BAPP AND PRION TRANSFECTED HUMAN MUSCLE CULTURES

在这项建议中，利用培养的人类肌肉感染 在腺病毒载体中引入BetaAPP或PrPC基因，我们将进行以下工作 目标： 目的1.确定BetaAPP或PrPc基因的长期过度表达 在正常人类肌肉培养中，i)动脉瘤和ii)神经支配，使用 腺病毒载体，将诱导IBM表型，包括a) 空泡化和IBM肌肉退变的其他方面b)PHF，c) 嗜血性淀粉样蛋白沉积和d)其他IBM- 特有的蛋白质。 目的2.确定肌肉纤维中的载脂蛋白E是否增加(通过 通过腺病毒载体或通过增加 从培养介质中暴露)将促进IBM的发展 培养的正常人肌肉同时存在β-APP和/或表型 PrPC基因过表达。 目标3A。确定挑战培养的h-IBM肌肉是动脉瘤还是 被额外拷贝的BetaAPP或PrPc基因支配会加速 IBM表型在它们中的协调发展并加剧了其 严重性，与a)培养的未感染的h-IBM肌肉和b)正常相比 培养的人类肌肉同时过表达BetaAPP和PrPc。 (这是必要的，因为培养的未感染的h-IBM肌肉纤维表达 IBM表型的某些方面，包括增加 BetaAPP和PrPc，但这只存在于20%-40%的肌肉纤维中 在任何给定的时间-受严重影响的纤维正在死亡，而其他纤维正在死亡 还没有表现出表型)。 目标3B。确定载脂蛋白E的积累(无论是通过遗传 通过腺病毒载体或通过暴露在培养物中表达 )将促进在培养的h-IBM中形成IBM表型 不含和含有过表达的BetaAPP或PrPc的肌纤维。 目标是3C。用腺病毒感染培养的h-IBM细胞 反义携带BetaAPP或PrPc基因将抑制 IBM表型的表现。 我们研究的广泛目标是更好地了解分子 导致h-和S-ibm增加积累的机制 成人正常情况下不结合外表达的蛋白质 肌肉，以及b)它们在疾病过程中的作用。 我们长期研究煤炭是为了了解分子致病机理 导致了h-和S-ibm，这最终可能导致治疗。

In this proposal, utilizing infection of cultured human muscle with BetaAPP or PrPC genes in adenovirus vector, we will pursue the following aims: Aim 1. Determine whether long-term overexpression of BetaAPP or PrPc genes in normal human muscle cultured i) aneurally and ii) innervated, using adenovirus vector, will induce the IBM-phenotype, including a) vacuolization and the other aspects of IBM muscle degeneration b) PHFs, c) congophilic amyloid deposits and d) accumulation of the other IBM- characteristic proteins. Aim 2. Determine whether increased ApoE in the muscle fiber (either by genetic overexpression through an adenovirus vector or by increased exposure from the culture medium) will facilitate development of the IBM phenotype in cultured normal human muscle concurrent with Beta APP and/or PrPc gene overexpression. Aim 3A. Determine whether challenging cultured h-IBM muscle aneurally or innervated with extra copies of the BetaAPP or PrPc gene will accelerate coordinated development in them of the IBM-phenotype and aggravate its severity, as compared to a) cultured uninfected h-IBM muscle and b) normal cultured human muscle with concurrently overexpressed BetaAPP and PrPc. (This is necessary because cultured uninfected h-IBM muscle fibers express some aspects of the IBM phenotype including increased accumulation of BetaAPP and PrPc, but this is present only in 20-40% of the muscle fibers at any given time -the severely affected fibers are dying while others do not yet express the phenotype). Aim 3B. Determine whether accumulation of ApoE (either by genetic expression thru an adenovirus vector or by exposure from the culture medium) will facilitate development of the IBM phenotype in cultured h-IBM muscle fibers without and with overexpressed BetaAPP or PrPc in them. Aim 3C. Determine whether infection of cultured h-IBM with adenovirus carrying BetaAPP or PrPc cDNA in antisense orientation will inhibit manifestation of the IBM phenotype. The broad objectives of our research are to better understand a) molecular mechanisms leading to increased accumulation in h- and s-IBM of a group of proteins that are normally not expressed extrajunctionally in adult human muscle, and b) their role in the disease process. Our long-term coals are to learn the molecular pathogenic mechanisms leading to h- and s-IBM which could eventually lead to treatment.