Cell Culture

项目总结(见说明)：核心C 细胞培养将为本项目提供细胞培养支持。我们使用的分析方法可以检测到 造血祖细胞处于分化的不同阶段，并能区分正常和 白血病集落形成细胞。这些分析非常适合于解决本计划中出现的一个主要问题 项目，即对本研究中所研究的各种抗白血病药物有反应的细胞的派生 P<0.01。这些检测将用于研究包括粒细胞巨噬细胞在内的外源性细胞因子的作用。 粒细胞集落刺激因子和c-Kit配体对伊马替尼诱导的CML祖细胞的保护作用 (IM；Gleevec)-使用对以下两种疾病敏感和耐药的患者的血液和骨髓细胞诱导凋亡 格列卫。这些研究将与项目4和项目5合作进行。此外，我们还将提供 支持项目5研究Lipocalin、反义24p3和bcr对Ph+细胞的影响 正常和BRC-ABL+早期和成熟祖细胞。我们将与项目3合作，研究是否 破坏对CML祖细胞的微环境专属保护将影响CML的敏感性 蛋白激酶抑制剂对集落形成细胞的作用以及Bcl2抑制剂是否会增强 蛋白激酶抑制剂。 因此，具体目标如下： 1)与Deininger博士(项目4)和Arlinghaus博士(项目5)合作，研究外源性刺激性细胞因子是否保护CML祖细胞免受IM和其他蛋白激酶抑制剂的凋亡影响 2)与Arlinghaus博士合作，研究慢病毒载体介导的抗24p3、抗NGAL抗体和BCR对CML细胞系、新鲜CML细胞和正常骨髓细胞的影响(项目5)。 3)研究阻断造血基质与CML克隆之间的相互作用如何影响正常和bcr-abl+祖细胞对蛋白酪氨酸激酶抑制剂的敏感性。我们将研究阻断SDF-1和CXCR4之间的相互作用的药物的效果，以及酪氨酸激酶和Bcl-2抑制剂的组合，使用造血基质培养、长期培养启动细胞(LTCIC)试验和器官生成试验的组合，与安德里夫博士合作(项目3)。

PROJECT SUMMARY (See instructions): Core C The cell culture will provide cell culture support for this Program Project. The assays we use can detect hematopoietic progenitors at various stages of differentiation and can distinguish between normal and leukemic colony-forming cells. These assays are well suited to address a major issue arising in this Program Project, namely the derivation ofthe cells responding to various anti-leukemic agents investigated in this P01. These assays will be used to investigate the role of extrinsic cytokines including granulocytemacrophage colony stimulating factor (GM-CSF) and c-Kit ligand in protecting CML progenitors from Imatinib (IM; Gleevec)-induces apoptosis using blood and marrow cells of both patients sensitive and resistant to Gleevec. These studies will be conducted in collaboration with Projects 4 and 5. Also, we will provide support to Project 5 in studying the effects of lipocalin and antisense 24p3 and BCR on Ph+ cell lines and on normal and brc-abl+ eariy and mature progenitors. In collaboration with Project 3 we will study whether disruption ofthe microenvironment-dedicated protection of CML progenitors will affect the sensitivity of CML colony-forming cells to protein kinase inhibitors and whether Bcl-2 inhibitors will potentiate the effects of protein kinase inhibitors. Thus, the Specific Aims are the following: 1) To investigate whether extrinsic stimulatory cytokines protect CML progenitors from apoptotic effect of IM and other protein kinase inhibitors in collaboration with Drs. Deininger (Project 4) and Arlinghaus (Project 5) 2) To study the effects of anti-24p3 and anti-NGAL antibodies and BCR (transduced by the lentivirus vector infection system) on CML cell lines and fresh CML and normal marrow cells in collaboration with Dr. Arlinghaus (Project 5). 3) To investigate how interruption ofthe interaction between the hematopoietic stroma and the CML clone would affect the sensitivity of normal and BCR-ABL+ progenitors to protein tyrosine kinase inhibitors. We will study the effect of agents that interrupt the interaction between SDF-1 and CXCR4, and the combination of tyrosine kinases and Bcl-2 inhibitors using a combination of hematopoietic stroma cultures, the long-term culture initiating cell (LTCIC) assay and donogenic assays, in collaboration with Dr. Andreeff (Project 3).