PO2 AND PH AS POTENT MODULATORS OF HEMATOPOIESIS

骨髓移植的造血细胞来源有 受相容捐献者供应的限制，病毒感染的可能性 感染，痛苦的骨髓采集程序，自体 (病人)因先前的治疗而耗尽的骨髓或患有白血病 受累，脐带内有少量祖细胞 血。造血祖细胞体外扩增系统的建立 可以增加有资格接受自体移植的患者数量 移植，允许使用脐带血造血细胞 重新填充成人，减少所需的骨髓量 移植，并允许采集外周血干和 祖细胞取代了骨髓采集过程。现在时 骨髓(造血细胞)长期培养方法的探讨 烧瓶中的基质层缺乏良好控制的生长环境。这个 拟议的研究将改善LTHC在骨骼中的应用前景 通过确定允许快速进行骨髓移植的条件 脐血造血祖细胞在灌流中的扩增 生物反应器，不需要基质细胞。消除间质 一层将消除对气孔细胞供体的需求，减少培养 时间，并简化培养条件。现有灌注室 适合于在基质层上培养细胞的将被修改为 无基质细胞手术加尼龙网片更好保留 非贴壁细胞。对骨髓环境的分析表明 祖细胞扩增的进一步改善可以通过以下方式获得 控制培养氧(O2)张力、pH和温度(T)， 并通过为LTHC开发更有效的细胞因子组合。 在氧气压力降低的情况下，祖细胞的扩增速度加快。这个 在含2.5-10%O2的饱和培养基中培养的效果将被检验 通过集落形成细胞的检测和原始的长期培养- 启动电池(LTC-IC)。负责增强性能的机制 在降低氧分压的情况下，将通过监测来研究造血 培养液中氧化物种和细胞因子的水平。细胞因子 混合IL-3/IL-6/干细胞因子，已被证明是成功的 扩增原始细胞，将作为基础配方进行试验 包括两者在内的其他细胞因子组合的有效性 阳性(IL-1，G-CSF)和阴性(肿瘤坏死因子-α，转化生长因子-β)调节因子 造血术。这些细胞因子组合将首先在 静态悬浮培养试验。最好的组合就是 在灌流条件下进行检查。虽然33摄氏度通常是 被认为比37摄氏度更适合静态LTHC，这可能是由于 到33摄氏度时营养物质消耗得不那么快，在这些罕见的- 养育文化。我们将比较35摄氏度和37摄氏度的灌流培养 对照33摄氏度培养对 祖细胞扩增。在LTHC中还没有检测到PH值效应 即使细胞外和/或细胞内的pH很重要 多种生物的增殖、功能和分化调节因子 细胞类型。在三种pH值从6.9到7.8的情况下培养 与pH值为7.35的对照培养相比较。

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, the painful bone marrow harvest procedure, autologous (patient) marrow that is depleted from prior therapy or has leukemic involvement, and the small number of progenitor cells in umbilical cord blood. An in vitro system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplantation, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow required for transplantation, and allow collection of peripheral blood stem and progenitor cells to replace the bone marrow harvest process. Present methods for long-term bone marrow (hematopoietic) culture (LTHC) on stromal layers in flasks lack a well-controlled growth environment. The proposed research will improve the prospects for use of LTHC in bone marrow transplantation by identifying conditions that allow for rapid expansion of cord blood hematopoietic progenitors in a perfusion bioreactor without the need for stromal cells. Eliminating the stromal layer will eliminate the need for a stomal cell donor, reduce culture time, and simplify culture conditions. Existing perfusion chambers suitable for culture of cells on a stromal layer will be modified for operation without stromal cells by adding a nylon mesh to better retain nonadherent cells. Analysis of the bone marrow environment suggests that further improvements in progenitor cell expansion may be obtained by manipulating the culture oxygen (O2) tension, pH, and temperature (T), and by developing more effective combinations of cytokines for LTHC. Progenitor cell expansion is enhanced at reduced O2 tensions. The effects of culture in medium saturated with 2.5-10% O2 will be examined via assays for colony-forming cells and the primitive long-term culture- initiating cells (LTC-IC). Mechanisms responsible for enhanced hematopoiesis at reduced O2 tension will be investigated by monitoring levels of oxidizing species and cytokines in the medium. The cytokine mixture IL-3/IL-6/stem cell factor, which has proved successful in expanding primitive cells, will be used as the base formulation to test the effectiveness of other cytokine combinations which will include both positive (IL-1,G-CSF) and negative (TNF-alpha, TGF-Beta) regulators of hematopoiesis. These cytokine combinations will be tested first in a static suspension culture assay. The best combinations will be then examined under perfusion conditions. While 33 degrees C is generally considered better than 37 degrees C for static LTHC, this may be due to less rapid nutrient depletion at 33 degrees C in these infrequently- fed cultures. We will compare perfusion cultures at 35 degrees C and 37 degrees C against the control 33 degrees C culture for effects on progenitor cell expansion. pH effects have not been examined in LTHC even though extracellular and/or intracellular pH are important regulators of proliferation, function and differentiation for a variety of cell types. Cultures at three pH values from 6.9 to 7.8 will be compared against the control culture at pH 7.35.