低強度間歇性超音波刺激增加神經膠細胞神經滋養 因子表現作用機轉之探討

Enhancement of Low-Intensity Pulsed Ultrasound on the Expression of Neurotrophic Factors in Glia Cells

蔣詩偉(Chiang, Shih-Wei)

指導教授 ： 林文澧

共同指導教授 ： 符文美(Wen-Mei Fu)

國立臺灣大學/工學院/醫學工程學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02579

摘要

低強度間歇性超音波提供非侵入式、非熱傳導的機械訊息傳遞，可以加速骨癒合速度、促進骨骼形成，然而超音波在中樞神經系統的機制目前仍然不很清楚，星狀細胞執行很多功能，包含血腦屏障的形成、神經組織的營養供給，並且在大腦修復和結痂的過程中扮演主要的角色，星狀細胞支持神經細胞是藉由分泌神經滋養因子，像是glial cell line-derived neurotrophic factor（GDNF）、brain-derived neurotrophic factor（BDNF）和vascular endothelial growth factor（VEGF），神經滋養因子可調控神經細胞之生長與存活，並可挽救受損的細胞，本篇論文則探討超音波的刺激對星狀細胞的影響，我們發現星狀細胞在超音波刺激後，會增加 GDNF、BDNF和VEGF 的表現，在機制方面，利用流式細胞儀分析可以觀察到超音波會增加 α2 及 β3 integrins在細胞上的表現，使用蛇毒蛋白 rhodostomin可以拮抗超音波刺激引起的神經滋養因子表現，另外phosphatidylinositol 3-kinase（PI3K）抑制劑LY294002、wortmannin以及 NF-κB 抑制劑 （PDTC）、IκB protease （TPCK）抑制劑也可以抑制超音波刺激引起的神經滋養因子表現，此外，超音波的刺激會促進 Akt、IκB、p65 的磷酸化並且促進 p50 和 p65 進入細胞核內，在動物實驗方面，我們也發現超音波會促進大鼠大腦海馬迴（hippocampus）中 GDNF和BDNF 的表現，總而言之，我們的結果証明超音波刺激會透過integrin/PI3K/Akt 及 NF-κB 這條訊息傳遞路徑而增加神經滋養因子的表現；我們也使用BV-2這株細胞研究超音波在微膠細胞的影響，微膠細胞在中樞神經系統的缺氧狀態下會形成活化態，之前的研究也已證實缺血性中風與神經細胞的死亡和缺氧與微膠細胞的活化有關，本論文則要研究超音波的刺激對活化微膠細胞的影響，我們發現在缺氧狀態下會引發 iNOS和 TNF-α 表現增加，另外我們也發現超音波刺激會抑制缺氧狀態下所引發的 iNOS 和 TNF-α 的表現。總結來說，我們證明超音波刺激會增加神經滋養因子的釋放，並且抑制活化微膠細胞 iNOS 和 cytokines 的產生，因此超音波刺激可能對中樞神經系統疾病是有所助益的。

關鍵字

超音波 ； 星狀細胞 ； 微膠細胞 ； 神經滋養因子 ； 誘導一氧化氮酶

並列摘要

Low-intensity pulsed ultrasound (LIPUS) is regarded as a non-invasive and non-thermal mechanotransduction which accelerates fracture healing, promotes bone formation and tissues regeneration. However, the mechanism and effect generated by ultrasound (US) in central nervous system (CNS) is not clear. Astrocytes exert many functions, including the formation of the blood-brain barrier, the provision of nutrients to the nervous tissue, and play a principal role in the repair and scarring process in the brain. Astrocytes support neuronal cells by secreting various kinds of neurotrophic factors such as the glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Neurotrophic factors play several important roles in the survival and development of neuronal cells. Here, we explored the influence of US stimulation on astrocytes. It was found that US stimulation increased mRNA expression levels of GDNF, BDNF and VEGF in cultured rat brain astrocyte (RBA-1). Flow cytometry analysis demonstrated that US increased cell surface expression of α2 and β3 integrins. Rhodostomin, a snake venom disintegrin, attenuated the expression of neurotrophic factors induced by US. Phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin antagonized the potentiating action of US. NF-κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also exerted similar inhibitory action on the effect of US. Furthermore, US stimulation increased the phosphorylation of Akt at Ser473 . US stimulation also induced phosphorylation of inhibitory subunit
(IκB) of nuclear factor-κB (NF-κB), phosphorylation of p65 and translocation of active p65 and p50 subunit from cytosol into the nucleus. It was also found that US stimulation increased mRNA expression levels of GDNF and BDNF in the hippocampus of Rats. Taken together, our results provide evidence that US stimulation increases GDNF, BDNF and VEGF expression via the integrin/PI3K/Akt and NF-κB signaling pathway. We also examined the effect of US on microglia by using BV-2 cell line. Exposure to hypoxia caused microglia activation and animal studies have shown that neuronal cell death is related to microglia activation following cerebral ischemia. Our preliminary results demonstrate that hypoxia induced iNOS and TNF-α expression in microglia. In addition, hypoxia-induced iNOS and TNF-α expression was attenuated by the pre-exposure to US. In conclusion, US enhanced neurotrophic factor release in astrocytes and inhibited iNOS and TNF-α production in hypoxic microglia. US may be thus helpful to the diseases of CNS.

並列關鍵字

Ultrasound ； Astrocytes ； Microglia ； neurotrophic factor ； iNOS

參考文獻

Adams JC and Watt FM (1993) Regulation of development and differentiation by the extracellular matrix. Development (Cambridge, England) 117(4):1183-1198.

Alexandrov AV, Demchuk AM, Burgin WS, Robinson DJ and Grotta JC (2004) Ultrasound-enhanced thrombolysis for acute ischemic stroke: phase I. Findings of the CLOTBUST trial. J Neuroimaging 14(2):113-117.

Chiu CY, Chen SH and Wang SH (2007) Effect of ultrasound doses on the amyloid-beta 25-35 induced PC12 apoptosis. Conf Proc IEEE Eng Med Biol Soc 2007:5838-5841.

Crisci AR and Ferreira AL (2002) Low-intensity pulsed ultrasound accelerates the regeneration of the sciatic nerve after neurotomy in rats. Ultrasound in medicine & biology 28(10):1335-1341.

Danen EH and Sonnenberg A (2003) Integrins in regulation of tissue development and function. The Journal of pathology 201(4):632-641.