[Screening of differentially expressed genes in the mouse hematopoietic stromal cells after long-term culture].

ABSTRACT

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 factors as 80% known growth factors have signal peptides. Our experimental results suggest that (a) based on the results of subtractive efficiency, the SSH could be a reliable method to screen differentially expressed genes; (b) gene expression may be regulated by multiple factors, even conditioning-dependent, in this experiment the genes expressed by bone marrow stromal cells are LTC-cultivation inducible; (c) it is possible to find interesting genes or special gene after relatively large-scale screen.