REGULATION OF OUTFLOW FACILITY BY GENE TRANSFER

描述：判断蛋白质贡献的一种有效方法 调节其表达的方法是将其引入细胞内， 细胞核酸，相同（有义）或互补的序列 在一个实施方案中，所述靶蛋白质包含与编码靶蛋白质的mRNA序列互补的（反义的）mRNA序列。 PI假说认为参与细胞骨架的基因表达 流出通道细胞中的动力学可影响房水流出 设施;操纵蛋白质编码的浓度， 这些基因将改变流出能力;复制缺陷 腺病毒载体将成功地将基因传递到流出途径 细胞;并且严格评估其功效和毒性， 递送系统将促进眼基因的潜在发展 青光眼的治疗 由于在体内和体外观察到的流出能力增加， 体外用微管解聚药物治疗后，我们希望 初步研究了B-微管蛋白、微管相关蛋白和 转录因子NF κ B。 PI将表征来自 这些基因家族在小梁网中表达。 她将 使用腺病毒转移技术使其过表达和低表达， 灌注猪和人眼的器官培养物中的特定区域。 她将 测量流出设施。 初步结果表明，复制缺陷型腺病毒 可能是理想的载体来研究我们的假设。 它们进入了 小梁网非常有效， 干扰细胞骨架网络，也不影响流出速率。 即使这些初始操作不改变房水流出， 研究仍应有助于对细胞 与房水流出有关的机制。 此外，完整的 该系统的开发，将分子生物学与生理学相结合， 测量，将允许测试未来的多个假设， 蛋白质/酶（受体、生长因子、离子通道、激酶.）的 已经被概念化为在房水生理学中重要。

DESCRIPTION: One effective means of judging the contribution of a protein to cellular function is to modulate its expression by introducing into the cell nucleic acid, sequences that are identical (sense) or complementary (antisense) to the mRNA sequence that encodes the target protein. The PI hypothesis is that expression of genes involved in cytoskeleton dynamics in cells of the outflow pathway can influence aqueous humor outflow facility; that manipulation of the concentration of the proteins coded by these genes will alter outflow facility; that replication deficient adenovirus vectors will successfully deliver genes to the outflow pathway cells; and that the rigorous evaluation of the efficacy and toxicity of this delivery system will advance the potential development of ocular gene therapy for glaucoma. Because of the increase in outflow facility observed both in vivo and in vitro after treatment with microtubule depolymerization drugs, we wish to initially investigate B-tubulins, microtubule-associated proteins and the transcription factor NFkB. The PI will characterize which isoforms from these gene families are expressed in the trabecular meshwork. She will employ adenovirus transfer technology to overexpress and underexpress their specific regions in organ culture perfused porcine and human eyes. She will measure outflow facility. Preliminary results indicate that replication-deficient adenoviruses are likely ideal vectors to investigate our hypotheses. They enter the cells of the trabecular meshwork very efficiently and, by themselves, do not interfere with the cytoskeleton network nor with the rate of outflow. Even if these initial manipulations do not alter aqueous outflow, this investigation should still contribute valuable insight about cellular mechanisms involved in aqueous humor outflow. Furthermore, the complete development of this system, coupling molecular biology with physiological measurements, will allow testing of future multiple hypotheses involving proteins/enzymes (receptors, growth factors, ion channels, kinases...) that have been conceptualized as being important in aqueous humor physiology.