EFFECT OF THERMOSENSITIZERS ON THERMOCHEMOTHERAPY IN BONE MARROW STEM CELLS

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的能力 PH(Phi)可提高肿瘤控制率。然而，慢性缺氧的区域 而由底物限制引起的酸中毒不仅存在于 肿瘤也存在于正常的骨髓中；而骨髓干细胞 对体温过高非常敏感。因此，它的影响 调节pHi的增敏剂对正常骨热响应的影响 必须对骨髓进行评估，因为骨髓可能是一种极限正常 组织。骨髓包含大量主要使用 糖酵解产生能量，并含有抵抗的细胞 暴露在20 mM乳酸盐中。这些特征表明，细胞 骨髓内可能适应低Phe。因此，它是假设的。 骨髓中可能含有适应低PHE的干细胞 这些细胞可能对高温敏感剂有反应 通过抑制H+交换来采取行动。此外，树干和树干的分布 骨髓中的祖细胞将对 体温过高和热敏剂的反应 抑制质子交换。这一假设将通过以下方式进行检验 确定：1)小鼠骨髓细胞是否适应低Phe 如果这种适应是特定于地点的；以及2)在多大程度上 阿米洛利(一种临床使用的利尿剂，可抑制Na+/H+反向通道)， Qercetin(一种抑制H+：乳酸结合的生物类黄素)，Dids(a 使Cl-/HCO3-交换器失活的二苯乙烯衍生物)或 高血糖与槲皮素联用可致敏多种骨髓 人群(即CFU-S12、CFU-GM、CFU-E和CFU-F)对高温的敏感性 在体或体外。细胞内的pH值将由荧光来确定 染料BCECF。用水浴原位加热胫骨骨髓 麻醉(戊巴比妥钠)C57BL/6小鼠的腿部，或在 浸泡组织培养管进行体外培养 有核的骨髓细胞。CFU-S将作为脾细胞集落在 受到致命辐射的宿主。CFU-GM将在软琼脂中培养 重组CSF-GM作为集落刺激因子来源的研究 在添加胎牛血清和CFU-E的完全培养基中生长 在含有促红细胞生成素的8%甲基纤维素中。这些研究将 证明正常骨髓中存在适应低Phe的细胞 而添加改变PHI的热敏剂将 优先使这些细胞对高温敏感。一种理解 骨髓微环境和干细胞分布的变化将改善我们的 了解这一独特的正常组织对细胞毒的反应 伤害，并找出可能的伤害机制 单纯热疗或与以下药物联合应用时的正常组织 干扰细胞质子控制。

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.