OBJECTIVETo observe the effect of Ly49A transfected mouse spleen cells on graft versus host disease (GVHD) and graft versus leukemia (GVL) effect after haploidentical allogeneic bone marrow transplantation in mice.

METHODSLy49A gene was transfected into spleen cells of C57BL/6 mice by retrovirus and the expression rate of Ly49A receptor was evaluated by flow cytometry. The murine model of haploidentical allogeneic acute GVHD was established by using C57BL/6(H - 2b) mouse as donor, and (BALB/c x C57BL/6) F1(H - 2d/b) (CB(6)F(1)) mouse as the recipient which was injected EL9611 cells before transplantation. After irradiation (TBI, (60)Co 10.5 Gy), the recipient received mixed graft of spleen cells and bone marrow cells to establish a GVHD model. The effects of Ly49A transfected spleen cells on GVHD and GVL post haploidentical allogeneic bone marrow transplantation were detected with this model.

RESULTSThe expression rate of Ly49A receptor was (42.20 +/- 4.87)%, (18.67 +/- 2.48)% and (18.73 +/- 3.82)% for pLXSN-Ly49A, pLXSN transfected and untransfected spleen cells respectively. Among haploidentical allo-BMT (C57BL/6(H - 2b)-->CB6F1(H - 2d/b)) groups, the survival time was (7.80 +/- 3.36) days for irradiation group; (21.70 +/- 2.87) days for cyclophosphomide therapy group; (29.40 +/- 6.43) days for mixed bone marrow cells and spleen cells transplantation group; (29.10 +/- 7.39) days for mixed bone marrow cells and pLXSN transfected spleen cells transplantation group and (45.00 +/- 12.38) days for mixed bone marrow cells and Ly49A transfected spleen cells transplantation group, which was much longer than that of any other groups (P = 0.000).

CONCLUSIONThe Ly49A transfected spleen cell transplantation could alleviate GVHD and retain GVL effect in the acute GVHD model post haploidentical allo-BMT.

Animals ; Antigens, Ly ; genetics ; immunology ; Bone Marrow Transplantation ; Cell Transplantation ; adverse effects ; Female ; Graft vs Host Disease ; etiology ; immunology ; mortality ; Graft vs Leukemia Effect ; immunology ; Lectins, C-Type ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; NK Cell Lectin-Like Receptor Subfamily A ; Receptors, NK Cell Lectin-Like ; Spleen ; cytology ; metabolism ; Survival Rate ; Time Factors ; Transfection

OBJECTIVETo probe into the function of stem cell antigen-1 (Sca-1) in cell proliferation and differentiation.

METHODSSca-(1+) and Sca-(1-) muscle derived cells were separated from C57BU6 mice by fluorescence-activated cell sorting and then cultured in vitro. After 5 days cells proliferative curve were drawn according CCK-8 experimental results. Western blot also were done to detect Sca(-1), MyoD and Myogenin expression in cultured Sca(-1+) and Sca-(1-) muscle derived cells.

RESULTSThe difference of the proliferative curve of Sca-(1+) and Sca-(1-) muscle derived cells cultured 3 days in vitro was not apparent, but Sca-(1-) muscle derived cells had a accelerated division rate in the follow days compared to the Sca-(1+) muscle derived cells. Sca-1 expression in both cells was not obvious. MyoD and Myogenin expression were stronger in Sca-(1+) than Sca-(1-) muscle derived cells.

CONCLUSIONSca-1 expression in muscle derived cells takes a period of time that related to the beginning and ending of the cell cycle.

