Regulation of self-renewal of hematopoietic stem cells by bone marrow stromal cells

骨髓基质细胞对造血干细胞自我更新的调控

• 批准号: 16390274
• 负责人: NAKAJIMA Hideaki
• 金额: $ 9.09万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390274/
• 关键词: Hematopoietic stem cell; Bone marrow stromal cell; Self-renewal; m Kirre; ISF; ShIF; 骨髄間質細胞; ストローマ; immune suppressor factor

In order to analyze m Kirre function in vivo, we set out to disrupt m Kirre in mouse genome. We screened BAC library to obtain genomic fragment containing m Kirre gene and isolated several genomic fragments. Restriction mapping of m Kirre genomic fragment combined with the computer data base search completed the genomic map of m Kirre locus. Targeting vector was designed to replace the first exon and large portion of the 5-promoter region with Neomycin cassette to disrupt m Kirre gene. Completed targeting vector was electroporated into E14 embryonic stem (ES) cells, and approximately 400 Neomycin-resistant clones were selected and processed to identify homologously recombinant clones by Southern hybridization. We obtained one correctly targeted clone, which was confirmed by hybridizing with 5'- and 3-probes, together with Neo-probe. We are currently making chimeric mice by injecting this clone into blastcysts from C57BL/6 mice.As for ISF, we performed cDNA microarray analysis of bone marrow stromal cell lines overexpressing ISF and ShIF. Comparing expression profiles of MS10 cells expressing ISF, ShIF or mock plasmids revealed that TIMP-3 and SFRP-1 were down-regulated by ISF overexpression. By re-expressing TIMP-3 or SFRP-1 genes in MS10/ ISF cells, the enhanced hematopoietic stem cell (HSC) supporting activity of MS10/ ISF cells reduced a level similar to that of parental MS10 cells. These results indicate that TIMP-3 and SFRP-1 play critical roles in enhanced HSC activity conferred by ISF.

为了分析m Kirre在体内的功能，我们着手干扰小鼠基因组中的m Kirre。对BAC文库进行筛选，获得含有m-Kirre基因的基因组片段，并分离出多个基因组片段。M-Kirre基因片段限制性内切酶图谱与计算机数据库检索相结合，完成了m-Kirre基因座的基因组图谱。设计靶向载体，用新霉素盒取代第一外显子和5-启动子的大部分区域，干扰m-KirRE基因。将已完成的靶向载体电击入E14胚胎干细胞，筛选出约400个新霉素抗性克隆，经Southern杂交鉴定同源重组克隆。我们获得了一个正确的靶向克隆，通过与5‘-和3-探针以及Neo-探针的杂交得到了证实。我们目前正在通过将该克隆注射到C57BL/6小鼠的囊胚中来制造嵌合小鼠。至于ISF，我们对高表达ISF和ShIF的骨髓基质细胞系进行了基因芯片分析。比较表达ISF、SHIF和模拟质粒的MS10细胞的表达谱，发现ISF过表达下调了TIMP-3和SFRP-1。通过在MS10/ISF细胞中重新表达TIMP-3或SFRP-1基因，MS10/ISF细胞增强的造血干细胞(HSC)支持活性降低到与亲代MS10细胞相似的水平。这些结果表明TIMP-3和SFRP-1在ISF增强HSC活性中起关键作用。

项目成果

Integrin αIIbβ3 induces the adhesion and activation of mast cells through interaction with fibrinogen

整合素αIIbβ3通过与纤维蛋白原相互作用诱导肥大细胞粘附和活化

• 发表时间: 2006
• 期刊: J.Immunol. 176
• 作者: Baba T;Mimura J;Nakamura N;Harada N;Yamamoto M;Morohashi K;Fujii-Kuriyama Y.;沖 俊彦;中島 秀明;中島 秀明;福地 由美;川島 敏行;沖俊彦
• 通讯作者: 沖俊彦

Dimerization of MLL fusion proteins and FLT3 activation synergize to induce multiple-lineage leukemogenesis

• DOI: 10.1172/jci200522725
• 发表时间: 2005-04-01
• 期刊: JOURNAL OF CLINICAL INVESTIGATION
• 影响因子: 15.9
• 作者: Ono, R;Nakajima, H;Nosaka, T
• 通讯作者: Nosaka, T

Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells

• DOI: 10.1016/j.bbrc.2005.11.146
• 发表时间: 2006-02-03
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Nakajima, H;Shibata, F;Kitamura, T
• 通讯作者: Kitamura, T