EFFECT OF THERMOSENSITIZERS ON THERMOCHEMOTHERAPY IN BONE MARROW STEM CELLS

热敏剂对骨髓干细胞热化疗的影响

• 批准号: 2755689
• 金额: $ 5万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2755689
• 关键词: acidity /alkalinity; amiloride; antiport; autoradiography; bone marrow; combination therapy; extracellular; gamma radiation; hematopoietic stem cells; intracellular; laboratory mouse; membrane transport proteins; misonidazole; neoplasm /cancer chemotherapy; neoplasm /cancer thermotherapy; quercetin; radiosensitizer; radiotracer; stilbene 2,4 diisocyanate; thermometry; tibia; tissue /cell culture; whole body irradiation effect

Thermosensitizers that alter the pH of the microenvironment of tumor cells (pHe) or alter the ability of tumor cells to regulate intracellular pH (pHi) may improve tumor control. However, regions of chronic hypoxia and acidosis, brought about by substrate limitation, exist not only in tumors but also in normal bone marrow; and bone marrow stem cells are exquisitely sensitive to hyperthermia. Therefore, the effects of sensitizers that modulate pHi on the thermal response of normal bone marrow must be assessed, since bone marrow may be a limiting normal tissue. Bone marrow contains numerous populations that primarily use glycolysis for energy production, and contains cells that are resistant to exposure to 20 mM lactate. These characteristics suggest that cells within marrow may be adapted to low pHe. Accordingly, it is hypothesized that bone marrow may contain stem cells that are adapted to low pHe and that these cells may be responsive to the hyperthermia sensitizers that act by inhibiting H+ exchange. Furthermore, the distribution of stem and progenitor cells in bone marrow will impose a differential response to hyperthermia and to the response of the thermal sensitizer that act by inhibiting proton exchange. The hypothesis will be tested by determining: 1) If elements of murine bone marrow are adapted to low pHe and if this adaptation is location specific; and 2) The extent to which amiloride (a clinically used diuretic which inhibits Na+/H+ antiport), quercetin (a bioflavinoid that inhibits the H+:lactate symport), DIDS ( a stilbene derivative that inactivates the Cl-/HCO3- exchanger) or hyperglycemia combined with quercetin can sensitize various marrow populations (i.e. CFU-S12, CFU-GM, CFU-E and CFU-F) to hyperthermia in situ or in vitro. Intracellular pH will be determined by the fluorescent dye BCECF. Tibial marrow will be heated in situ by waterbath immersion of the legs of anesthetized (pentobarbital) C57bl/6 mice, or heated in vitro by immersion of tissue culture tubes containing suspensions of nucleated marrow cells. CFU-S will be cultured as spleen colonies in lethally irradiated hosts. CFU-GM will be cultured in soft agar with recombinant CSF-GM as the source of colony stimulating factor, CFU-F will be grown in complete medium supplemented with fetal calf serum and CFU-E in 8% methylcellulose with erythropoietin. These studies will demonstrate that cells exist in normal marrow that are adapted to low pHe and that the addition of the thermosensitizers that alter pHi will preferentially sensitize these cells to hyperthermia. An understanding of the marrow micromilieu and stem cell distribution will improve our understanding of the response of this unique normal tissue to cytotoxic injury, and identify the possible mechanisms of injury imposed in a normal tissue by hyperthermia alone or in combination with agents that interfere with cellular proton control.

改变肿瘤微环境pH值的热敏剂 细胞（pHe）或改变肿瘤细胞调节细胞内 pH（pHi）可改善肿瘤控制。 然而，慢性缺氧区域 和酸中毒，由底物限制，不仅存在于 肿瘤，但也在正常骨髓;和骨髓干细胞， 对高温非常敏感 因此， 调节pHi对正常骨热反应的敏化剂 必须评估骨髓，因为骨髓可能是一个限制性的正常 组织. 骨髓中含有大量的细胞， 糖酵解产生能量，并含有细胞， 暴露于20 mM乳酸盐中。 这些特征表明， 骨髓内的细胞可能适应低pH。 因此，假设 骨髓可能含有适应低pH值的干细胞， 这些细胞可能对高温敏化剂有反应， 抑制H+交换。 此外， 骨髓中的祖细胞将对 热疗和热致敏剂的反应， 抑制质子交换。 该假设将通过以下方式进行检验： 确定：1）鼠骨髓的成分是否适应于低pHe 以及这种适应是否是特定于位置的;以及2） 阿米洛利（一种临床上使用的利尿剂，其抑制Na+/H+反向转运）， 槲皮素（一种抑制H+：乳酸盐共转运的生物类黄酮）、DIDS（一种 使Cl-/HCO 3-交换剂失活的芪衍生物）或 高血糖联合槲皮素可增敏多种骨髓 CFU-S 12、CFU-GM、CFU-E和CFU-F）对高温的影响 原位或体外。 细胞内pH将通过荧光测定法测定。 染料BCECF。 通过水浴浸泡原位加热胫骨骨髓 麻醉（戊巴比妥）C57 bl/6小鼠的腿部，或在 通过浸没含有以下悬浮液的组织培养管进行体外培养 有核骨髓细胞 CFU-S将在中培养为脾集落 致命辐射的宿主CFU-GM将在软琼脂中培养， 重组CSF-GM作为集落刺激因子的来源， 在补充有胎牛血清和CFU-E的完全培养基中生长 8%甲基纤维素和促红细胞生成素 这些研究将 表明正常骨髓中存在适应低pH的细胞 并且改变pH的热敏剂的添加将 优先使这些细胞对高温敏感。 的理解 骨髓微环境和干细胞分布的变化将改善我们的 了解这种独特的正常组织对细胞毒性的反应， 伤害，并确定可能的机制，伤害强加在一个 通过单独的热疗或与 干扰细胞质子控制。