Study On Reconstructing Hematopoietic Function Of LDR-mobilized Peripheral Stem Cells

Low-dose radiation (LDR)-induced hormesis has been extensively studiedfor the last two decades . Exposure of cells in vitro or in vivo to LDR has beenfound to increase cellular metabolic activities, including DNA, RNA, and proteinsynthesis, as well as to enhance DNA repair activity and antioxidant capacity.This stimulating effect was termed "hormesis." The finding that LDR induceslymphocyte and splenocyte proliferation in vitro and in vivo had promotedprevious studies on LDR-induced hormesis and the adaptive response in thehematopoietic system .In 2001,wangguanjun and tanyehui work team in ourlaboratory provided mouse with low-dose radiation by deep X-ray,CFU-GM andBFU-E were cultured in methylcellulose semi-solid culture system , levels ofGM-CSF and IL-3 were assayed by ELISA and mRNA levels ofGM-CSF ,G-CSF , IL-3 by in situ hybridization , narrow line hybridization andNorthern blot. Results ①The in vitro yields of CFU-GM and BFU-E fromradiated mice was higher than that from the control. ②The serum protein level ofGM-CSF increased obviously than that of controls;③mRNA levels of GM-CSFand G-CSF were also increased. These result suggested that there is hormesiseffect on hematopoietic system induced by low dose radiation ,which may berelated to the increasing of cytokines and 75-mGy x-rays induced a maximalstimulation for bone marrow HPC proliferation. Moreover they still continuedtheir work in the related field,and they found that LDR-stimulated bone marrowHPC proliferation results in a peripheral blood mobilization in 2004.This is aimportant discovery,which may be a new method of mobilizationing bone marrowHPC to peripheral blood for PBSCT and partial injection in fault part.Atpresent ,HSC has been already appied to treatment of many diseases asfollowing:①malignant hematopathy such as leukemia,lymphoma,myeloma andsevere aplastic anemia et.al.②malignant tumor of other system such as lungcancer,breast carcinoma and ovarian cancer.③hereditary metabolic disease suchas mucopolysaccharidosis and lipidoses.④autoimmune disease such assystemic lupus erythematosus,rheumatoid arthritis and cirrhosis.⑤vasculopathysuch as pedal damage induced by diabetes,ischemic cardiomyopathy andarteriosclerosis obliterans.So LDR has a wider development prospect. BecausePBCST has many merits compared with bone marrow stem celltransplantation,the use of hematopoietic progenitor cells collected from peripheralblood has replaced the method of collection from bone marrow for autologousHPC transplantation in clinics. The predominant key is how to obtain adequateperipheral blood stem cells in order to reconstruct haemotopoietic function of therecipients. At present ,a variety of mobilization agents, including chemotherapydrugs ,cytokines and glucocorticoid ,are used to obtained adequate peripheralblood stem cells.Any approach has also respectively indication ,advantage anddisadvantage.Therefore,searching a new mobilization method has become a focuswhich people pay close attention to.On account of the foundings thatLDR-stimulated bone marrow HPC prolifetation result in a peripheral bloodmobilization ,we proposed to explore whether these LDR-stimulated andperipherally mobilized HPCs are functionally normal as other mobilizingagents.So we designed the following experiment .LDR-mobilized HPCs werefunctionally evaluated by examination of the repopulation of peripheral bloodcells in lethally irradiated recipient mice after transplantation with LDR-treateddonor peripheral HPCs.Materials and methods:We adopted PBSCT means to certified whetherLDR-induced peripheral stem cells can repopulate the heamatopoieticfunction.Meanwhile,we use spleen colonies count and PCR which detects specificstrap of Sex-determining region of the Y chromosome for observation index.Forfunctional evaluation of LDR-mobilized HPCs,50 female BALB/C mice and 50male BALB/C mice were used.First peripheral mononuclear cells were preparedfrom five groups of the 50 male donor mice.The five groups included:A group(10mice treated evety 12 hours with a subcutaneous injection of G-CSF for 4 days ata total dose of 300μg/kg/d).B group ((10 mice treated evety 12 hours with asubcutaneous injection of G-CSF for 4 days at a total dose of 150μg/kg/d).Cgroup (10 mice given a combination of treatments from 150μg/kg/d and LDR).Dgroup (10 mice irradiated by 75 mGy LDR and sacrificed 3 days later).E group(10 mice without any treatment as donor controls). Each mouse was sacrificed atindicated times for the collection of peripheral blood to prepare mononuclear cellsas HPC donor. Female mice (recipients) were exposed to whole-body lethalradiation with 8 Gy -rays and received transplants of donor peripheralmononuclear cells (1.5x106 cells/recipient) by tail vein injection 4 to 6 hours later.WBC, animal survival, and CFUs-S were examined at indicated times. ForCFUs-S examination, spleens were harvested and fixed in Bouin'ssolution.Macroscopic nodules or colonies formed in spleens of lethally irradiatedmice after intravenous injection of donor cells provided a measure of viablepluripotent HPCs . Spleen colonies formed 11 days or later after irradiation aremeasurements of viable pluripotent hematopoietic stem cells, since the coloniesscored earlier than 11 days derived mainly from stem cells committed toerythropoiesis . Eventually we adopt PCR to explore cell origin of these spleencolonies ,looking on Sex-determining region of the Y chromosome as cytogeneticssign.Result: After transplantation, peripheral white blood cells of recipients wereexamined by collection of whole blood from mouse tail vein at indicated times .Peripheral WBCs of recipients in group A were found to significantly decrease onday 4 and day 7 (to about 12 to 10%of the day-0 level) and continually decreaseduntil day 13 after lethal irradiation . However, although all of the peripheral WBCnumbers decreased to a very low level (10% of day 0) on day 7, they weregradually recovered on day 10 and day 13 after lethal irradiation in other groups.The efficiency of WBC recovery in the LDR/150-G-CSF group is almost same asthat of 300μg /kg/day G-CSF. On day 14 after transplantation, animal survival inthe control group was 20%, while survivals in other groups were 80 to 100% .More importantly, mice in the LDR/150-G-CSF and 300μg /kg/day G-CSF(300-G-CSF) groups both showed 100% survival . Furthermore, the number ofCFUs-S in the survival recipient spleens significantly increased in LDR alone andin LDR/150-G-CSF as compared to control group. The effect of CFUs-S in theLDR/150-G-CSF group was the same as that in the 300μg /kg/day G-CSF group,and LDR group was the same as that in the 150μg /kg/day G-CSF group.Andthe effect of CFUs-S and WBC recovery in both of A and C group is respectivelybetter than that in Band D group.Eventually PCR consequence indicated that all ofthe spleen colonies has specific strap of Sex-determining region of the Ychromosome.Conclusion:LDR-mobilized peripheral stem cells also repopulateheamotopoietic function of mices as the same as G-CSF.