以Bβ1 基因轉殖鼠建立並分析其海馬迴的組織切片培養

Establishment and Characterization of hippocampal slice culture from Bβ1 transgenic mice

馬智剛(Chih-Kang Ma)

指導教授 ： 謝秀梅

國立臺灣師範大學/理學院/生命科學研究所/碩士(2010年)

摘要

脊髓小腦共濟失調症第12型(SCA12)是由於PPP2R2B基因5’端未轉譯區(UTR)的CAG重覆片段擴增所造成，該基因是一個腦神經專一性表現的去磷酸化酶protein phosphatase 2A (PP2A)中的調控β次單元。該CAG擴增可能造成PPP2R2B基因的過度表現，因而提高了PP2A的活性，這樣的改變可能因而造成神經細胞的失能與死亡，也是目前推論的SCA12的可能致病機理。我們實驗室已經成功的建立Bβ1過度表現的基因轉殖小鼠，也在這些小鼠中觀察到Purkinje 細胞丟失與神經膠細胞些微的增生(gliosis)。在本研究中，我們繼續的在分子層面分析這些老鼠的致病機轉。我們在行為測試發現，Bβ1過表現小鼠，並沒有明顯呈現出焦慮與運動失調的現象，反而表現出較正常組更為好的成績，唯獨在實驗中的排遺程度遠較對照組有顯著提高的結果。此外我們了解PP2A的受質之ㄧ是維繫微管胞器穩定( microtubule- associated)的重要蛋白，也就是Tau蛋白，在這樣的關係上我們認為Bβ1與阿茲海默氏症(Alzheimer’s disease, AD)可能有某種程度的相關性。有鑒於此，為了未來AD與SCA12相關性的研究方向，我們試著建立成鼠的大腦海馬迴(hippocampus)的切片培養(slice culture)。Bβ1過表現基因轉殖小鼠的腦切片培養可以提供我們便捷的平台以了解SCA12在分子層面的作用機理與篩選治療方式。目前能夠確定的是操作的培養技術上可以穩定的讓成鼠的海馬迴離體培養18天後，亦保有60%以上的細胞存活率，雖然沒有觀測到Bβ1過表現小鼠與正常小鼠的切片培養有差異存在，但在實驗技術的開發上的成果，將能提供我們對於Bβ1失調在離體實驗中更多的了解。

關鍵字

脊髓小腦共濟失調症第12型 ； 海馬迴 ； 成鼠腦切片培養

並列摘要

Spinocerebellar ataxia type 12 (SCA12) is associated with CAG repeat expansion of the 5’UTR of PPP2R2B gene isoform 1 (Bβ1), which encodes a brain-specific regulatory subunit of the protein phosphatase 2A (PP2A). SCA12’s pathogenesis was hypothesized that CAG repeat expansion increased PPP2R2B expression, which altered PP2A activity and induced neuronal dysfunction and death. We have established Bβ1 overexpression transgenic mice and found that Purkinje cell loss, gliosis and some emotional alteration were shown in these mice. In this study, we continued characterize these mice. In addition, substrates of PP2A including microtubule-associated protein tau, which has a close connection with Alzheimer’s disease (AD). To have a quick and easy platform for further study the Bβ1 impact on the SCA12 and AD diseases, we tried to establish an adult or young adult slice culture from mouse hippocampus. Slice culture from the Bβ1 transgenic mice enable us to investigate the molecular pathology and screen potential therapeutic compounds for SCA12. Our behavior analysis showed that Bβ1 transgenic mice (line 20) had a better performance in rotarod and lower anxiety in EPM tasks. The only noticeable abnormality of transgenic mice is that they had significant increasing in fecal output during the rotarod analysis. For slice culture, we have successfully established a stable testing platform by using adult mouse hippocampal tissues. We could maintain the slice survival rate over 60% at DIV18. This ex vivo system should enable us to gain more information about the deregulation of Bβ1.

並列關鍵字

SCA12 ； hippocampus ； slice culture

參考文獻

Arai AC, Orwig N (2008) Factors that regulate KiSS1 gene expression in the hippocampus. Brain Res 1243:10-18.

Bahl S, Ahmed I, Mukerji M (2009) Utilizing linkage disequilibrium information from Indian Genome Variation Database for mapping mutations: SCA12 case study. J Genet 88:55-60.

Brera B, Serrano A, de Ceballos ML (2000) beta-amyloid peptides are cytotoxic to astrocytes in culture: a role for oxidative stress. Neurobiol Dis 7:395-405.

Butterfield DA, Drake J, Pocernich C, Castegna A (2001) Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid beta-peptide. Trends Mol Med 7:548-554.

Celerier E, Ahdepil T, Wikander H, Berrendero F, Nyberg F, Maldonado R (2006) Influence of the anabolic-androgenic steroid nandrolone on cannabinoid dependence. Neuropharmacology 50:788-806.