A Single-Cell Immunofluorescence Method for the Division Patterns Research of Mouse Bone Marrow-Derived Hematopoietic Stem Cells.

How hematopoietic stem cells (HSCs) maintain the balance of self-renewal and differentiation could be partially ascribed to asymmetric and symmetric division patterns. However, a simple and effective method to detect stem cell division patterns is lacking. In this study, we introduce a strategy to describe stem cells division patterns with high spatial resolution at the single-cell level. We show that the fate determinant, Numb, exhibits low expression levels in HSCs that increase upon the initiation of differentiation. Using this single-cell immunofluorescence technique, we found that HSCs mainly undergo symmetric self-renewal in the presence of only stem cell factor, but with the addition of trombopoietin this division pattern is transformed into a symmetric commitment dominant mode in vitro. In addition, our study indicated that the division pattern cannot be defined by cell size or the nuclear/cytoplasm ratio. These findings collectively demonstrate that this single-cell immunofluorescence technique provides a new biological strategy in stem cell division research, and can be more widely applied given its flexibility, easy operability, and inexpensiveness.

[Role of Vascular Niche of Bone Marrow in the Development of MLL-AF9 Induced Acute Myclaid Leukemia].

OBJECTIVE: To investigate the role of bone marrow vascular niche in the development of MLL-AF9 acute myeloid leukemia (AML). METHODS: Transplantation experiments were performed to establish non-radiated MLL-AF9 AML model; the half-bone immunofluorescence staining and tile-scan imaging of two-photon confocal microscopy were used to obtain the data of 3 main bone marrow niche cells; flow cytometry analysis was performed to characterize leukemia cells in different anatomical sites. RESULTS: In the early stage of MLL-AF9 AML, the proportion of leukemia cells in the metaphysis of the femur was significantly higher than that in the diaphysis. The detection of apoptosis and proliferation rate of leukemia cells showed that the percentage of leukemia cells in metaphysis significantly decreased, and the proliferation (S/G2/M phase) was also significantly more active. These different features of leukemia cells may relate with different bone marrow microenvironment. The image data of 3 major components of bone marrow niche (endothelial cells, endosteum, megakaryocytes) showed different distribution of blood vessels in metaphysis and diaphysis. Furtherly comparing the spatial distance between leukemia cells and endothelial cells, endosteum, megakaryocytes indicated that leukemia cells are closer to the blood vessels, suggesting the important role of blood vessels in the development of leukemia. Glucose uptake assays and intracellular ROS detection showed that the supportive role of blood vessels for leukemia cells did not related with nutrient metabolism pathway. CONCLUSION: The vascular niche plays an important role in the development of leukemia, and does not relate with the transport of nutrients and the elimination of metabolic waste, instead, which may relate with perivascular cytokines or other vascular functions.

[Role of Rheb in Human Acute Myeloid Leukemia].

OBJECTIVE: To investigate the role of Rheb (mTOR activator) in AML development by measuring Rheb expression in bone marrow of adult AML patients and in AML cell line HL-60. METHODS: Real-time PCR assay was used to measure the Rheb mRNA expression in 27 AML patients and 29 ITP patients as control. The relationship between Rheb mRNA expression and age, AML subtype, fusion gene, splenomegaly, hepatomegaly and survival of AML patients was analyzed and compared. In addition, HL-60 cell line over-expressing Rheb was established, and the HL-60 cells and HL-60 cells with overexpression of Rheb were treated with Ara-C of different concentrations, the proliferation level was detected by CCK-8 method, and the IC50 was calculated. RESULTS: The mRNA level of Rheb in AML patients was similar to that in ITP patients (control). Interestingly, higher expression of Rheb was associated with better survival and was sensitive to Ara-C treatment. However, the expression level of Rheb was not associated with age, AML subtype, fusion gene, and hepatomegaly of patients. Lower expression level of Rheb was associated with splenomegaly. In vitro analysis of HL-60 line indicated that overexpression of Rheb could increased the cell sensitivity to Ara-C treatment (IC50=0.54 µmol/L) and caused HL-60 cell apoptosis. CONCLUSION: The lower Rheb expression is a poor prognostic indicator for AML patients, which is associated with AML splenomegaly, the patients and HL-60 cells with low expression of Rheb are insensitive to Ara-C treatment.

