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ABSTRACT: The purpose of this study was to establish the phospho-specific flow cytometry (phospho-flow) to detect the phosphorylated signaling proteins of leukemia cells and to evaluate its useful value in leukemia study. The bone marrow of leukemia children was collected, and treated by phospho-flow of extracted mononulear cells (MNC) and phospho-flow of directly fixed bone marrow (BM) respectively. In phospho-flow of extracted MNC, the MNC extracted from BM were fixed and permeabilized, then were cultured with P-AKT and P-ERK1/2, finally were analyzed by flow cytometry. In phospho-flow of directly fixed BM, the BM was treated with fixation/lysis buffer and 90% methanol, then were incubated with the surface CD antibody, P-AKT and P-ERK1/2, finally the treated BM cells were analyzed by flow cytometry. The results showed that the positive rates of P-AKT and P-ERK1/2 in MNC treated by phospho-flow of extracted MNC of 26 leukemia children were 46.2% and 30.8% respectively, while the positive rates of P-AKT and P-ERK1/2 in BM treated by phospho-flow of directly fixed BM were 50.0% and 38.5% respectively. The comparison of positive rates of P-AKT and P-ERK1/2 between the 2 treatment protocol showed no difference (p > 0.05). It is concluded that the phospho-flow of directly fixed BM established by our laboratory can be used to analyze the signaling proteins of leukemia cells.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 10/2011; 19(5):1176-9.
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ABSTRACT: To study the clinical significance of Toll-like receptor 4 (TLR4) in children with severe sepsis.
A prospective control study was performed. All cases were enrolled from pediatric department of the First Affiliated Hospital of Guangzhou Medical College, and they were divided into severe sepsis group (14 patients) who were diagnosed to have severe sepsis or septic shock in intensive care unit (ICU), pneumonia group (10 cases) with diagnosis of bronchial pneumonia, and healthy control group (10 healthy children). Venous blood samples of 2 ml were collected at admission, the level of TLR4 was detected by flow cytometry .At the same time, the changes in serum interleukin (IL-6, IL-10 ) and tumor necrosis factor-α (TNF-α) levels were determined by enzyme linked immunoadsorbent assay (ELISA).
In severe sepsis group, the contents of TLR4 [(71.56±15.32)%], IL-6 [(1.98±1.55) ng/L], IL-10 [(88.20±61.23) ng/L] and TNF-α [(104.08±85.36) ng/L] were significantly higher than those in pneumonia group [(50.07±26.36)%, (0.93±0.16) ng/L, (41.42±7.02) ng/L, (48.96±6.40) ng/L] and healthy control group [(39.43±17.43)%, (0.94±0.43) ng/L,(43.73±22.68) ng/L, ( 49.94± 18.47) ng/L, all P<0.05). But there was no significant difference in the contents of TLR4, IL-6, IL-10 and TNF-α between pneumonia group and healthy control group (all P>0.05).
This study suggests that TLR4 might be critically involved in the development of sepsis, and changes in TLR4 expression are parallel with levels of proinflammatory cytokines, including IL-6, TNF-α, and IL-10. The combination of TLR4 and proinflammatory cytokines would serve as the predictive parameters in early diagnosis and severity evaluation of sepsis in children.
Zhongguo wei zhong bing ji jiu yi xue = Chinese critical care medicine = Zhongguo weizhongbing jijiuyixue 08/2011; 23(8):475-7.
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ABSTRACT: This study was purposed to investigate the changes of CD4(+) CD25(+) regulatory T cells and NK cells in peripheral blood of acute leukemia children at different stages, the function of immune system and the possible roles of the CD4(+) CD25(+) regulatory T cells as well as NK cells in leukemia immunity. The number and proportion of CD4(+) CD25(+) regulatory T cells and NK cells were detected by flow cytometry in the peripheral blood of 53 acute leukemia children, including 25 patients in new diagnosis and 28 patients in continuous complete remission (CCR), and were compared with that of 20 normal children. The results indicated that the mean proportion of CD4(+) CD25(+) CD127(+) in CD4(+) T cells of peripheral blood in newly diagnosed patients, patients with CCR and normal children were (9.55 +/- 2.41)%, (8.54 +/- 2.51)% and (6.25 +/- 0.85)% respectively, the mean proportions of CD4(+)CD25(+)CD127(+) in newly diagnosed patients and patients with CCR were higher than that in normal children, the mean proportion of CD4(+)CD25(+)CD127(+) in newly diagnosed patients were higher than that in patients with CCR (p < 0.05). At the same time, the NK cell count in patients with acute leukaemia decreased as compared with normal control, while after achieving CCR, the NK cell count in patients were also less than that in normal control (4.11 +/- 3.87% and 10.41 +/- 7.20% vs 14.06 +/- 5.95%, p < 0.05). It is concluded that the application of CD4(+), CD25(+) and CD127(+) to detect regulatory T cells is a simple, reproductive and accurate method, and the CD4(+) CD25(+) CD127(+) T cells can better reflect the proportion of CD4(+)CD25(+) regulatory T cells. The increase of regulatory T cells and decrease of NK cells in pediatric patients with acute leukemia indicate that the function of NK cells may be depressed. Treg T cells play a role in occurrence and development of leukemia, and are involved in down-regulating NK cell function.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 06/2010; 18(3):709-13.
