1.Cytotoxicity of trichloroethylene in keratinocytes involving alterations of mitochondrial function and ultrastructure.
Qi-xing ZHU ; Tai MA ; Tong SHEN ; Yun YU ; Liang-ping YE ; Rui DING ; Jun-feng YU
Chinese Journal of Industrial Hygiene and Occupational Diseases 2007;25(5):263-266
OBJECTIVETo explore mechanism of dermal toxicity of trichloroethylene(TCE).
METHODSNormal human keratinocytes (KC) were isolated from foreskins of healthy donors undergoing circumcision by two-step trypsin digestion and cultured in serum-free medium. Cells were treated with medium, 1% acetone (volume fraction) 0.125, 0.500 or 2.000 mmol/L TCE for different time (4, 8, 12 or 24) hours. After treatment, MTT assay and ATPase activity detected, inhibition ratio of mitochondrial enzyme was calculated according to optical density (A) value of MTT assay. Mitochondrial membrane potential (MMP) was detected by flow cytometry FCM after being stained with Rhodamine123 (Rh123). Morphological changes were also observed through transmission electron microscope (TEM).
RESULTSCellular viability and ATPase activity declined with dose of TCE, while inhibition ratio of mitochondrial enzyme increased with dose of TCE. FCM results showed that after treatment with 2.000 mmol/L TCE, fluorescence density of Rh123 decreased quickly from 18.73 +/- 0.45(0 h) to 8.20 +/- 0.66(8 h) (P < 0.01). After 8 h, fluorescence density maintained at the level equal to that of 8 h (fluorescence density of Rh123 were 8.20 +/- 0.36 and 8.20 +/- 0.40 for 12 and 24 h respectively, compared with that for 8 h group, P > 0.05). The results also showed that MMP diminished with dose of TCE. Under TEM, mitochondria in TCE-treated group appeared extensive swelling and vacuolar degeneration with less matrix and obscure or vanished mitochondria cristae but in control group, mitochondrial structure was integrated, with uniform matrix and visible mitochondria cristae.
CONCLUSIONSTCE could inhibit mitochondrial metabolic enzyme, reduce ATP production, diminish MMP, and destroy ultrastructure of mitochondria in KC, all these contributing to the cytotoxicity of TCE.
Cell Survival ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Humans ; Keratinocytes ; drug effects ; metabolism ; ultrastructure ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Microscopy, Electron, Transmission ; Mitochondria ; drug effects ; metabolism ; ultrastructure ; Trichloroethylene ; toxicity
2.Analysis of sodium benzoate biotoxicity by atomic force microscope.
Mingqian HU ; Jiongkun WANG ; Jiye CAI ; Yangzhe WU ; Xiaoping WANG
Chinese Journal of Biotechnology 2008;24(8):1428-1432
Atomic force microscope (AFM) was used to study biotoxicity of food preservative sodium benzoate (SB) at the single cellular level. Lymphocyte morphology and membrane ultrastructure treated with SB at different concentrations and time were analyzed visually. As compared to the normal lymphocyte, the cell morphology and membrane was significantly changed and its ultrastructure was also complicated. After treated with SB, the Rp-v, Rq, Ra and Z values were changed. The statistical analysis of lymphocytes after treated with SB was studied, and discussed its mechanism.
Animals
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Cell Membrane
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ultrastructure
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Lymphocytes
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drug effects
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pathology
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Mice
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Mice, Inbred BALB C
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Microscopy, Atomic Force
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Sodium Benzoate
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toxicity
3.Interaction between necroptosis and apoptosis in MC3T3-E1 cell death induced by dexamethasone.
Min FENG ; Ruirui ZHANG ; Pei YANG ; Kunzheng WANG ; Hui QIANG
Journal of Southern Medical University 2019;39(9):1030-1037
OBJECTIVE:
To investigate the relationship between necroptosis and apoptosis in MCET3-E1 cell death induced by glucocorticoids.