[Applications of Microscopic Imaging and Analysis Technology in Studies of Neutrophil Movement and Phagocytosis].

OBJECTIVE: To analyze and evaluate the application of spinning disk confocal microscopy and imaging analysis software in movement and phagocytosis of neutrophils. METHODS: Neutrophils were isolated from bone marrow by centrifugation on discontinuous Percoll gradient, and then were stained with PE Gr-1 antibody and mixed with FITC-labeled Zymosan A bioparticles. Multichannel time-lapse videos were captured by using the spinning disk confocal microscopy. The result was analyzed by using volocity and ImageJ software, the parameters associated with movement and phagocytosis of neutrophils were analyzed, including morphological changes, cell tracking, pseudopod dynamics, binding and phagocytosis index. RESULTS: Most neutrophils would be polarized in response to Zymosan particles during a short time. Binding and phagocytosis process occured in forty minutes. CONCLUSION: A method of precisely quantifying the movement and phagocytosis of neutrophils using microscopic imaging and imaging analysis technique has been set up successfully. Using this method, biological activity and function of neutrophils can be evaluated visually and rapidly. The physiologically rapid response to Zymosan particles can be applied to the neutrophils function research in the future.

[Identification and characterization of porcine bocavirus episomes].

To verify that the circular forms of bocavirus genome exist in their host, bocavirus episomes were detected in fecal samples of healthy piglets using a semi-nested PCR method. Two species of porcine bocaviruses (PBoVG2-episome and PBoVG3-episome) were identified for the first time. The relevant terminal sequences of the noncoding region (405 and 511 nt, respectively) were also obtained. Sequence analyses and secondary structure prediction indicated that the PBoVG2-episome was more similar to that of human bocavirus 3 (HBoV3) but the PBoVG3-episome was quite different from that of other members of the genus Bocavirus. Discovery of episomal forms of porcine bocaviruses (PBoV) suggested that PBoV, like HBoV, used a different replication mechanism from other parvoviruses. The sequencing of episome Inverted Terminal Repeats (ITRs) also contributes to a possible alternative strategy for constructing infectious molecular clones of bocavirus in a future study.

Genome characterization of a novel porcine bocavirus.

Using a high-throughput DNA sequencing method, one DNA sequence (contig01006), suspected to belong to a novel porcine bocavirus (PBoV), was found with a high rate of detection (19.6 %) in fecal samples from healthy piglets. Moreover, a novel PBoV (tentatively named PBoV3C) with a nearly complete genome sequence (5235 bp) was identified. PBoV3C exhibits typical genome characteristics of bocaviruses and shows the highest genomic sequence identity (78 % to 81 %) to PBoV3A/B (PBoV3/4-UK) and PBoV3D/E (PBoV3/4-HK), respectively. Phylogenetic and recombination analysis indicated high diversity, prevalence and complexity among the PBoVs. The phospholipase A2 (PLA2) site of VP1 and the secondary structure of VP2 of PBoV3C were also analyzed. Additionally, we propose a uniform method of PBoV nomenclature based on the VP1 gene.

[A novel method for multiplex detection of gastroenteritis-associated viruses].

To develop and optimize a simultaneous detection method of RotavirusA, Norovirus GI, GII, Sapovirus, human astrovirus, enteric adenoviruses and HBoV2 with GenomeLab GeXP analysis system. The sensitivity was verified to be 10(4) copies/microL with plasmids containing the viral targets in triplicate on different days, and no cross-reaction with enterovirus71, human Parechovirus and PicobirnavirusII was observed. Finally, we successfully developed a high throughout, rapid and maneuverable multiplex RT-PCR assay for simultaneous detection of seven viruses related with viral gastroenteritis, which provide a novel method for the molecular diagnosis of diarrhea-associated virus.