Cloning Of Three Candidate Murine Hematopoietic Genes And Screeningin Their Roles In Regulation Of Bone Marrow Regeneration

Hematopoietic stem cells（HSC） transplantation has demonstrated to be valuble therapy in the clinical treatment of hemotopietic diseases, chemotherapy induced bone marrow （BM） toxicity, and some genetic disease therapies. Due to shortage of donors, its clinical usage has been restricted. Identification of genetic controls of BM regeneration will aid ex vivo expansion of HSC and accelerate recovery of damaged BM .Using gene microarray techonology we have analyzed the gene expression profiles during 5-fluorouracil （5-Fu） induced BM regeneration. Several important known hematopoietic regulatory genes as well as 12 functional unkown genes encoding secreted polypeptides were identified. The expression patterns of three genes, AK008108, BC052844 and NM₀29537, showed remarkable feature of first increasing and then declining during BM regeneration, which suggest their roles in regulation of BM regeneration. To screen their roles in regulation of BM regeneration, all three genes were individually overexpressed in normal as well as 5-flurouracil （5-Fu） induced BM regeneration mouse model. Their roles in regulation of BM regeneration were analyzed by examining the peripheral blood cell counts, BM cellularity, and BM histology.Three genes were cloned by RT-PCR from mouse BM total RNA, and constructed into eukaryotic expessive vector pcDNA3.1 to create three expression plasmids of pcDNA3.1-AK008108, pcDNA3.1-BC052844 and pcDNA3.1-NM₀29537. Each plasmid was electroporated into mouse tibia muscle to overexpress the secreted protein in vivo. Peripheral white blood cells and BM cells were counted on specific day. Mouse femurs were harvested and embedded in paraffin and sectioned. Selected tissue were processed for hematoxylin and eosin （H&E） staining for histological observation. In the 5-Fu induced BM regeneration model, 7 days after plasmid electroporation mice were injected with a singal intravenous dose of 5-Fu to examine their roles in regulation of BM regeneration.The results and conclusion of our work are as follows: In the normal mice received plasmid injections, the numbers of BM cells and peripheral blood cells of the AK008108 and BC052844 group were significantly increased compared to the control pcDNA3.1 group; BM hematopoiesis was activated in the AK008108 and BC052844 group with increased number and size of BM sinusoids. In the 5-Fu induced BM regeneration mouse model, the mice injected with 5-Fu after AK008108 and BC052844 electroporation showed significant slower recovery of peripheral blood cells than those electroporated with pcDNA3.1 empty plasmid. The NM₀29537 electroporated mice did not show significant difference from the control. The results suggest that AK008108 and BC052844 positively regulate the regeneration of bone marrow, and provide new strategies treating chemotherapy induced BM suppression and ex vivo expansion of hematopoietic stem/progenitor cells.