Effect of Inducible Antioxidants on Hemoglobin Toxicity

超出所提供的空间。出血是许多外伤性脑脊液损伤的并发症，约占中风的20%。在随后的几个小时内，红细胞溶解并将其内容物释放到血管外空间。最丰富的蛋白质释放是血红蛋白（Hb）。越来越多的实验证据表明，细胞外Hb的氧化毒性有助于出血性中枢神经系统损伤的发病机制。此外，由于其病程较长，Hb毒性可能是治疗干预的理想靶点。因此，进一步深入了解细胞机制和预防这种毒性似乎是可取的。培养的神经元对Hb非常脆弱，但星形胶质细胞通过一种需要蛋白质合成的机制来抵抗Hb。初步实验表明，这种差异可以部分解释为两种诱导抗氧化剂的作用：血红素加氧酶(HO)-1和铁蛋白。前者可被Hb迅速诱导，并可能促进星形胶质细胞中富l铁蛋白的合成。相反，Hb降低神经元中富l铁蛋白的表达；作为血红素分解的产物释放的铁可能是有毒的。该项目将解决HO和铁蛋白在细胞培养和体内模型中的作用。HO-1的过表达将通过基因转移在胶质、神经元或混合培养中实现；将建立活性、血红素介导的活性氧形成与细胞死亡之间的关系。然后比较野生型、HO-1敲除小鼠和HO-2敲除小鼠制备的培养物对Hb或血红蛋白的细胞脆弱性。使用特异性识别H-或l -铁蛋白的抗体，铁蛋白亚基含量将在基线和对这些培养中Hb的反应中进行评估。与血红素铁结合并促进血红素铁摄取的HasA的表达也将被确定。重组H或l -铁蛋白将构建富H和富l -铁蛋白异聚物。神经元和胶质通过受体介导的内吞作用摄取这些异聚物，将有助于研究H:L比率对细胞对激素介导损伤的易感性的影响。最后，将野生型、HO-1或HO-2敲除小鼠和HO-1过表达转基因小鼠的壳核注射Hb或coflagenase，以诱导内源性出血。然后在注射后12-96小时的规定时间点对周围神经元丢失、DMA切割和caspase-3激活进行量化。性能<《(年代;(组织、城市、州}v费城托马斯杰弗逊大学胡桃街1020号,PA 19107关键人员 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. Hemorrhage complicates many traumatic injuries to the CMSand about 20% of strokes. Over subsequent hours, erythrocytes lyse and release their contents into the extravascular space. The most abundant protein released is hemoglobin (Hb). A growing body of experimental evidence suggests that the oxidative toxicity of extracellular Hb contributes to the pathogenesisof hemorrhagic CNS injury. Moreover, because of its prolonged time course, Hb toxicity may be an ideal target for therapeutic intervention. Further insight into the cellular mechanisms and prevention of this toxicity therefore seems desirable. Cultured neurons are highly vulnerable to Hb, but astrocytes are resistant via a mechanism that requires protein synthesis. Preliminary experimentssuggest that this discrepancy may be explained in part by the effects of two inducible antioxidants: heme oxygenase(HO)-1 and ferritin. The former is rapidly induced by Hb and may facilitate synthesis of L-rich ferritin in astrocytes. In contrast, Hb decreases the expression of L-rich ferritin in neurons; iron released as a product of heme breakdown may then be toxic. ¿ This project will address the role of HO and ferritin in cell culture and in vivo models. Overexpression of HO-1 will be accompished in glial, neuronal, or mixed cultures via gene transfer; the relationship between activity, heme-mediated reactive oxygen species formation, and cell death will be established. Cellular vulnerability to Hb or hemin will then be compared in cultures prepared from wild-type, HO-1 knockout, and HO-2 knockout mice. Using antibodies that specifically recognize H- or L-ferritin, the subunit content of ferritin will be assessed at baseline and in response to Hb in these cultures. Expression of HasA, which binds to and may facilitate heme iron uptake, will also be determined. H and L-rich ferritin heteropolymers will be constructed from recombinant H or L-ferritin. Neuronal and glial uptake of these heteropolymers via receptor-mediated endocytosis will allow investigation of the effect of the H:L ratio on cellular vulnerability to herne-mediated injury. Finally, the putamen of wild type, HO-1 or HO-2 knockout, and transgenic mice that overexpress HO-1 will be injected with Hb, or with coflagenase to induce an endogenous hemorrhage. Surrounding neuronal loss, DMA cleavage, and caspase-3 activation will then be quantified at defined time points 12-96 hours after injection. PERFORMANCE S<nTE(S; (organization, city, state} v Thomas Jefferson University 1020 Walnut Street Philadelphia, PA 19107 KEY PERSONNEL ========================================Section End===========================================