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.

Role of Abro1 in LPS-induced acute lung injury / 军事医学

Objective To investigate the effect of Abro1 on acute respiratory distress syndrome(ARDS)/acute lung injury(ALI)in mice.Methods Abro1 knock-out(KO)mice and wild type(WT)mice were both randomly divided into two groups for intratracheal instillation of lipopolysaccharide(LPS)or normal saline.At 6 or 24 hours after treatment, the pathological changes in lung tissue were observed by HE staining.At 6 hours after treatment,inflammatory immune cells and cytokines production(IL-6)in the bronchoalveolar lavage fluid were examined.Myeloperoxidase(MPO)and the mRNA level of IL-6 in the lung tissue were compared.Results At 24 hours after treatment, compared with WT mice treated with LPS,Abro1 KO mice showed a significantly lower lung injury score.At 6 hours after treatment,Abro1 depletion resulted in reduced levels of inflammatory immune cell infiltration and cytokines production(IL-6)in the bronchoalveolar lavage fluid(P<0.05).In addition,the MPO content and the mRNA level of IL-6 in the lung tissue were much lower than those in WT mice treated with LPS for 6 hours(P<0.05).Conclusion Abro1 deficiency can attenuate LPS-induced ARDS/ALI.

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>

Optimization of conditions for human erythropoiesis in human-mouse xenotransplant model / 中国实验血液学杂志

This study was purposed to investigate the conditions for improving human-mouse xenograft and the erythroid differentiation of human hematopoietic stem cells (HSC) in the xenotransplant model. The engraftments of different mouse strains (NOD/SCID or NOD/SCID/IL2rγ(null)), schemes of irradiation (single-time or 2-times radiation; Co(60)γ-ray or X-ray) and strategies of CB CD34(+) cells ex vivo culture time and lentivirus infection were compared. The results showed that at 4 weeks after transplantation, the ratio of hCD45 positive cells in bone marrow of NOD/SCID/IL2rγ(null) mice increased to (51.4 ± 13.9)%, and erythroid precursor could be detected. All of the mice receiving X-ray irradiation for 2 times (a dose of 1 Gy, then the second of 1.5 Gy, with an interval of 15 min) survived. Fresh isolated CB CD34(+) cells were cultured and infected with lentivirus for 72 h and then transplanted into receptor mouse. After 4 weeks, higher engraftment [hCD45 (51.4 ± 13.9)%] and better erythroid development [hCD71(+) GPA(+) (5.98 ± 3.46)%] were observed. It is concluded that NOD/SCID/IL2rγ(null) mice receiving X-ray irradiation for 2 times and were injected with fresh isolated CB CD34(+) cells cultured and infected with lentivirus ex vivo within 72 h show a better xenograft and erythroid development.

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.