METHODS:
MC3T3-E1 cells were incubated with 10-6 mol/L dexamethasone followed by treatment with the apoptosis inhibitor z-VAD-fmk (40 μmol/L) or the necroptosis inhibitor necrostatin-1 (40 μmol/L) for 2 h. At 72 h after incubation with dexamethasone, the cells were harvested to determine the cell viability using WST-1 assay and the rate of necrotic cells using annexin V/PI double staining; the percentage of apoptotic cells was determined using Hoechst staining. The mitochondrial membrane potential and the level of ATP in the cells were also evaluated. Transmission electron microscopy was used to observe the microstructural changes of the cells. The expressions of RIP-1 and RIP-3 in the cells were detected by Western blotting.
RESULTS:
At a concentration of 10-6 mol/L, dexamethasone induced both apoptosis and necroptosis in MC3T3- E1 cells. Annexin V/PI double staining showed that inhibition of cell apoptosis caused an increase in cell necrosis manifested by such changes as mitochondrial swelling and plasma membrane disruption, as shown by electron microscopy; Hoechst staining showed that the percentage of apoptotic cells was significantly reduced. When necroptosis was inhibited by necrostatin-1, MC3T3-E1 cells showed significantly increased apoptosis as shown by both AV/PI and Hoechst staining, and such changes were accompanied by changes in mitochondrial membrane potential and ATP level in the cells.
CONCLUSIONS
In the process of dexamethasone-induced cell death, necroptosis and apoptosis can transform reciprocally accompanied by functional changes of the mitochondria.
3T3 Cells
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Adenosine Triphosphate
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Animals
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Apoptosis
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Cell Death
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drug effects
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Dexamethasone
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Membrane Potential, Mitochondrial
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Mice
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Microscopy, Electron
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Mitochondria
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ultrastructure
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Necrosis
4.In vitro Antibacterial and Morphological Effects of the Urushiol Component of the Sap of the Korean lacquer tree (Rhus vernicifera Stokes) on Helicobacter pylori.
Ki Tae SUK ; Hyun Soo KIM ; Moon Young KIM ; Jae Woo KIM ; Young UH ; In Ho JANG ; Soo Ki KIM ; Eung Ho CHOI ; Myong Jo KIM ; Jung Soo JOO ; Soon Koo BAIK
Journal of Korean Medical Science 2010;25(3):399-404
Eradication regimens for Helicobacter pylori infection have some side effects, compliance problems, relapses, and antibiotic resistance. Therefore, alternative anti-H. pylori or supportive antimicrobial agents with fewer disadvantages are necessary for the treatment of H. pylori. We investigated the pH-(5.0, 6.0, 7.0, 8.0, 9.0, and 10.0) and concentration (0.032, 0.064, 0.128, 0.256, 0.514, and 1.024 mg/mL)-dependent antibacterial activity of crude urushiol extract from the sap of the Korean lacquer tree (Rhus vernicifera Stokes) against 3 strains (NCTC11637, 69, and 219) of H. pylori by the agar dilution method. In addition, the serial (before incubation, 3, 6, and 10 min after incubation) morphological effects of urushiol on H. pylori were examined by electron microscopy. All strains survived only within pH 6.0-9.0. The minimal inhibitory concentrations of the extract against strains ranged from 0.064 mg/mL to 0.256 mg/mL. Urushiol caused mainly separation of the membrane, vacuolization, and lysis of H. pylori. Interestingly, these changes were observed within 10 min following incubation with the 1 x minimal inhibitory concentrations of urushiol. The results of this work suggest that urushiol has potential as a rapid therapeutic against H. pylori infection by disrupting the bacterial cell membrane.
Anti-Bacterial Agents/chemistry/*pharmacology/therapeutic use
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Catechols/chemistry/*pharmacology/therapeutic use
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Cell Membrane/drug effects/ultrastructure
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Helicobacter Infections/drug therapy
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Helicobacter pylori/*drug effects/ultrastructure
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Humans
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Microbial Sensitivity Tests
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Microbial Viability/drug effects
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Molecular Structure
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Rhus/*chemistry
5.Influence of enterococci on human sperm membrane in vitro.
Hua QIANG ; Ming-Sen JIANG ; Jian-Yin LIN ; Wei-Min HE
Asian Journal of Andrology 2007;9(1):77-81
AIMTo study the influence of enterococci on human sperm membrane in vitro.
