發(fā)布時間：2018-05-10 06:27

  本文選題：蛛網(wǎng)膜下腔出血 + 早期腦損傷　； 參考：《浙江大學》2015年博士論文

【摘要】：背景與目的： 蛛網(wǎng)膜下腔出血(subarachnoid hemorrhage, SAH)僅占所有腦卒中病例的5%,但是具有較高的死亡率和致殘率,因此其診治仍然是每一個神經(jīng)外科醫(yī)師所面臨的最棘手的問題之一。 早期腦損傷(early brain injury, EBI)是指SAH后急性期(0-3天)腦組織發(fā)生的一系列病理生理改變導致的繼發(fā)性腦損傷。越來越多的研究已表明EBI的發(fā)生與嚴重程度和SAH的預(yù)后密切相關(guān)。而神經(jīng)元的凋亡作為EBI中的關(guān)鍵環(huán)節(jié),近年來已成為SAH研究中的熱點。相關(guān)實驗性研究已證明在SAH動物模型中抗神經(jīng)元凋亡可有效地改善神經(jīng)功能缺失。 原肌球蛋白相關(guān)激酶B(Tropomyosin-related kinase B, TrkB)是體內(nèi)多種神經(jīng)營養(yǎng)因子的高親和性受體。近年來大量研究表明,TrkB相關(guān)信號通路與多種中樞神經(jīng)系統(tǒng)疾病的病理生理機制密切相關(guān)。在缺血性腦卒中動物模型中,激活TrkB可有效地減輕早期腦損傷并改善預(yù)后。然而,TrkB在SAH中的作用卻從未進行過深入的研究。 N-乙酰血清素(N-acetyl serotonin, NAS)是體內(nèi)由血清素轉(zhuǎn)化為褪黑素時的中間產(chǎn)物。N-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-2-oxopiperidine-3-carboxamide (HIOC)作為NAS的一種新型衍生物,相對于NAS來說,能夠更有效地選擇性激活TrkB受體。然而HIOC卻從未用于卒中動物模型的研究。 因此,本研究的目的是為了揭示HIOC在實驗性SAH模型中通過激活TrkB受體,從而減輕SAH后早期腦損傷并改善預(yù)后的潛在神經(jīng)保護作用。 材料與方法： 本實驗共使用156只成年雄性Sprague-Dawley (SD)大鼠。采用血管內(nèi)穿刺法誘導蛛網(wǎng)膜下腔出血模型。SAH造模前24小時經(jīng)側(cè)腦室注射TrkB小干擾RNA(small interfering RNA, siRNA)和對照小干擾RNA (scramble siRNA)。SAH造模后3小時經(jīng)側(cè)腦室注射HIOC和腦源性神經(jīng)營養(yǎng)因子(brain-derived neurotrophic factor, BDNF),并比較兩組間療效。在預(yù)后研究中評估SAH出血量評分和神經(jīng)功能學評分；在機制研究中,通過免疫蛋白印記法(western blot)檢測穿刺側(cè)大腦半球ΓrkB,磷酸化TrkB (p-TrkB),磷酸化細胞外信號調(diào)節(jié)激酶(phosphorylated extracellular signal regulated kinase, p-ERK), B淋巴細胞瘤-2(B-cell lymphoma-2,Bcl-2)和裂解半胱氨酸天冬氨酸蛋白酶3(cleaved caspase-3, CC3)等蛋白水平的表達；通過免疫熒光共染驗證TrkB在神經(jīng)元上表達；采用末端標記法(Terminal deoxynucleotidyl transferase-mediated uridine5'-triphosphate-biotin nick end-labeling, TUNEL)染色觀察神經(jīng)元凋亡水平。 結(jié)果： 1.SAH前24小時側(cè)腦室注射TrkB siRNA下調(diào)了大腦半球TrkB蛋白的表達,減少了SAH后24小時TrkB的激活水平和抗凋亡蛋白Bcl-2的表達,并加重了神經(jīng)功能缺失。 2.SAH后經(jīng)側(cè)腦室注射HIOC可有效地減輕神經(jīng)功能缺失。使用高劑量(30ug)的HIOC治療比低劑量(6ug)更好地改善了預(yù)后評分。3.SAH造模后經(jīng)側(cè)腦室注射HIOC可通過激活TrkB/ERK信號通路,減少神經(jīng)元凋亡。造模前24小時側(cè)腦室注射TrkB siRNA后阻斷了該信號通路的激活從而抵消了HIOC的神經(jīng)保護作用,對照siRNA的使用不會影響HIOC的神經(jīng)保護作用。 4.在SAH后側(cè)腦室注射HIOC和BDNF均可有效地激活TrkB。在SAH后6小時,兩者的效用無顯著差別；而在SAH后24小時,HIOC比BDNF具有更強的抗凋亡作用。 結(jié)論： 在SAH模型后注射HIOC可通過激活TrkB/ERK信號通路減少SAH后的早期腦損傷。HIOC可能成為今后腦卒中治療的一種具有應(yīng)用前景的藥物。
[Abstract]:Background and purpose:
Subarachnoid hemorrhage (subarachnoid hemorrhage, SAH) accounts for only 5% of all cerebral apoplexy cases, but has a high mortality and disability rate, so the diagnosis and treatment of the subarachnoid hemorrhage is still one of the most difficult problems facing every neurosurgeon.