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ABSTRACT: To evaluate the role of glycogen synthase kinase-3beta (GSK-3beta) in the induced differentiation of human glioblastoma cells.
Cell proliferation was determined by bromodeoxyuridine (BrdU) incorporation assay. The protein level of p-GSK-3beta, GSK-3beta, glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) were determined using Western blots. The overexpression of mutant GSK-3beta was analyzed by immunocytochemistry.
The biotoxin cholera toxin is capable of inducing differentiation of U87-MG human glioblastoma cells, which is characterized by morphological changes to astrocytic phenotype, increase in differentiation marker protein GFAP and decrease in proliferation. GSK-3beta activation is induced during this differentiation. Small interfering RNA against GSK-3beta suppresses the induced-differentiation in U87-MG cells. Conversely, overexpression of a constitutively active form of human GSK-3beta (pcDNA3-GSK-3beta-S9A) mutant leads to differentiation of U87-MG cells.
Our findings suggest that GSK-3beta plays an important role in astrocytic differentiation of human glioblastoma cells and may be a novel therapeutic target in the malignant tumor.
Acta Pharmacologica Sinica 02/2010; 31(3):355-60. · 1.95 Impact Factor
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ABSTRACT: This study was aimed to investigate the conditions of culturing in vitro mesenchymal stem cells (MSCs) derived from bone marrow of children with acute leukemia and the biological characteristics of MSCs from leukemia children. The bone marrow MSCs of acute leukemia children were isolated by density gradient centrifugation combined with adherent segregating method and cultured in DMEM/F12. The morphology of Wright stained MSCs was observed under inverted microscope. Cell surface markers were analyzed with flow cytometry. The growth characteristic features of cultured MSCs was measured with MTT method. Induced adipogenic and osteogenic differentiation of MSCs in appropriate induction media was observed. The results indicated that BM-MSCs of acute leukemia children could be successfully cultured in vitro in appropriate conditions. At 24 hours of culture the MSCs began to adhere to wall, grew in colony and appeared in different shapes. As the culture lasted, the MSCs proliferated continuously and shaped in fusiform. After 2 - 3 weeks of culture, MSCs covered the bottom of culture flask. The analysis of growth feature showed that MSCs were in latency for 3 days, and then entered into growth period. After 8 days of culture the growth of MSCs showed to be in plateau stage. The shape of MSCs in 1st and 2nd generation showed to be heterogeneous but the 3rd generation to be homogeneous with long-fusiform. Cells were arranged in shape of whirlpool or radiation. The surface marker analysis showed that the MSCs were positive for CD105, CD29, CD13, but negative for CD34, CD45, CD14 and HLA-DR. The MSCs from leukemia children could be induced into adipocytes and osteocytes in appropriate conditions. It is concluded that (1) MSCs derived from children with acute leukemia can be successfully cultured and passaged in vitro; (2) MSCs from leukemia children not received chemotherapy are more successfully cultured in vitro than those received chemotherapy; (3) the common biological characteristics of MSCs from children with acute leukemia are same as the MSCs from healthy person.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 07/2009; 17(3):734-8.
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ABSTRACT: Pregnant adult C57BL/6J mice were randomly assigned to four groups: one sham and three irradiated groups that were exposed to β-irradiation from tritiated water (HTO) by a single intraperitoneal injection on Day 12.5 of gestation. The offspring received cumulative doses of 0.036, 0.071, and 0.213 Gy, respectively. The litters were observed for postnatal growth (body weight, brain weight), the development of four physiologic milestones (pinna detachment, eye opening, testes decent, vaginal opening), the acquisition age of several reflexes (cliff avoidance, air righting) and sensory functions (auditory startle, thermal reflex), movement and coordination functions and activity (pivoting, foot splay, continuous corridor activity), learning and memory performance (shock avoidance, conditioning reflex), and the density of CA1–CA4 hippocampal pyramidal neurons. Modest but significant dose-dependent neuronal death and functional impairment were seen in both 0.071 and 0.213 Gy groups. In conclusion, even prenatal low-dose β-radiation may impair murine central nervous system (CNS) development suggesting the potential importance of minimizing environmental exposure during human pregnancy.
Neurotoxicology and Teratology.