METHODSEjaculated human sperm were artificially infected with beta-hemolytic or non-beta-hemolytic enterococci at the bacteria: sperm ratio of 50:1 at 37 degrees . Sperm membrane integrity was examined after incubation for 1, 3 and 5 h by hypoosmotic swelling (HOS) test and electron microscopy.
RESULTSSperm infected with beta-hemolytic enterococci had lower HOS scores compared with non-beta-hemolytic strains or uninfected control (P < 0.01). The HOS test scores of sperm infected with beta-hemolytic enterococci increased in the presence of phosphatidylcholine, an inhibitor of hemolysin. Non-beta-hemolytic strains showed no significant difference in swelling rate, compared with the control group (P > 0.05). It was shown by electron microscopy that beta-hemolytic enterococci caused significant rupture of human sperm membrane.
CONCLUSIONBeta-hemolytic enterococci caused human sperm membrane injury, and might be mediated by the hemolysin of enterococci.
Cell Membrane ; drug effects ; microbiology ; Ejaculation ; Enterococcus ; physiology ; Feces ; microbiology ; Humans ; Male ; Phosphatidylcholines ; pharmacology ; Reference Values ; Spermatozoa ; drug effects ; microbiology ; ultrastructure
6.Nidogen Plays a Role in the Regenerative Axon Growth of Adult Sensory Neurons Through Schwann Cells.
Hyun Kyoung LEE ; In Ae SEO ; Duk Joon SUH ; Hwan Tae PARK
Journal of Korean Medical Science 2009;24(4):654-659
We previously reported that nidogen is an extracellular matrix protein regulating Schwann cell proliferation and migration. Since Schwann cells play a critical role in peripheral nerve regeneration, nidogen may play a role in it via regulation of Schwann cells. Here, we demonstrate direct evidence that nidogen induces elongation of regenerative axon growth of adult sensory neurons, and that the effect is Schwann cell dependent. Continuous infusion of recombinant ectodomain of tumor endothelial marker 7, which specifically blocks nidogen function in Schwann cells, suppressed regenerative neurite growth in a sciatic nerve axotomy model. Taken together, it is likely that nidogen is required for proper regeneration of peripheral nerves after injury.
Animals
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Axotomy
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Cell Movement
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Cell Proliferation
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Male
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Membrane Glycoproteins/*physiology
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Membrane Proteins/pharmacology
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*Nerve Regeneration
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Nerve Tissue Proteins/pharmacology
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Neurites/drug effects/*physiology/ultrastructure
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Rats
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Rats, Sprague-Dawley
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Recombinant Proteins/pharmacology
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Schwann Cells/cytology/*physiology
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Sensory Receptor Cells/*physiology
7.Investigation of apoptosis mechanism of arsenic trioxide on oral squamous cell carcinoma.
Chinese Journal of Stomatology 2003;38(1):20-23
OBJECTIVETo probe the possible mechanism of growth-inhibitory and apoptosis of oral squamous cell carcinoma by arsenic trioxide.
METHODSThe induction of apoptosis in two tongue squamous carcinoma cells treated by arsenic trioxide was investigated. The morphology changes of the cells was observed under electron microscope. The mitochondrial transmembrane potential was detected using rhodamine 123 and flow cytometry. The cell cycle was detected by flow cytometry, and p16, p53, BCL-2, Caspase-3, and PARP changes were examined by western blot.
RESULTS1. The antiproliferative effect on the oral squamous carcinoma cells by arsenic trioxide was carried out through two ways: induction of apoptosis and toxicity damage. 2. Activation of the caspase-3 and PARP, while no changes of p16, p53, BCL-2 occurred. 3. Mitochondrial transmembrane potential collapse and G(2)-M stagnation were correlated with apoptosis of oral squamous cell carcinoma.
