發(fā)布時間：2018-05-21 13:06

  本文選題：經鼻給藥 + 運動神經元病��； 參考：《河北醫(yī)科大學》2014年碩士論文

【摘要】：目的：運動神經元病（motor neuron disease，MND）是一組病因未明的選擇性侵犯脊髓前角細胞、腦干運動神經元、皮層錐體細胞及錐體束的慢性進行性神經變性疾病。肌萎縮側索硬化(amyotrophic lateral sclerosis，ALS)是運動神經元病中發(fā)病率最高的一種類型，是選擇性累及脊髓前角細胞、腦干運動神經核以及大腦運動皮質的進行性致死性神經變性病，以同時累及上、下運動神經元為特點[1-2]�；颊叨嘣谑状纬霈F癥狀后的3～5年內因肌肉逐漸萎縮無力、呼吸衰竭而死亡。目前，臨床上除利魯唑可以有限的延緩患者病程外，尚無有效治療手段[3]。干細胞移植治療、基因治療等新方法尚在研究中[4-6],家族性ALS占5～10％，其中20％與銅／鋅超氧化物歧化酶(SODl)基因突變有關。 神經生長因子（nerve growth factor,NGF）由Levi Montalcini于20世紀50年代首次在小鼠肉瘤細胞中發(fā)現。NGF具有維持神經干細胞的存活，促進神經干細胞的增殖、分化及遷移，抑制細胞凋亡，營養(yǎng)神經元，保護和修復受損神經等效應[7]。NGF可促進體外培養(yǎng)的神經前體細胞分化成未成熟的神經元和星形膠質細胞。NGF有促進神經干細胞存活、增殖、分化及定向遷移等作用，有助于中樞神經系統損傷后神經的再生及修復,為MND治療帶來了希望。由于肝臟的首過效應和血腦屏障(Blood-brain barrierBBB)的阻擋，經典的靜脈及皮下給藥途徑并未使藥物最大程度的發(fā)揮效應。SOD1—G93A轉基因小鼠是國際上公認、代表家族性ALS的動物模型。所以，本實驗研究采用經鼻給予SOD1小鼠不同濃度的NGF觀察其腦內海馬齒狀回的BrdU、Nestin的表達及神經元的數量的方法，，評價其對SOD1轉基因小鼠內源性神經干細胞的增殖調節(jié)作用，旨在尋找一種可以使NGF繞過BBB并在中樞靶部位迅速達到最佳效應濃度的新型給藥途徑。 方法： 1實驗模型及分組：100天雌性非轉基因正常對照組小鼠6只，分為A組；國際公認的100天（癥狀期）SOD1-G93A雌性轉基因小鼠36只(均由中國醫(yī)學科學院實驗動物研究中心提供)，隨機分為:B組（空白對照組）6只；C組（經鼻給藥組）30只。再按照經鼻給予NGF不同濃度，將經鼻給藥組分為：C1組（15mg/kg濃度組）；C2組（20mg／kg濃度組）、C3組（25mg／kg濃度組）、C4組（30mg／kg濃度組）、C5組（35mg／kg濃度組），每組6只。 2給藥途徑及方法：A組及B組經鼻給予生理鹽水，C組經鼻給予NGF，給藥量每次給予5ul，間隔2min再次給藥，根據各組分別給予總量A:15ul、B:15ul、C1:15ul、C2:20ul、C3:25ul、C4:30ul、C5:35ul。用同樣給藥方法連續(xù)給3天藥。 3各組隨機取一半標本通過免疫組織化學染色以顯示內源性神經干細胞及腦內增殖細胞。進行圖像分析并計數Nestin和Brdu陽性細胞數目，分析比較A組、B組、C組之間腦內神經細胞增殖及內源性神經干細胞增殖和分化的變化情況。 4另一半標本腹腔內注射戊巴比妥鈉（40mg/Kg）對小鼠進行麻醉后，斷頭取腦，腦組織中分離出海馬組織，采用Western-Blot法檢測Nestin和Brdu蛋白表達水平。 結果： 1. H.E.及Masson染色結果顯示：A組中，海馬齒狀回存在較少BrdU陽性細胞。B組皮質均有較多的Nestin和Brdu陽性細胞，同時雙側的海馬齒狀回區(qū)也有Nestin陽性細胞，但該區(qū)域Brdu陽性細胞數量相對較少；但C1-C5組Nestin和Brdu陽性細胞數目均有明顯增多(P0．05)；當NGF濃度達到25mg／kg以后，隨著濃度增加，Nestin和Brdu陽性細胞增殖數目較以前增殖不明顯。 2.Western Blot相關蛋白基因變化：Western blot檢測結果顯示：正常對照組腦內有一定水平的Nestin和Brdu蛋白表達；B組小鼠相對于正常對照組小鼠海馬區(qū)齒狀回Nestin和Brdu蛋白表達較高。C組中隨著經鼻給藥劑量逐漸加大，Nestin和Brdu蛋白表達也隨之逐漸增高，但C4、C5組蛋白表達較C3組有所減弱，差異有顯著性意義(P0．01)。 結論： 1.癥狀期SOD1轉基因小鼠腦內發(fā)現有內源性神經干細胞增殖，說明本實驗操作效果明顯。 2.NGF對SOD1轉基因小鼠腦內作用與其給藥量密切相關。低給藥量時(15ul)，主要表現為對腦內某些神經元的保護作用，對內源性神經干細胞增殖作用較強，當給藥量大于25ul以上時，對神經干細胞的增殖作用不明顯，當給藥量達到35ul時，小鼠可產生明顯的不良反應癥狀。
