MEGAKARYOCYTE DIFFERENTIATION

巨核细胞分化

• 批准号: 2228407
• 负责人: MICHAEL A LIEBERMAN
• 金额: $ 22.54万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2228407
• 关键词: DNA footprinting; RNase protection assay; antisense nucleic acid; cell differentiation; gene expression; genetic library; genetic promoter element; genetic regulation; hematopoiesis; megakaryocytes; messenger RNA; model; northern blottings; oligonucleotides; polymerase chain reaction; protooncogene; tissue /cell culture; transcription factor; transfection

The long term objective of this research program is to significantly enhance our understanding of megakaryopoeisis at the molecular level. Three specific aims are proposed. The first is to document the role of the c-ski proto-oncogene in megakaryopoeisis. Preliminary studies in several human hematopoietic tumor cell lines showed that expression of the c-ski proto-oncogene is specifically induced during in vitro megakaryocyte differentiation but not during erythroid or myeloid differentiation. The importance of ski expression in mediating megakaryocyte differentiation will be tested by inhibiting its activity in various hematopoietic cell lines through use of antisense oligonucleotides, antisense RNA, ribozymes, and a dominant-negative mutant of ski. Additionally, overexpression of ski in the same cell lines will be examined to determine if ski expression, by itself, is sufficient to induce the initial stages of megakaryocytic differentiation. The ski protein appears to be a transcription factor that binds specific responsive elements in DNA, and these sequences have been located in the promoter regions of four platelet/megakaryocytic specific genes, as well as in the upstream region of the gene encoding the erythroid/megakaryocytic transcription factor GATAI. Therefore, the role of ski-responsive elements in these cell lines will also be examined, through the use of appropriate reporter constructs. The second specific aim is to identify, in a model cell line, CHRF-288-11, mRNAs whose expression changes during megakaryocyte differentiation. These will be identified using two recently developed PCR-based cDNA cloning techniques; differential display, and a gene expression screen. Once obtained, these cDNAs will be used for the experiments described in the third specific aim, which is to determine if their expression is necessary and/or sufficient for differentiation to occur. These experiments will employ overexpression and inhibition of expression techniques similar to the ones outlined in the first specific aim, in which the role of the c-ski protooncogene was assessed. In this manner genes important in regulating megakaryopoeisis will be identified allowing their mechanism(s) of action to be determined. With this information in hand, it may become feasible to design agents to artificially manipulate the expression of these genes, which has important implications both for treating diseases in which platelet production is impaired, and for engraftment of megakaryocytes after bone marrow transplantation.

这项研究计划的长期目标是， 提高我们对巨核生物在分子水平上的理解。 提出了三个具体目标。第一是记录 巨核细胞增殖中的c-ski原癌基因。初步研究 几种人造血肿瘤细胞系显示， c-ski原癌基因在体外被特异性诱导 巨核细胞分化，但不在红系或髓系分化 分化滑雪表达在调解中的重要性 巨核细胞分化将通过抑制其活性来测试 在各种造血细胞系中通过使用反义 寡核苷酸、反义RNA、核酶和显性负性 滑雪的变种 此外，在同一细胞中， 行将被检查，以确定是否滑雪表达，本身， 足以诱导巨核细胞的初始阶段 分化ski蛋白似乎是一种转录因子 结合DNA中特定的反应元件，这些序列 已经定位在四个基因的启动子区域， 血小板/巨核细胞特异性基因，以及在上游 编码红细胞/巨核细胞转录的基因区域 因素GATAI。因此，滑雪响应元件在这些中的作用 细胞系也将通过使用适当的 报告构建体。第二个具体目标是在模型中识别 细胞系CHRF-288-11，其表达在 巨核细胞分化这些将使用两个 最近开发的基于PCR的cDNA克隆技术;差异 显示器和基因表达屏幕。一旦获得，这些cDNA将 用于第三个具体目标中描述的实验， 是确定它们的表达是否是必要的和/或足够的， 分化发生。这些实验将采用过表达 和抑制表达的技术类似的概述 在第一个具体目标中，c-ski原癌基因的作用 进行了评估。以这种方式，基因在调节 将确定巨核细胞生成， 行动有待确定。掌握了这些信息， 可行的设计代理人人工操纵的表达， 这些基因对于治疗和治疗都具有重要意义 血小板生成受损的疾病，以及用于植入 骨髓移植后的巨核细胞。