Animals ; Antigens, Ly ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Membrane Proteins ; Mice ; Muscle, Skeletal ; Myogenin ; Stem Cells

The present study was aimed to elucidate the expression features of lymphoid differentiation antigens and their clinical implications in acute myeloid leukemia (AML). Immunophenotypes were examined by indirect immunofluorescence method with monoclonal antibodies (McAb) in 62 patients with AML. The results showed that 11 cases of AML were found to express lymphoid differentiation antigen Ly(+)AML in addition to myeloid antigen expression. There was no significant difference in clinical manifestation and blood test between the groups of Ly(+)AML and Ly(-)AML when they were newly diagnosed. Only one case gained long-term remission using standard chemotherapy. It is concluded that Ly(+)AML cells seem to be not sensitive to conventional chemotherapy, however, a treatment protocol for both ALL and AML can improve the curative effects. The overexpression of CD34(+) may be responsible for the relatively low level of long term survival rate in Ly(+)AML patients.
Adolescent ; Adult ; Aged ; Antigens, Differentiation ; analysis ; Antigens, Ly ; analysis ; Female ; Fluorescent Antibody Technique, Indirect ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; drug therapy ; immunology ; pathology ; Male ; Middle Aged ; Prognosis

Humans with chronic psychological stress are prone to develop multiple disorders of body function including impairment of immune system. Chronic psychological stress has been reported to have negative effects on body immune system. However, the underlying mechanisms have not been clearly demonstrated. All immune cells are derived from hematopoietic stem cells (HSC) in the bone marrow, including myeloid cells which comprise the innate immunity as a pivotal component. In this study, to explore the effects of chronic psychological stress on HSC and myeloid cells, different repeated restraint sessions were applied, including long-term mild restraint in which mice were individually subjected to a 2 h restraint session twice daily (morning and afternoon/between 9:00 and 17:00) for 4 weeks, and short-term vigorous restraint in which mice were individually subjected to a 16 h restraint session (from 17:00 to 9:00 next day) for 5 days. At the end of restraint, mice were sacrificed and the total cell numbers in the bone marrow and peripheral blood were measured by cell counting. The proportions and absolute numbers of HSC (LinCD117Sca1CD150CD48) and myeloid cells (CD11bLy6C) were detected by fluorescence activated cell sorting (FACS) analysis. Proliferation of HSC was measured by BrdU incorporation assay. The results indicated that the absolute number of HSC was increased upon long-term mild restraint, but was decreased upon short-term vigorous restraint with impaired proliferation. Both long-term mild restraint and short-term vigorous restraint led to the accumulation of CD11bLy6C cells in the bone marrow as well as in the peripheral blood, as indicated by the absolute cell numbers. Taken together, long-term chronic stress led to increased ratio and absolute number of HSC in mice, while short-term stress had opposite effects, which suggests that stress-induced accumulation of CD11bLy6C myeloid cells might not result from increased number of HSC.
Animals ; Antigens, Ly ; metabolism ; Bone Marrow Cells ; cytology ; CD11b Antigen ; metabolism ; Cell Proliferation ; Hematopoietic Stem Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Restraint, Physical ; Stress, Psychological