[Abstract]:Objective: motor neuron disease (MND) is a group of unknown causes of selective invasion of the spinal cord anterior horn cells, brainstem motoneurons, cortical pyramidal cells and pyramidal bundles of chronic progressive neurodegenerative disease. Amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS) is the highest incidence of motor neuron disease. A type of progressive neurodegenerative neurodegenerative disease involving the spinal cord anterior horn cells, the brainstem motor nucleus, and the motor cortex of the brain, which is involved in the simultaneous involvement of the lower motor neurons in the [1-2]. patients in the 3~5 years after the first symptom of the onset of the atrophy of the muscles and the death of respiratory failure. In addition to the limited delay in the course of the patient's disease, there is no effective treatment for [3]. stem cell transplantation. The new method of gene therapy, such as gene therapy, is still in the study of [4-6], and familial ALS accounts for 5 to 10%, of which 20% are related to the mutation of copper / zinc superoxide dismutase (SODl) gene.
Nerve growth factor (NGF) was first found in mouse sarcoma cells by Levi Montalcini in 1950s that.NGF has the survival of neural stem cells, promoting the proliferation, differentiation and migration of neural stem cells, inhibiting apoptosis, nourishing neurons, protecting and repairing damaged nerve effects [7].NGF can promote in vitro. The differentiation of cultured neural precursor cells into immature neurons and astrocytes.NGF can promote the survival, proliferation, differentiation and directional migration of neural stem cells, which can help the regeneration and repair of the nerve after the central nervous system injury, and bring hope for the treatment of MND. The first effect of the liver and the blood brain barrier (Blood-brain Barr) IerBBB), the classical vein and subcutaneous administration pathway did not make the maximum effect of the drug.SOD1 G93A transgenic mice as an internationally recognized animal model representing familial ALS. Therefore, this experiment studied the BrdU of the dentate gyrus of the intracerebral sea, the expression of Nestin, and the NGF of different concentrations of the SOD1 mice given to SOD1 mice. The number of neurons is used to evaluate the proliferation regulation of endogenous neural stem cells in SOD1 transgenic mice, aiming to find a new way to make NGF bypass BBB and quickly reach the best effect concentration at the central target site.
Method錛

本文編號：1919317