CONCLUSIONS1. Tubulins and mitochondria may be the chief action position of arsenic trioxide, which is the start-up factors of mechanism. 2. Activation of the caspase-3 proteolytic pathway may be one of the pivotal ways of apoptosis procedure induced by arsenic trioxide.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Blotting, Western ; Carcinoma, Squamous Cell ; drug therapy ; metabolism ; pathology ; Caspase 3 ; Caspases ; metabolism ; Cell Cycle ; drug effects ; DNA, Neoplasm ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Intracellular Membranes ; drug effects ; physiology ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; physiology ; Mouth Neoplasms ; drug therapy ; metabolism ; pathology ; Oxides ; pharmacology ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Tumor Cells, Cultured ; drug effects ; ultrastructure ; Tumor Suppressor Protein p53 ; metabolism
8.Effects of aminophylline on proliferation and apoptosis in Raji lympho-blastoid cell line.
Ze-Lin LIU ; Zuo-Ren DONG ; Xue-Jun ZHANG ; Fu-Xu WANG ; Jing-Ci YANG ; Wei-Dong MA ; Xing-Yan DU ; Li YAO
Journal of Experimental Hematology 2003;11(1):45-49
The aim of this study was to investigate whether and how phosphodiesterase (PDE) inhibitors modulate the proliferation, cell cycle and apoptosis in lymphoma cells. The effects of aminophylline (AM), a non-specific PDE inhibitor, on Raji cells were explored in vitro. MTT assay, light and transmission electron microscopy and annexin V staining were used to observe cell proliferation, morphologic changes and apoptosis rate in AM-treated cells, and FCM and RT-PCR techniques were adopted to detect the effect on cell cycle, the expression of cyclin B1 and Bcl-2 and mitochondrial transmembrane potential in AM-treated cells. The results showed that AM inhibited the growth of Raji cells in a concentration-dependent manner. Morphologic observations showed apoptosis changes in AM-treated cells, including cytoplamic shrinkage, cytoplasmic bubbling, karyopyknosis and nuclear fragmentation. FCM and RT-PCR detection showed that AM intervention increased the fraction of annexin V(+) cells, reduced the value of mitochondrial transmembrane potential, induced S phase arrest, and down-regulated the expression of Bcl-2 at both mRNA and protein level and cyclin B1 protein in a concentration-dependent manner. It is concluded that PDE inhibitor aminophylline may induce Raji cell growth inhibition, S phase arrest, apoptosis via down-regulation of Bcl-2 and reduction of mitochondrial transmembrane potential.
Aminophylline
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pharmacology
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Apoptosis
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drug effects
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Burkitt Lymphoma
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drug therapy
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genetics
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pathology
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Cell Division
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drug effects
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Cyclin B
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genetics
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metabolism
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Cyclin B1
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Dose-Response Relationship, Drug
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Flow Cytometry
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Gene Expression Regulation, Neoplastic
;
drug effects
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Humans
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Intracellular Membranes
;
drug effects
;
physiology
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Membrane Potentials
;
drug effects
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Mitochondria
;
drug effects
;
physiology
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Phosphodiesterase Inhibitors
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pharmacology
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Proto-Oncogene Proteins c-bcl-2
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genetics
;
metabolism
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RNA, Messenger
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drug effects
;
genetics
;
metabolism
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S Phase
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Tumor Cells, Cultured
;
drug effects
;
metabolism
;
ultrastructure
9.Effect of bone morphogenic protein 7 on nephrin expression and distribution in diabetic rat kidneys.
Hou-qin XIAO ; Wei SHI ; Yong ZHANG ; Yong-zheng LIANG
Journal of Southern Medical University 2009;29(4):671-675
OBJECTIVETo evaluate the effect of bone morphogenic protein 7 (BMP-7) on nephrin expression and distribution in diabetic rat kidneys.
METHODSTwenty rats with diabetes mellitus (DM) induced by streptozotocin (STZ) injection were randomly divided into DM model group and BMP-7 treatment group, with another 10 normal rats serving as the normal control group. The rats in BMP-7 group received intraperitoneal human recombinant BMP-7 injections at 30 microg/kg twice a week for 24 consecutive weeks, while normal saline was administered in rats of the other two groups. Blood glucose and 24 hour urinary protein and creatinine (Ccr) were measured at 8, 16 and 24 weeks, and the rats were sacrificed at 24 weeks to obtain the renal tissues for detecting the expression and distribution of nephrin using immunofluorescence assay and RT-PCR and for examining the expressions of transforming growth factor-beta1 (TGF-beta1) and WT1 using immunohistochemistry.
