Dynamics of Proliferation and Differentiation after Transplantation of Hematopoietic Cells in Mouse / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of Listeria monocytogenes infection on hematopoietic stem and progenitor cell (HSPC) composition, cell cycle and cell colony-forming ability in mouse bone marrow.@*METHODS@#The C57BL/6J mice were divided into infected group and control group. The mice in injected group were infected intraperitoneally with 6.7×10 CFU Listeria monocytogenes,while the mice in control group were injecfed with PBS of same volume.The serum levels of IFNγ were detected at different time points. After 24 hours, the HS/PC composition, cell cycle and cell colony-forming ability in bone marrow of mice were measured, and the difference between the control group and the infected group was statistically analyzed.@*RESULTS@#Serum IFNγ levels peaked at 24 hours after infection with Listeria monocytogenes. After 24 h, the proportion of LSK, LSK in S phase, and short-term hematopoietic stem cells (ST-HSC) in the infected group were significantly higher than those in the control group (P＜0.001), long-term hematopoietic stem cells (LT-HSC) and the proportion of LT-HSC in S phase were significantly increased (P＜0.01), and the cell colony-forming ability of bone marrow significantly decreased (P＜0.01). [WTHZ]Conclusion: [WTB1]After infection with Listeria monocytogenes, bone marrow hematopoietic stem cells enter the proliferative state from rest, the cell colony-forming ability decreases, suggesting that Listeria monocytogenes infection can cause hematopoietic stem cell depletion.

Construction of BRCC3 gene knockout mice and preliminary analysis of the pheno-type / 军事医学

Objective To construct BRCC3(BRCA1/BRCA2-containing complex subunit 3)gene knockout mice and preliminarily study the phenotypes.Methods Using the Cas9/sgRNA-Mediated genome Editing, the BRCC3 knockout mouse models were constructed.Genomic DNAs of mouse tail tissues were extracted and identified, the genotypes of mice were determined at the DNA level,and RNAs and proteins of tissues, such as the heart, liver, spleen, lung, kidney of mice were extracted and the expression of BRCC3 gene was detected by real-time-PCR and Western blotting(WB).The trend of relative body mass change and indexes that might affect the growth development and metabolism were observed. Major organs were hematoxylin-eosin(HE)stained and observed.The routine blood test of peripheral blood of mice was conducted.Results The mouse model of BRCC3 knockout was successfully constructed.BRCC3 knockout mouse survived and were fertile, indexes of blood lipid and liver function were normal, organs were not degenerative and indexes of peripheral blood in routine blood test were all in the normal range.The relative body mass of BRCC3 knockout mice was higher than that of wild type mice,and the level of serum cholesterol was increased.Conclusion BRCC3 may be involved in relative body mass regulation and cholesterol metabolism in mice.

Construction of Protein Phosphatase 2A Catalytic Subunit β (PPP2Cβ) Overexpression Lentiviral Vector and Its Effect on K562 Erythroid Differentiation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To construct the ovexpression lentivirus vector of PPP2Cβ, the catalytic subunit of protein phosphatase 2A, so as to obtain high-titer packaged lentivirus particles, and to examine the effect of PPP2Cβ on the erythroid differentiation Methods: The CDS of PPP2Cβ was cloned into the second generation of lentivirus vector FUGW, which should be used to co-transfect HEK 293T cells with the lentiviral expression vector and packaging vectors including pMD2G and pSPAX2. Lentiviruses were harvested at 36 and 48 hours after transfection. Titers of viral stock were determined by using flow cytometric analysis. The Western blot was performed to detect the expression level of PPP2Cβ in K562 cells transinfected with the lentiviruses. Benzidine staining and real-time PCR analysis were used to assess the erythroid differentiation of K562 cells.</p><p><b>RESULTS</b>The PPP2Cβ overexpressing lentivirus vectors were constructed, the high-titer lentiviral particles were obtained, and then the PPP2Cβ overexpression K562 cell line was established and promote erythroid differentiation of K562 cells.</p><p><b>CONCLUSION</b>This study suggests that overexpression PPP2Cβ can promote K562 cell erythroid differentiation.</p>

Lentivirus packaging, concentration and infection of CD34~+ cells from umbilical blood / 军事医学科学院院刊

Objective:To methodologically establish the lentivirus granule packaging, concentration and infection against CD34~+ cells from umbilical blood. Methods:The lentivirus system of the 3~(rd) generation was used to produce the virus. Ultrafiltration and ultracentrifugation were employed to concentrate virus. Several treatments were used to improve virus infection including in vitro amplification culture, facilitation of rest cells into cell cycle, promotion of cell adhesion and immobilization during infection, and repeat infection methods. Results:CD34~+ cells were not obviously changed by checking the expression level of CD34 marker on the cell surface after 48 h culture. After two-step concentration, virus titer was increased up to 5.06×10~7/ml, and the infection rate against CD34~+ cells from umbilical blood was increased up to 37.7%.Conclusion:Lentivirus supernatant with over 10~7/ml titer can be obtained using the above methods. Efficient infection against CD34~+ cells from umbilical blood can be achieved.

Mitogenic effects of growth and differentiation factors on rat liver stem cell WB-F344 in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the relationship between the proliferation, differentiation of rat hepatic stem like cell line WB-F344 and cytokines in vitro.</p><p><b>METHODS</b>(3)H thymidine labelling of new synthesized DNA was used to examine the mitogenic responsiveness of WB-F344 cells to cytokines, western blot was used to study the expression of cytokines receptors on hepatic stem cells, and apoptotic cells were detected by Flow cytometry.</p><p><b>RESULTS</b>WB-F344 cells showed a proliferative response to the cytokines of hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), Insulin at the dose of 80 ng/ml, and the relative cpm values are 982.95, 906.32, 863.98 and 968.67 respectively, while non response to interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) at the same dose, and an inhibition or apoptosis response to transforming growth factor-beta (TGF-beta) at 80 ng/ml with a 26.89% apoptotic rate. Western blot showed that there were HGF, EGF, FGF, TGF-beta receptors expressed on WB-F344 cells. When WB-F344 cells were cultured in the differential system (DMEM, 10% Fcs, HGF 10 to approximately 50 ng/ml, EGF 20 ng/ml, Insulin 1 microg/ml, Dex 1 micromol/L), the cells could differentiated into hepatocytes. In addition, HGF could scattered WB-F344 cells.</p><p><b>CONCLUSION</b>The proliferation and differentiation of liver stem cells are regulated by various cytokines which may play an important role when liver is damaged seriously.</p>