长期培养后小鼠造血基质细胞差异表达基因的筛选  

Screening of Differentially Expressed Genes in the Mouse Hematopoietic Stromal Cells after Long-Term Culture

作　　者：张咏[1] 崔春萍[1] 鱼咏涛[1] 李玉珍 魏汉东[1] 任爱辉[1] 赵士富[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850

出　　处：《中国实验血液学杂志》2002年第3期177-182,共6页Journal of Experimental Hematology

基　　金：国家"973"资助项目 编号G19990 5 3 90 3

摘　　要：为了进一步研究长期培养体系 (long termculture ,LTC)中造血基质细胞调控造血的分子基础及其作用机制 ,并发现新型造血调控因子 ,以LTC体系培养后小鼠骨髓造血基质细胞 (LTC St)为被扣除杂交组 ,以未经培养的小鼠骨髓细胞 (BMC)为扣除杂交组 ,采用抑制性扣除杂交法 (SSH)进行杂交 ,将杂交产物克隆并测序 ,进行生物信息学分析。结果 :获得了 131个差异表达EST克隆 ,这些克隆代表了 2 6种已知或部分已知的不同基因和 7条无任何线索的全新基因 ,5条具有完整开放阅读框架 ,无功能线索的新基因中 3条有信号肽。结论 :通过本实验得到了LTC体系中特异性表达的一个消减文库 ,获得了几条可能与造血调控相关的新型基因或EST 。Hematopoietic stromal cells, being the essential ingredient of the hematopoietic microenvironment, play very important roles in the control and regulation of self renewal, proliferation and differentiation of hematopoietic stem cells(HSC) via complex interactions of cell cell, cell humoral and cell extracellular matrix. Evidence from in vivo experiment has proved that HSC derived from normal mice could reconstitute hematopoiesis of mice with HSC defects but failed to reconstitute hematopoiesis of those mice with microenvironment defects, showing the importance of hematopoietic microenvironment in the maintenance of hematopoiesis in vivo. A well known long term culture(LTC) system established by Dexter demonstrated in another way that stromal cell layer in the system could support ex vivo hematopoiesis for several months, even more than one year under the optimal conditions. It, however, has not been demonstrated that what is the key elements and in which way the ex vivo hematopoiesis could be maintained for so long time. As the inventions for the large scale screening methodologies the suppression subtractive hybridization(SSH) was chosen for the screening differentially expressed genes expressed by LTC cultured stromal cells but not by the uncultured bone marrow cells(BMC). mRNA extracted from both cultured adherent cells(tester) and BMC (driver) were hybridized according to the protocol provided by CLONTECH. Total of 130 clones differentially expressed by cultured cells were randomly picked up and 106 ESTs were obtained after sequencing. They represent 26 identical or similar genes and 7 novel genes after the bioinformatics analysis. 5 of the novel genes with the entire open reading frame, without functional clues, have been cloned into the mammalian expression vectors and the functions of them in the control of proliferation and differentiation of HSC will be further exploring. The most interesting discovery is that 3 novel genes have signal peptides, implying the potential discovery of novel growth fac

关 键 词：骨髓造血基质细胞 长期培养 差异基因表达 抑制性差异杂交 造血功能 

分 类 号：R331.2[医药卫生—人体生理学]