OBJECTIVE: To investigate the role and mechanism of Ly6C^high monocyte in mice with ventilator-induced lung injury (VILI). METHODS: Forty-eight healthy male SPF C57BL/6 mice were divided into spontaneous breathing group (n = 8), normal tidal volume (VT) group (VT was 8 mL/kg, n = 8), and high VT group (VT was 20 mL/kg, n = 32). The mice in the high VT group were subdivided into 1, 2, 3 and 4 hours subgroups, with 8 mice in each subgroup. All mice underwent direct tracheal intubation, those in the spontaneous breathing group maintained spontaneous breathing, and those in the normal VT group and high VT group were mechanically ventilated with different VT. After ventilation for 4 hours, bronchoalveolar lavage fluid (BALF) was collected to determine total protein, and the levels of inflammatory factors including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were determined by enzyme-linked immune sorbent assay (ELISA). The lung tissues were harvested to determine the wet/dry (W/D) ratio, and lung tissue injury was assessed in terms of lung histopathologic examination after hematoxylin-eosin (HE) staining under the light microscope. The protein expressions of monocyte chemotactic protein-1 (MCP-1) and CC-chemokine receptor 2 (CCR2) in lung tissues were determined by Western Blot. Flow cytometry was used to detect the proportion of Ly6C^high monocyte in lung tissue. RESULTS: The histopathology of lung tissue structures was normal in the spontaneous breathing group and the normal VT group. Inflammatory reaction began to appear at 2 hours of high VT ventilation, and inflammatory reaction was gradually aggravated with the time extension. Compared with the spontaneous breathing group, the total protein, TNF-α, and IL-1β levels in BALF, the lung W/D ratio and MCP-1 expression were increased from 2 hours of high VT ventilation [total protein in BALF (g/L): 1.05±0.13 vs. 0.58±0.11, TNF-α in BALF (ng/L): 116.86±16.14 vs. 38.27±8.00, IL-1β in BALF (ng/L): 178.98±10.41 vs. 117.56±23.40, lung W/D ratio: 5.76±0.27 vs. 4.98±0.39, MCP-1/GAPDH: 0.87±0.19 vs. 0.29±0.12, all P < 0.05], and CCR2 expression and the proportion of Ly6C^high monocyte was significantly increased from 3 hours of high VT ventilation [CCR2/GAPDH: 0.84±0.19 vs. 0.24±0.11, Ly6C^high monocyte proportion: (9.01±2.47)% vs. (1.06±0.35)%, both P < 0.05], and they all showed an increased tendency with the time extension. There was no significant difference in the parameters mentioned above among the spontaneous breathing group, normal VT group and high VT ventilation 1-hour group. CONCLUSIONS Ly6C^high monocytes are involved in VILI, which aggravate VILI by activating the MCP-1/CCR2 axis.
Animals ; Antigens, Ly/metabolism* ; Lung ; Male ; Mice ; Mice, Inbred C57BL ; Monocytes ; Rats ; Rats, Sprague-Dawley ; Tidal Volume ; Tumor Necrosis Factor-alpha ; Ventilator-Induced Lung Injury

BACKGROUND: Both the Trendelenburg position and pneumoperitoneum with carbon dioxide have been reported to increase intracranial pressure (ICP) and to alter cerebral blood flow or cerebral blood volume. Also anesthetic agents have variable effects on cerebral hemodynamics and ICP. The present study was conducted to determine whether regional cerebral oxygen saturation (rSO2) values differ between propofol and sevoflurane anesthesia during laparoscopic surgery in the Trendelenburg position. METHODS: Thirty-two adult women undergoing gynecological laparoscopic surgery were divided into sevoflurane and propofol groups. rSO2 values were recorded at 10 min after induction in the neutral position (Tpre), 10 min after the pneumoperitoneum in the Trendelenburg position (Tpt) and 10 min after desufflation in the neutral position (Tpost). For analysis of rSO2, we did ANOVA and univariate two-way ANCOVA with covariates being mean arterial pressure and end tidal carbon dioxide tension. RESULTS: Between sevoflurane and propofol groups, the change in rSO2 was significantly different even after ANCOVA. rSO2 at Tpt (76.3 +/- 5.9% in sevoflurane vs 69.4 +/- 5.8% in propofol) and Tpost (69.5 +/- 7.1% in sevoflurane vs 63.8 +/- 6.6% in propofol) were significantly higher in the sevoflurane group compared with the propofol group. In the propofol group, rSO2 at Tpost was significantly lower than at Tpre (71.1 +/- 4.8%) and cerebral oxygen desaturation occurred in two patients (14.3%). CONCLUSIONS: Significantly lower rSO2 values were observed in the propofol group during gynecological laparoscopic surgery. The possibility of cerebral oxygen desaturation should not be overlooked during propofol anesthesia even after desufflation of the abdomen in the neutral position.
Abdomen ; Adult ; Anesthesia ; Anesthetics ; Antigens, Ly ; Arterial Pressure ; Blood Volume ; Carbon Dioxide ; Female ; Head-Down Tilt ; Hemodynamics ; Humans ; Hypoxia, Brain ; Intracranial Pressure ; Isoantigens ; Laparoscopy ; Methyl Ethers ; Oxygen ; Pneumoperitoneum ; Propofol ; Prostaglandins, Synthetic ; Spectroscopy, Near-Infrared

OBJECTIVETo investigate the anti-aging effect of ginsenoside R1 in serial transplantation of hematopoietic stem cells and progenitor cells.

