Role Of SPARC In Regulating Hematopoiesis

ObjectiveStem cell niche is the specified microenvironment where stem cell reside, it affects the self-renewal, proliferation and differentiation of the hematopoietic stem cell directly. In order to explore the role of SPARC, an extracellular matrix which is expressed abundantly in bone marrow microenvironment in regulating hematopoiesis, we analyzed hematopoietic tissue and hematopoietic development characteristics of SPARC gene deletion mice and its wide-type, the effort of exogenous SPARC on HSC expansion, migration.Methods1. Breed and propagate the introduced heterozygote mice. Crosses between heterozygotes produced three genotype offsprings:heterozygotes, homozygous and wild-type. The genome DNA extracted from the tails of filial generation mice and PCR method was employed to amplify the SPARC and Neo gene fragments. Then, we analyzed the specificity of PCR primers through digesting PCR products with restriction endonuclease. Additionally, the expression of SPARC protein was detected by Western blotting to further confirmed the reliability of PCR methods for genotype identification of SPARC mutant mice.2. First, compare the trabecular bone number in the tibia,volume and structure of the spleen, peripheral blood cell count in homozygous and wild-type; Then, test the content of hematopoietic stem cells and progenitor cells in homozygous and wild-type via Flow cytometry and LTC-IC, colony culture technology; At last, detect the effects of exogenous SPARC on CD34+ cell expansion and migration.Results1. Electrophoresis results revealed that the molecular weight of PCR product was in accordance with the expected gene fragment. The results of restriction endonuclease enzyme digestion and Western blotting confirmed the reliability of PCR methods for genotype identification of SPARC knock out mice.2. In Sparc-null mice trabecular bone number per unit volume decreased 34.5%, trabecular separation increased 65.6% compared to wild-type mice. The spleens weight and the spleen to body weight ratios were larger in the Sparc-null mice; HE stained of spleen sections in the Sparc-null mice have show the white pulp was expansion in comparison to the widy-type spleens.Sparc-null mice showed significantly lower WBC counts, HGB, HCT in the Peripheral Blood compared to wild-type mice; Although RBC were lower in Sparc-null mice, differences were not statistically significant; The percentage of KSL(c-kit+, sca-1+, lin-) populations and the total number of KSL were also decreased in the Sparc-null mice were decreased. Long-term culture initiating cell colony formation significantly reduced in the Sparc-null mice. SPARC null mice showed a significantly impaired ability to form erythroid burst-forming units (BFU-E). However, no significant differences were found in the formation of granulocyte/monocyte colony-forming units (CFU-GM). Suicide rate of bone marrow cell were increased in Sparc-null mice. CD34+cells were cultured in HSC culture medium supplemented with cytokine combination (SCF,TPO,FL and G-CSF) with or without SPARC for 7 days, with SPARC the proliferation efficiency much higher than without SPARC(82.5±4.20 VS 31.5±3.31); Transwell test show with SPARC the migration.of CD34+ increased 1.34 fold.ConclusionPCR methods can identify the genotype of the mice with SPARC gene mutant precisely. Not only the developmental anomaly of hematopoietic tissue and hematopoietic development exit but also the hematopoietic stem/progenitor cells decreased in Sparc-null mice. Exogenous SPARC can promote the migration and proliferation of CD34+.