Gene Therapy Reduction of Radiotherapy Esophagitis

描述(申请人提供)：本资助申请旨在证明 分割照射致急性食道损伤的机制是通过 关键的食道干细胞(ESCs)因脂质过氧化(LP)而丧失，以及 这一过程是由血凝素(HA)的表达调节的 在这些细胞中进行表位标记的人MnSOD转基因。我们开发了一部小说 小鼠Sca1-Lin-Thy1+ESCs分化为 细胞角蛋白阳性的鳞状细胞或粘蛋白阳性的腺细胞 活着。我们已经证明了来自绿色荧光蛋白阳性(GFP+)的ESCs 小鼠静脉注射后直接细胞剂量反应方式的移植 注射和重新填充受照射的鳞状组织和腺体组织区域 GFP阴性(GFP-)受者的食道和小肠。 我们已经在C3H/HeNsd和C57BL/6J小鼠身上证明了单一或分离的 放射治疗会导致食管炎和发病前体重减轻25% 死亡的标志是30天，虽然有显著的细胞凋亡，但 毒性与脂质过氧化(LP)和硫醇耗竭有关 体内的食道。我们已经表明，照射后ESC数量减少， 但如果小鼠接受食道内注射血凝素，则会被保存 (HA)-MnSOD-质粒/脂质体(PL)复合体。第一 特定的目的是测试过氧化脂质介导细胞死亡的假设 分割照射的食道在体内和在移植器官细胞培养中的应用 在试管中。第二个特定目的是测试LP会导致 照射后的胚胎干细胞。第三个具体目标是检验这样一个假设 将骨髓干细胞移植作为ESCs的另一来源将 提高照射后组织的恢复能力。在每个目标中，我们将测试 总的假设是中和辐射诱导的ROS产生 通过给予表位标记HA-MnSOD-PL降低ESCs中的LP 放射性食管炎和晚期狭窄。方法包括：分步 照射，用彗星试验测量ROS，LP和DNA链断裂， Apotag法检测细胞凋亡，免疫组织化学P53定位增强 PL原位和食道器官外植体移植ESCs和HA-MnSOD的研究 培养；流式细胞仪分选和连续平板法分离ESCs；以及 纯化的未辐照绿色荧光蛋白阳性细胞(GFP+)共培养 经辐射的GFP阴性(GFP-)ESCs。这些研究应该确定 放射性食管炎的关键细胞，明确了其发病机制 辐射致死与抗氧化剂转基因的防护潜力 心理治疗。

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.