METHODHSC/HPC aging model in vivo was established through the Sca-1 (+) HSC/HPC serial transplantation of male donor mice that had been separated and purified by the magnetic-activated cell sorting method. The female recipient mice that had been radiated with lethal dose of 60Co gamma ray were divided into four groups: the control group, the aging group, the Rg1-treated aging group and the Rg1 anti-aging group. The expression of Sry genes in bone marrow cells of recipient mice was analyzed by fluorescence quantitative PCR, in order to determine the source of hematopoietic reconstruction cells, observe the survival time and the recovery of the hematology of peripheral blood, and study the reconstruction of the hematopoietic function of recipient mice, the hematopoietic recovery promoted by Rg1, the culture of CFU-Mix of hemopoietic progenitor cells, the cell cycle analysis and aging-related SA-beta-Gal staining analysis on biological characteristics of Sca-1 (+) HSC/HPC aging, and the effect of Rg1 in vivo regulation on Sca-1 + HSC/HPC aging.

RESULTThe hematopoietic reconstruction cells of female recipient mice were derived from male donor mice. With the serial transplantation, the 30-day survival rate and the hematology in peripheral blood of recipient mice decreased. Sca-1 (+) HSC/HPC showed aging characteristics: the ratio of cells in G0/G1 phase and the positive rate of SA-beta-gal staining increased, whereas the number of CFU-Mix decreased. Compared with the aging group of the same generation, Rg1 -treated aging group and Rg1 anti-aging group showed higher 30-day survival rate and WBC, HCT, PLT and CFU-Mix, and lower cell ratio in Sca-1 (+) HSC/HPC G0/G1 stage and positive rate of SA-beta-gal staining. The Rg1 anti-aging group showed more significant changes than the Rg1 -treated aging group.

CONCLUSIONGinsenoside Rg1 has the effect of delaying and treating Sca-1 (+) HSC/HPC aging during the serial transplantation. Rg1 's anti-aging effect is superior to its effect of treating aging.

Aging ; drug effects ; metabolism ; Animals ; Antigens, Ly ; genetics ; metabolism ; Cell Cycle ; drug effects ; Female ; Ginsenosides ; pharmacology ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; cytology ; drug effects ; metabolism ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL

OBJECTIVETo investigate the effect of SIRT6/NF-κB signal axis in delaying hematopoietic stem/progenitor cell senescence with ginsenoside Rg1, in order to provide theatrical and experimental basis for looking for methods for delaying HSC senescence.

METHODSca-1 + HSC/HPC was isolated by magnetic cell sorting (MACS) and divided into five groups: the normal control group, the aging group, the positive control group, the Rg1 anti-senescence group, and the Rg1-treated group. Senescence-associated β-galactosidase (SA-β-Gal) staining, cell cycle analysis and hemopoietic progenitor cell mix (CFU-Mix) were adopted to determine the effect Rg1 in delaying or treating Sca-1 + HSC/HPC senescence biology. The mRNA and protein of senescence regulation molecules SIRT6 and NF-KB were examined by realtime fluorescence quantitative PCR (FQ-PCR) and western blotting.

RESULTCompared with the senescence group, the Rg1 anti-senescence group and the Rg1-treated group showed lower percentage in SA-β-Gal-stained positive cells, decreased cell proportion in G1 phase, increased number of CFU-Mix, up-regulated in SIRT6 mRNA and protein expression, down-regulation in NF-KB mRNA and protein expression. The Rg1 anti-senescence group showed more evident changes in indexes than the Rg1-treated group.

CONCLUSIONRg, may inhibit Sca-1 + HSC/HPC senescence induced by t-BHP by regulating SIRT6/NF-KB signal path.

