本文選題：經(jīng)鼻給藥 + 運(yùn)動(dòng)神經(jīng)元病��； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：運(yùn)動(dòng)神經(jīng)元�。╩otor neuron disease，MND）是一組病因未明的選擇性侵犯脊髓前角細(xì)胞、腦干運(yùn)動(dòng)神經(jīng)元、皮層錐體細(xì)胞及錐體束的慢性進(jìn)行性神經(jīng)變性疾病。肌萎縮側(cè)索硬化(amyotrophic lateral sclerosis，ALS)是運(yùn)動(dòng)神經(jīng)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(xì)胞、腦干運(yùn)動(dòng)神經(jīng)核以及大腦運(yùn)動(dòng)皮質(zhì)的進(jìn)行性致死性神經(jīng)變性病，以同時(shí)累及上、下運(yùn)動(dòng)神經(jīng)元為特點(diǎn)[1-2]�；颊叨嘣谑状纬霈F(xiàn)癥狀后的3～5年內(nèi)因肌肉逐漸萎縮無(wú)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(wú)有效治療手段[3]。干細(xì)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(guān)。 神經(jīng)生長(zhǎng)因子（nerve growth factor,NGF）由Levi Montalcini于20世紀(jì)50年代首次在小鼠肉瘤細(xì)胞中發(fā)現(xiàn)。NGF具有維持神經(jīng)干細(xì)胞的存活，促進(jìn)神經(jīng)干細(xì)胞的增殖、分化及遷移，抑制細(xì)胞凋亡，營(yíng)養(yǎng)神經(jīng)元，保護(hù)和修復(fù)受損神經(jīng)等效應(yīng)[7]。NGF可促進(jìn)體外培養(yǎng)的神經(jīng)前體細(xì)胞分化成未成熟的神經(jīng)元和星形膠質(zhì)細(xì)胞。NGF有促進(jìn)神經(jīng)干細(xì)胞存活、增殖、分化及定向遷移等作用，有助于中樞神經(jīng)系統(tǒng)損傷后神經(jīng)的再生及修復(fù),為MND治療帶來(lái)了希望。由于肝臟的首過效應(yīng)和血腦屏障(Blood-brain barrierBBB)的阻擋，經(jīng)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(yīng)。SOD1—G93A轉(zhuǎn)基因小鼠是國(guó)際上公認(rèn)、代表家族性ALS的動(dòng)物模型。所以，本實(shí)驗(yàn)研究采用經(jīng)鼻給予SOD1小鼠不同濃度的NGF觀察其腦內(nèi)海馬齒狀回的BrdU、Nestin的表達(dá)及神經(jīng)元的數(shù)量的方法，，評(píng)價(jià)其對(duì)SOD1轉(zhuǎn)基因小鼠內(nèi)源性神經(jīng)干細(xì)胞的增殖調(diào)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(dá)到最佳效應(yīng)濃度的新型給藥途徑。 方法： 1實(shí)驗(yàn)?zāi)Ｐ图胺纸M：100天雌性非轉(zhuǎn)基因正常對(duì)照組小鼠6只，分為A組；國(guó)際公認(rèn)的100天（癥狀期）SOD1-G93A雌性轉(zhuǎn)基因小鼠36只(均由中國(guó)醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)動(dòng)物研究中心提供)，隨機(jī)分為:B組（空白對(duì)照組）6只；C組（經(jīng)鼻給藥組）30只。再按照經(jīng)鼻給予NGF不同濃度，將經(jīng)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(jīng)鼻給予生理鹽水，C組經(jīng)鼻給予NGF，給藥量每次給予5ul，間隔2min再次給藥，根據(jù)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(jī)取一半標(biāo)本通過免疫組織化學(xué)染色以顯示內(nèi)源性神經(jīng)干細(xì)胞及腦內(nèi)增殖細(xì)胞。進(jìn)行圖像分析并計(jì)數(shù)Nestin和Brdu陽(yáng)性細(xì)胞數(shù)目，分析比較A組、B組、C組之間腦內(nèi)神經(jīng)細(xì)胞增殖及內(nèi)源性神經(jīng)干細(xì)胞增殖和分化的變化情況。 4另一半標(biāo)本腹腔內(nèi)注射戊巴比妥鈉（40mg/Kg）對(duì)小鼠進(jìn)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(cè)Nestin和Brdu蛋白表達(dá)水平。 結(jié)果： 1. H.E.及Masson染色結(jié)果顯示：A組中，海馬齒狀回存在較少BrdU陽(yáng)性細(xì)胞。B組皮質(zhì)均有較多的Nestin和Brdu陽(yáng)性細(xì)胞，同時(shí)雙側(cè)的海馬齒狀回區(qū)也有Nestin陽(yáng)性細(xì)胞，但該區(qū)域Brdu陽(yáng)性細(xì)胞數(shù)量相對(duì)較少；但C1-C5組Nestin和Brdu陽(yáng)性細(xì)胞數(shù)目均有明顯增多(P0．05)；當(dāng)NGF濃度達(dá)到25mg／kg以后，隨著濃度增加，Nestin和Brdu陽(yáng)性細(xì)胞增殖數(shù)目較以前增殖不明顯。 2.Western Blot相關(guān)蛋白基因變化：Western blot檢測(cè)結(jié)果顯示：正常對(duì)照組腦內(nèi)有一定水平的Nestin和Brdu蛋白表達(dá)；B組小鼠相對(duì)于正常對(duì)照組小鼠海馬區(qū)齒狀回Nestin和Brdu蛋白表達(dá)較高。C組中隨著經(jīng)鼻給藥劑量逐漸加大，Nestin和Brdu蛋白表達(dá)也隨之逐漸增高，但C4、C5組蛋白表達(dá)較C3組有所減弱，差異有顯著性意義(P0．01)。 結(jié)論： 1.癥狀期SOD1轉(zhuǎn)基因小鼠腦內(nèi)發(fā)現(xiàn)有內(nèi)源性神經(jīng)干細(xì)胞增殖，說(shuō)明本實(shí)驗(yàn)操作效果明顯。 2.NGF對(duì)SOD1轉(zhuǎn)基因小鼠腦內(nèi)作用與其給藥量密切相關(guān)。低給藥量時(shí)(15ul)，主要表現(xiàn)為對(duì)腦內(nèi)某些神經(jīng)元的保護(hù)作用，對(duì)內(nèi)源性神經(jīng)干細(xì)胞增殖作用較強(qiáng)，當(dāng)給藥量大于25ul以上時(shí)，對(duì)神經(jīng)干細(xì)胞的增殖作用不明顯，當(dāng)給藥量達(dá)到35ul時(shí)，小鼠可產(chǎn)生明顯的不良反應(yīng)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(hào)：1919317