鐢，

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