以聚氰基丙烯酸正丁酯奈米粒子調控神經滋養因子-3基因釋放和治療出血性中風

Polybutylcyanoacrylate Nanoparticle-Mediated Neurotrophin-3 Gene Delivery for Treating Intracerebral Hemorrhage

鐘秋燕(Chiu-Yen Chung)

指導教授 ： 郭勇志

國立中正大學/工學院/化學工程學系/博士(2013年)

摘要

中文摘要

為探討由聚氰基丙烯酸丁酯(Polybutylcyanoacrylate, PBCA)奈米粒子(nanoparticles, NPs)和神經滋養因子-3與缺血感應元素所組成的非病毒載體調解基因運送系統，對於治療出血性腦中風(hypertensive intracerebral hemorrhage, ICH)老鼠的能力。在本篇論文的第一部分，我們利用聚氰基丙烯酸丁酯奈米粒子調解神經滋養因子-3 (neurotrophin-3, NT-3)基因進入誘導式多能幹細胞(induced pluripotent stem cell, iPSCs)分化成神經細胞的研究，以確認基因構築的功能性。結果顯示，聚氰基丙烯酸丁酯奈米粒子能有效的吸附神經滋養因子-3基因，因此使得粒子粒徑增加且表面帶負電荷。此外，對誘導式多能幹細胞處理聚氰基丙烯酸丁酯奈米粒子/神經滋養因子-3基因複合體，由於誘導式多能幹細胞的分化，導致NT-3, TrkC, NH-H, NSE和PSD95蛋白質的表現增加。結論，氰基丙烯酸丁酯奈米粒子能提高神經滋養因子-3對誘導式多能幹細胞的轉染效率，進而促使誘導式多能幹細胞分化成神經細胞，因此可以作為基因載體。
在本篇論文的第二部分，我們以膠原蛋白酵素誘導老鼠腦出血的方式，模擬人類高血壓腦中風出血。我們設計一個含有神經滋養因子-3基因，缺氧感應元素(hypoxia-responsive element, HRE)，及cytomegalovirus (cmv)啟動區 (promotor)的質體 (plasmid)，經由聚氰基丙烯酸丁酯奈米粒子的覆載進入腦內，可啟動缺氧誘發因子-1 (hypoxia inducible factor-1, HIF-1)/缺氧感應元素系統，在誘發腦出血後產生大量的神經滋養因子-3。研究結果顯示，聚氰基丙烯酸丁酯奈米粒子/神經營養因子-3-缺氧感應元素基因複合體之生物毒性非常低。對缺氧的誘導式多能幹細胞處理聚氰基丙烯酸丁酯奈米粒子/神經滋養因子-3-缺氧感應元素基因複合體，由於誘導式多能幹細胞的分化，致使NT-3, TrkC和MAP-2蛋白質的表現增加。此外，聚氰基丙烯酸丁酯奈米粒子可以使神經營養因子-3-缺氧感應元素基因在體外避免酵素降解，而達到保護基因的目的，在體內又能使神經營養因子-3-缺氧感應元素基因通過腦血流屏障(blood-brain barrier, BBB)，進入腦細胞，並於出血性腦中風後，表現神經營養因子-3蛋白，抑制AIF, cleavaged caspase-3和DNA fragmentation的產生，並增加神經細胞的存活。結論，聚氰基丙烯酸丁酯奈米粒子會影響出血性腦中風老鼠治療的結果，因此適合作為腦部基因治療之載體。

關鍵字

腦血流屏障 ； 缺氧感應元素 ； 缺氧誘發因子-1 ； 神經滋養因子-3 ； 聚氰基丙烯酸丁酯 ； 腦出血 ； 奈米粒子

並列摘要

The aim of this study was to show the capability of a non-viral vector-mediated gene delivery system comprising Polybutylcyanoacrylate (PBCA) nanoparticles (NPs) and cmvNT-3-HRE in treating hypertensive intracerebral hemorrhage (ICH) rats. In the first part of studies, we went to identify the functional of gene construct, PBCA NP-mediated neurotrophin-3 (NT-3) gene delivery for differentiating induced pluripotent stem cell (iPSCs) into neuron. The data indicate that PBCA NPs could efficiently grasp NT-3 gene, thereby increase the particle size and confer a negative surface charge. In addition, the treatments with PBCA NP/NT-3 complexes enhanced the expression of NT-3, TrkC, NH-H, NSE, and PSD95 by differentiating iPSCs. In conclusion, PBCA NPs can serve as an efficient gene delivery vector in enhancing transfection of iPSCs with cmvNT-3 to prompt their differentiation toward neurons.
In the second part of studies, the rats subjected to the collagenase-induced ICH were used to mimic hypertensive intracerebral hemorrhage in human. We construct a NT-3 expression plasmid containing the hypoxia-responsive element (HRE) and a cytomegalovirus (cmv) promotor. PBCA NP-NT-3 plasmid containing HRE is applied to activate the hypoxia inducible factor-1 (HIF-1)/HRE system of gene regulation and used to produce NT-3 proteins after ICH. The data indicate that the PBCA NPs/cmvNT-3-HRE complexes are very low toxicity. Treatments with PBCA NP/cmvNT-3-HRE complexes increased the expression of NT-3, TrkC, and MAP-2 by differentiating iPSCs under hypoxia condition. Furthermore, the PBCA NPs can protect cmvNT-3-HRE from degradation in vitro, sustain across the blood-brain barrier (BBB) in vivo, enhance the NT-3 protein expression, inhibit the AIF, cleavaged caspase-3 and DNA fragmentation expression, and increase neuros survival after hemorrhagic stroke. In conclusion, the PBCA NPs are justified as an appropriate system for gene delivery in the brain because of it possibly influences therapeutic outcome of ICH rats.

並列關鍵字

polybutylcyanoacrylate ； blood-brain barrier ； hypoxia-responsive element ； neurotropnin-3 ； hypoxia inducible factor-1 ； intracerebral hemorrhage ； nanoparticle

參考文獻

1. Schellinger PD, Fiebach JB, Hoffmann K, Becker K, Orakcioglu B, Kollmar R, et al. Stroke MRI in intracerebral hemorrhage: is there a perihemorrhagic penumbra? Stroke 2003;34:1674–9.

2. Wang YX, Yan A, Ma ZH, Wang Z, Zhang B, Ping JI, et al. Nuclear factor-B and apoptosis in patients with intracerebral hemorrhage. J Clin Neurosci 2011;1392–5.

3. Matsushita K, Meng W, Wang X, Asahi M, Asahi K, Moskowitz MA, et al. Evidence for apoptosis after intracerebral hemorrhage in rat striatum. J Cereb Blood Flow Metab 2000;20:396–404.

4. Lu A, Tang Y, Ran R, Ardizzone TL, Wagner KR, Sharp FR. Brain genomics of intracerebral hemorrhage. J Cereb Blood Flow Metab 2006;26:230–52.

5. Wang J, Dor’e S. Inflammation after intracerebral hemorrhage. J Cereb Blood Flow Metab 2007;27:894–908.