BAPP AND PRION TRANSFECTED HUMAN MUSCLE CULTURES

在该提议中，利用感染培养的人肌肉， 为了在腺病毒载体中引入BetaAPP或PrPC基因，我们将进行以下研究： 目的： 目标1.确定BetaAPP或PrPc基因是否长期过表达 在正常人肌肉培养i）尿道和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肌肉变性的其他方面B）PHF，c） 嗜中性淀粉样蛋白沉积和d）其他IBM- 特征蛋白质 目标二。确定肌肉纤维中ApoE的增加是否（通过 通过腺病毒载体的基因过表达或通过增加 暴露于培养基）将促进IBM的发展 在培养的正常人肌肉中与β APP和/或 PrPc基因过表达。 目标3A。确定是否挑战培养的h-IBM肌肉， 受BetaAPP或PrPc基因额外拷贝支配的细胞， 协调发展，在他们的IBM表型，并加剧其 严重程度，与a）培养的未感染h-IBM肌肉和B）正常 同时过表达BetaAPP和PrPc的培养人肌肉。 (This是必要的，因为培养的未感染的h-IBM肌纤维表达 IBM表型的某些方面，包括 BetaAPP和PrPc，但仅存在于20-40%的肌纤维中 在任何给定的时间-严重受影响的纤维正在死亡，而其他人做 尚未表达表型）。 目标3B确定ApoE的蓄积（无论是通过遗传 通过腺病毒载体表达或通过从培养物中暴露 培养基）将促进培养的h-IBM中IBM表型的发育 不含和含有过表达BetaAPP或PrPc的肌纤维。 瞄准3C确定腺病毒是否感染培养的h-IBM 以反义方向携带BetaAPP或PrPc cDNA将抑制 这是IBM Photype的一部分。 我们研究的广泛目标是更好地理解a）分子 机制导致增加积累的h-和s-IBM的一组 在成年人中通常不表达的蛋白质 肌肉，和B）它们在疾病过程中的作用。 我们长期研究煤的分子致病机理 导致了h-和s-IBM，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.