Early brain injury (EBI) refers to secondary brain damage caused by a series of pathophysiological changes in the brain tissue after SAH (0-3 days). More and more studies have shown that the occurrence and severity of EBI are closely related to the prognosis of SAH. The withering of neurons, as a key link in EBI, has become a SAH in recent years. Experimental studies have demonstrated that anti neuronal apoptosis in SAH animal models can effectively improve neurological deficits.
The promyosin related kinase B (Tropomyosin-related kinase B, TrkB) is a high affinity receptor for various neurotrophic factors in the body. In recent years, a large number of studies have shown that TrkB related signaling pathways are closely related to the pathophysiological mechanism of various central nervous system diseases. In the animal model of ischemic stroke, the activation of TrkB can be effectively reduced. Mild early brain injury and improved prognosis. However, the role of TrkB in SAH has not been thoroughly studied.
N- acetyl serotonin (N-acetyl serotonin, NAS) is a new derivative of.N-[2- (5-hydroxy-1H-indol-3-yl) ethyl]-2-oxopiperidine-3-carboxamide (HIOC), a intermediate product of the transformation from serotonin to melatonin, as a new derivative of NAS, which is more effective than NAS for selectively activating TrkB receptors. However, HIOC has never been used for death. The study of the animal model in the middle.
Therefore, the aim of this study is to reveal the potential neuroprotective effect of HIOC in the experimental SAH model by activating the TrkB receptor, thus reducing the early brain damage after SAH and improving the prognosis.
Materials and methods:
156 adult male Sprague-Dawley (SD) rats were used in this experiment. The intravascular puncture method was used to induce the subarachnoid hemorrhage model.SAH to be injected with TrkB small interference RNA (small interfering RNA, siRNA) and control small interference RNA (scramble siRNA) 24 hours before the model of the model of the subarachnoid hemorrhage, and 3 hours after the injection of the lateral ventricle and brain source God. Brain-derived neurotrophic factor (BDNF) and comparison of the efficacy between the two groups were compared. In the prognostic study, the SAH hemorrhage score and the neurologic score were evaluated. In the mechanism study, the immunoglobulin imprinting (Western blot) was used to detect the cerebral hemisphere in the cerebral hemisphere of gamma rkB, phosphorylated TrkB (p-TrkB), and phosphorylated extracellular signal regulation. Kinase (phosphorylated extracellular signal regulated kinase, p-ERK), the expression of B lymphocytoma -2 (B-cell lymphoma-2, Bcl-2) and Lysic cysteine aspartic proteinase 3, expressed on the neuron by immunofluorescence CO staining; Ynucleotidyl transferase-mediated uridine5'-triphosphate-biotin nick end-labeling (TUNEL) staining was used to observe neuronal apoptosis.
Result錛，

本文編號：1868194