脊髓小腦運動失調症小鼠及Purkinje細胞模式之建立

脊髓小腦運動失調症( spinocerebellar ataxia,SCA )為一群異質性的三或五核苷擴增的神經退化性疾病，患者小腦、腦幹、脊幹及周邊神經系統等出現漸進式的退化。大部分的SCA(如一、二、三、六、七、十七型等)源自於致病基因轉譯區內CAG三核苷的擴增，導致突變的蛋白沉積在細胞內，對神經細胞造成傷害。SCA8基因表現於腦部，此RNA上並無開放的解讀架構(open reading frame)。SCA8 RNA的5’端和KLHL1 RNA的5’端互補(自轉譯起始處至第一個intron)，即兩個基因共用一段DNA序列只是方向相反。KLHL1蛋白的結構和腦專一表現的NRBP及KLHL2蛋白非常相似，故其功能可能和肌動蛋白細胞骨架的組成相關。由於兩者皆表現於小腦，顯示SCA8 RNA可作為一antisense-RNA，來調節KLHL1基因的表現。目前已知SCA8/KLHL1在人類及小鼠各組織及細胞株的表現情形，與先前的研究顯示有些許差異，我們也利用In-situ hybridization來確認SCA8/KLHL1在小腦及睪丸表現的位置。此外，由SCA8病人之臨床表徵判斷很可能是小腦神經細胞之退化所造成，所以我們將小腦專一性表現之啟動子攜帶綠色螢光蛋白(GFP)基因之表現質體轉入具多能性之P19 embryo carcinoma cells，在做誘導分化之過程中，可偵測綠螢光蛋白之表現而建立小腦主要的神經細胞Purkinje細胞株，利用這種特定細胞株將可進一步探討SCA8的致病機轉。另外我們也已利用Purkinje專一表現之啟動子帶領nitroreductase基因之DNA片段建立誘導模式的基因轉殖鼠，nitroreductase可藉由prodrug CB1954之處理而產生有毒衍生物以專一性傷害小腦，因此可模擬類似小腦萎縮的病症，利用這些轉殖鼠我們將進一步應用於治療相關疾病的基因及藥物的研究。

關鍵字

脊隨小腦運動失調症； Purkinje Cells ； L7/pcp2 ； nitroreductase

並列摘要

Autosomal dominant spinocerebellar ataxias (SCA) are a clinically and genetically heterogeneous group of neurodegenerative disorders primarily affecting the cerebellum. Several forms (SCA1, SCA2, SCA3, SCA6, SCA7, SCA17) share unstable CAG repeat expansions in the coding region that are translated into polyglutamine tracts. The polyglutamine tract is thought to exert its effect through a toxic gain of function of the corresponding protein. Spinocerebellar ataxia type 8 (SCA8) is reported to be caused by an unstable CTG repeat expansion in the 3’ untranslated region of a novel gene, KLHL1AS, on chromosome 13q21.However, KLHL1AS dose not encode protein, and was proposed to play an anti-sense regulatory role on the sense strand gene, KLHL1. To seek for a proper model for the study of SCA8, we first identified the expression pattern of KLHL1 and KLHL1AS in various tissues of both human and mice. In-situ analyses were also performed to further characterize the cellular level of expression in cerebellum and testes. We also constructed an expression plasmid in which an EGFP reporter gene was driven by a Purkinje-specific promoter. Purkinje cell lineage will be sorted through P19 cells transfected with this construct and differentiated in-vitro. In addition to the cell model, a transgenic mouse model with inducible nitroreductase is also under investigation. With the established cell or animal models we will be able to create an ideal environment for further studying the molecular mechanisms underlying the pathogenesis of SCA, including SCA8.

並列關鍵字

spinocerebellar ataxia ； Purkinje Cells ； L7/pcp2 ； nitroreductase

參考文獻

Baader SL, Schilling K.(1996).Glutamate receptors mediate dynamic regulation of nitric oxide synthase expression in cerebellar granule cells. J Neurosci. 16(4):1440-9.

Cellini E, Nacmias B, Forleo P, Piacentini S, Guarnieri BM, Serio A, Calabro A, Renzi D, Sorbi S. (2001).Genetic and clinical analysis of spinocerebellar ataxia type 8 repeat expansion in Italy. Arch Neurol. 58(11):1856-9

Chen S, Wu K, Zhang D, Sherman M, Knox R, Yang CS.(1999).Molecular characterization of binding of substrates and inhibitors to DT-diaphorase: combined approach involving site-directed mutagenesis, inhibitor-binding analysis, and computer modeling.Mol Pharmacol.56(2):272-8.

Clark AJ, Iwobi M, Cui W, Crompton M, Harold G, Hobbs S, Kamalati T, Knox R, Neil C, Yull F, Gusterson B.(1997).Selective cell ablation in transgenic mice expression E. coli nitroreductase. Gene Ther. 4(2):101-10.

Cormack BP, Valdivia RH, Falkow S.(1996).FACS-optimized mutants of the green fluorescent protein (GFP).Gene. 173(1 Spec No):33-8.

國際替代計量

脊髓小腦運動失調症小鼠及Purkinje細胞模式之建立

未授權

主題瀏覽