Gene Therapy Reduction of Radiotherapy Esophagitis

描述（由申请人提供）：本赠款申请旨在证明， 分次照射引起食管急性损伤的机制是通过 脂质过氧化（LP）导致关键食管干细胞（ESC）丢失，以及 这一过程受到血凝素（HA）表达的调节， 表位标记的人MnSOD转基因在这些细胞。我们已经开发出一种新颖 分化为 细胞角蛋白阳性鳞状或粘蛋白阳性腺细胞在体外和在 vivo.我们已经证明，从绿色荧光蛋白阳性（GFP+） 小鼠以直接细胞剂量-反应方式移植， 注射并重新填充受辐照的鳞状和腺体组织区域 食管和小肠的GFP阴性（GFP-）受体。 我们已经在C3 H/HeNsd和C57 BL/6 J小鼠中证明， 辐射产生食管炎和> 25%的体重减轻作为发病前 30天死亡的标志物，虽然有显著的细胞凋亡， 毒性与脂质过氧化（LP）和巯基耗尽有关， 体内食管。我们已经表明，ESC数量减少照射后， 但如果小鼠接受食道内注射血凝素， 在照射之前，将（HA）-MnSOD-质粒/脂质体（PL）复合物置于细胞中。第一 一个特定的目的是检验过氧化脂质介导细胞死亡的假设， 分割照射食管的体内和体外器官细胞培养 体外第二个具体目标检验了LP导致损失的假设。 照射后的ESC。第三个具体目标检验了以下假设： 骨髓干细胞移植作为胚胎干细胞的另一来源， 改善照射后组织恢复。在每个目标中，我们将测试 总的假设是， 通过施用表位标记的HA-MnSOD-PL， 放射性食管炎和晚期狭窄。方法包括：分级 照射，通过彗星试验测量ROS、LP和DNA链断裂， Apotag检测细胞凋亡，免疫组化定位p53增加， PL对ESCs和HA-MnSOD的原位及移植食管组织的影响 培养;用于分离ESC的FACS分选和连续平板接种技术，以及 共培养纯化的、未经辐照的绿色荧光蛋白阳性（GFP+） ESCs与经照射的GFP阴性（GFP-）ESCs。这些研究应确定 参与放射性食管炎的关键细胞，定义了 辐射杀伤和抗氧化转基因保护潜力 疗法

DESCRIPTION (provided by applicant): This grant application seeks to prove that the mechanism of acute esophageal injury in fractionated irradiation is through loss of critical esophageal stem cells (ESCs) by lipid peroxidation (LP), and that this process is modulated by expression of a hemagglutinin (HA) epitope-tagged human MnSOD transgene in these cells. We have developed a novel assay for murine Sca1-lin-Thy1+ ESCs which differentiate to cytokeratin-positive squamous or mucin-positive glandular cells in vitro and in vivo. We have shown that ESCs from green fluorescent protein-positive (GFP+) mice engraft in a direct cell dose-response fashion following intravenous injection and repopulate squamous and glandular tissue areas in irradiated esophagus and small intestine, respectively, of GFP-negative (GFP-) recipients. We have demonstrated in C3H/HeNsd and C57BL/6J mice that single or fractionated irradiation produces esophagitis and >25 percent weight loss as a premorbid marker of death by 30 days, and that while there is significant apoptosis, the toxicity is associated with lipid peroxidation (LP) and thiol depletion in esophagus in vivo. We have shown that ESC numbers decrease after irradiation, but are preserved if mice receive intraesophageal injection of hemagglutinin (HA)-MnSOD-plasmid/liposome (PL) complex prior to irradiation. The first specific aim tests the hypothesis that peroxidized lipids mediate cell death in fractionated irradiated esophagus in vivo and in explanted organ cell culture in vitro. The second specific aim tests the hypothesis that LP causes loss of ESCs after irradiation. The third specific aim tests the hypothesis that transplantation of bone marrow stem cells as another source of ESCs will improve tissue recovery after irradiation. In each aim, we will test the overall hypothesis that neutralization of irradiation-induced ROS production and LP in ESCs by administration of an epitope-tagged HA-MnSOD-PL decreases irradiation esophagitis and late stricture. Methods include: fractionated irradiation, measurement of ROS, LP, and DNA strand breaks by Comet assay, Apotag assay for apoptosis, immunohistochemical localization of p53 increase in ESCs and of HA-MnSOD delivered by PL in situ and in esophagus organ explant culture; FACS sorting and serial plating techniques for isolation of ESCs, and coculture of purified, unirradiated green fluorescent protein-positive (GFP+) ESCs with irradiated GFP-negative (GFP-) ESCs. These studies should identify the critical cells involved in irradiation esophagitis, define the mechanism of irradiation killing and potential for protection by antioxidant transgene therapy.