RESULTSCompared with the normal control group, the DM model group showed significantly increased 24 hour urinary protein, kidney to body weight ratio and TGF-beta1 expression, but had lowered Ccr, glomerular podocyte number and nephrin expression. The linear distribution of nephrin along the capillary loops as found in the normal control group became granular in the kidney of diabetic rats. The rats in BMP-7 group showed less urinary protein excretion, lower TGF-beta1 expression and greater glomerular podocyte number than those in the DM group, and the expression and distribution of nephrin remained normal in the kidney.
CONCLUSIONAdministration of BMP-7 can significantly suppress the down-regulation of nephrin expression and maintain its normal distribution in the podocytes in diabetic rats possibly in association with a direct suppression of TGF-betasignaling.
Animals ; Blood Glucose ; metabolism ; Body Weight ; drug effects ; Bone Morphogenetic Protein 7 ; pharmacology ; Cell Count ; Diabetes Mellitus ; genetics ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Humans ; Kidney ; drug effects ; metabolism ; pathology ; ultrastructure ; Male ; Membrane Proteins ; genetics ; metabolism ; Microscopy, Electron, Transmission ; Organ Size ; drug effects ; Podocytes ; drug effects ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism
10.In vivo administration of Fms-like tyrosine kinase-3 ligand effectively stimulates lung dendritic cell expansion in mice.
Hong-Wei WANG ; Jiang-Yang LU ; Lin WANG ; Guang TIAN
Chinese Medical Journal 2012;125(14):2562-2567
BACKGROUNDDendritic cells (DCs) are the most important professional antigen presenting cells that play a key role in initiating adaptive immune responses. The depletion and dysfunction of DCs contribute to the development of immunodeficiency or immunoparalysis in some lung diseases. In the present study, we investigated the effects of Fms-like tyrosine kinase-3 ligand (Flt3L) administration in vivo on lung DCs expansion to provide an experimental basis of Flt3L used as a potential therapeutic agent for the related lung disorders.
METHODSBalb/c mice were randomly divided into Flt3L group (n = 10) and control group (n = 10). Each mouse in the Flt3L group received subcutaneous administration of Flt3L at a dose of 10 µg once daily for nine consecutive days. Lung histology was observed, and CD11c and CD205 were immunologically labeled in lung tissue sections. Low-density lung cells were separated by density gradient centrifugation, and then subsets and MHC-II/I-A(d) expression of DCs were analyzed by flow cytometry.
RESULTSIn the Flt3L group the number and density of DC-like cells were markedly increased compared with the control group, mainly distributed in the alveolar septa. Immunological labeling in situ found that there were significantly higher numbers of CD11c(+) and CD205(+) DCs in lung mesenchymal tissue (P < 0.05), where they formed a denser reticular formation. Flow cytometry analysis demonstrated that the proportions of myeloid CD11c(+)CD11b(+) DCs and plasmacytoid CD11c(+)CD45R/B220(+) DCs in the low-density lung cells in the Flt3L group were significantly higher compared with the control group; showing 3.17- and 3.3-fold increase respectively (P < 0.05). The proportion of CD11c(+) DCs expressing MHC-II/I-A(d+) was significantly increased, with a 2.7-fold increase as compared with the control group (P < 0.05).
CONCLUSIONSFlt3L administration in vivo induces lung DCs expansion, favoring myeloid and plasmacytoid DC subsets, which are phenotypically more mature. Flt3L may be useful in the therapy to augment immune function of the lung.
Animals ; Antigens, CD ; metabolism ; Cells, Cultured ; Dendritic Cells ; drug effects ; metabolism ; ultrastructure ; Flow Cytometry ; Immunohistochemistry ; Lectins, C-Type ; metabolism ; Lung ; drug effects ; metabolism ; ultrastructure ; Male ; Membrane Proteins ; pharmacology ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron, Transmission ; Minor Histocompatibility Antigens ; Random Allocation ; Receptors, Cell Surface ; metabolism