Animals ; Antigens, Ly ; analysis ; Cellular Senescence ; drug effects ; Female ; Ginsenosides ; pharmacology ; Hematopoietic Stem Cells ; drug effects ; Male ; Membrane Proteins ; analysis ; Mice ; Mice, Inbred C57BL ; NF-kappa B ; physiology ; Signal Transduction ; physiology ; Sirtuins ; physiology

OBJECTIVETo compare frequencies of natural killer (NK) cell subsets and their surface expression of the NKG2D receptor in patients with primary biliary cirrhosis (PBC), and to determine the correlation between expression of MICA on monocytes and function-associated receptors on the NK cells of PBC patients.

METHODSTwenty patients with PBC and 18 healthy donors were included in the study. Peripheral blood samples anticoagulated with heparin were labeled with the following antibody combinations: anti-CD45/anti-CD14/anti-MICA, antiCD3/anti-CD56/anti-CD16/anti-NKG2D. Frequencies of MICA-positive monocytes, NK cell subsets, and NK cells with surface expression of NKG2D were measured with flow cytometry. Correlation of MICA expression on monocytes with NKG2D expreesion on NK cells was assessed through linear correlation and regression analysis.

RESULTSThe PBC patients had significantly lower percentages of NK cells than the healthy donors (6.8%+/-2.9% vs.16.4%+/-3.4%, P =0.000<0.05). In the PBC patients, the percentage of CD56-positive NK ceils was significantly higher than that of CD16-positive NK cells (4.2%+/-2.8% vs.1.4%+/-0.7%, P=0.003<0.05). The PBC patients also had significantly higher percentage of NKG2D surface expressing CD56-positive NK cells than the healthy donors (79.4%+/-10.2% vs.64.8%+/-10.7%, P=0.000<0.05). The PBC patients and healthy donors showed no statistically significant differences in percentages of NKG2D surface expressing CDl6-positive NK (70.1%+/-12.9% vs.61.1%+/-5.9%, P=0.078>0.05). MICA was seldom detected on normal monocytes (2.6%+/-1.9%), but present for 51.6%+/-16.2% of monoeytes from the PBC patients (P =0.000<0.05). There was a significant difference in frequency of CD14/MICA double-positive monocytes between the healthy donors and PBC patients. No correlation of MICA expression on monocytes with NKG2D expression on NK cells was found.

CONCLUSIONPBC patients have lower levels of NK cells in peripheral blood than their healthy counterparts. PBC patients also have higher levels of the CD56+ NK cell subset and cells with surface expression of the activated NKG2D receptor. It appears that PBC patients have a greater level of CD14+MICA+ peripheral blood mononuclear cells. NK cells may be affected by the PBC-related monocytes and participate in disease pathogenesis through immune regulation.

Flow Cytometry ; Humans ; Killer Cells, Natural ; Liver Cirrhosis, Biliary ; NK Cell Lectin-Like Receptor Subfamily K

OBJECTIVETo investigate the effect of Xuefu Zhuyu Decoction (XFZYD) on the number, phenotype, cell cycle and colony formation of bone marrow hematopoietic stem cells (HSC) in mice.

METHODSKunming mice were randomly divided into 4 groups: the control group, the low- (3.25 g/kg), middle- (6.5 g/kg) and high-dose (13.0 g/kg) XFZYD groups. After they were medicated by gastrogavage respectively with saline or corresponding dose of XFZYD for 7 days, their bone marrow HSC were separated and counted. The phenotype Sca and cell cycle of HSC were detected by flow cytometer, and the colony formation was determined with semisolid methyl media culture.

RESULTSNo obvious difference in the number of mononuclear cell, suspended cell and colony production was found among all the groups (P > 0.05); while the expression of CD34 and Sca-1 increased in the low-dose XFZYD group, but in the middle-dose XFZYD group increase only showed in Sca-1 expression.

CONCLUSIONXFZYD plays a role of removing blood stasis and promoting regeneration through improving hematopoietic function by means of increasing the number and enhancing the function of premature HSC.

Animals ; Antigens, CD34 ; biosynthesis ; Antigens, Ly ; biosynthesis ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Count ; Cells, Cultured ; Colony-Forming Units Assay ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hematopoietic Stem Cells ; cytology ; drug effects ; metabolism ; Male ; Membrane Proteins ; biosynthesis ; Mice ; Random Allocation