ABSTRACT
[ABSTRACT]AIM:ToinvestigatetheroleofquercetinintheapoptosisofhumanbreastcancercelllineMCF-7 and the association with Fas/Fas ligand (FasL) pathway.METHODS: The apoptosis model of MCF-7 cells was estab-lished by the induction with quercetin .The morphological characteristics of apoptotic MCF-7 cells were observed under transmission electron microscope .The apoptotic rates and alternation of mitochondrial membrane potential (Δψm ) in the MCF-7 cells were measured by flow cytometry using fluorescein labeled Annexin V-FITC/PI and JC-1, respectively.FasL neutralizing antibody was applied to block the apoptosis .The expression of Fas/FasL on the cells was detected by immuno-fluorescence technique and flow cytometry , respectively.The influence of SB203580 (an inhibitor of p38 MAPK) on the expression of Fas/FasL was also examined by flow cytometry .The protein levels of p 38 MAPK and p-p38 MAPK were de-termined by Western blot .RESULTS: The phenomenon of nuclear condensation and marginalization in the MCF -7 cells treated with quercetin at 80.0 μmol/L for 48 h was observed under transmission electron microscope .Compared with the control cells , theΔψm was decreased by 17.4%, 44.3% and 68.9% in the MCF-7 cells treated with quercetin at 80.0μmol/L for 24 h, 48 h and 72 h, respectively .The apoptotic rates of MCF-7 cells treated with quercetin at 80.0 μmol/L for 24 h, 48 h and 72 h were (10.2 ±3.3)%, (28.9 ±7.5)%and (39.2 ±8.9)%, respectively.However, the apop-totic rates were decreased to (8.2 ±2.8)%, (19.2 ±5.3)% and (22.5 ±6.9)% after the cells were pretreated with FasL neutralizing antibody , respectively .When MCF-7 cells were treated with quercetin for 24 h, 48 h and 72 h, Fas/FasL expression rates were increased in a time-dependent manner , which were largely inhibited by SB203580.The protein level of p38 MAPK was not changed obviously , but the protein level of p-p38 MAPK was significantly increased at 48 h and 72 h.CONCLUSION: Quercetin up-regulates the expression of Fas/FasL on MCF-7 cells, and induces apoptosis via Fas/FasL pathway .Meanwhile , p-p38 MAPK is potentially critical signaling molecule for up-regulating the expression of Fas/FasL.
ABSTRACT
AIM:To investigate the role of damaged mitochondria in dendritic cell ( DC) apoptosis induced by Vibrio vulnificus (Vv) and its possible mechanism.METHODS: DC2.4 cells were co-cultured with Vv 1.1758 strain. Fluorescent probes DCFH-DA and Fluo-8-AM were used to detect reactive oxygen species ( ROS) and intracellular Ca 2+concentration in the invaded cells , respectively .The cellular apoptotic rates and mitochondrial membrane potential (Δψm ) were measured by flow cytometry.The expression of nuclear factor-kappa B p65 (NF-κB p65) and tumor necrosis factor-al-pha (TNF-α) was detected by Western blotting.RESULTS:Vv 1.1758 induced DC2.4 cell apoptosis.Vv 1.1758 bacte-ria invaded into the DC2.4 cells by binding with cellular membrane though the end of the body .In the invaded DC2.4 cells, the visible mitochondrial damage, elevated ROS and intracellular Ca2+levels, and declinedΔψm were presented.Af-ter 1 h of co-culture, NF-κB p65 began to rise and reached the peak at 5 h, and then slightly decreased at 6 h.The TNF-αlevel increased after 2 h of co-culture and reached the peak at 6 h.CONCLUSION:The damaged mitochondria play an important role in DC apoptosis induced by Vv , and its possible mechanism may associate with the elevation of ROS and in-tracellular Ca2+level, and the declined Δψm.Meanwhile, NF-κB p65 and TNF-αare potential critical signaling molecules in the process of apoptosis .
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of phospholipase C(PLC) in cytoskeleton rearrangement of mouse dendritic cells invaded by Mycobacterium tuberculosis.</p><p><b>METHODS</b>Mouse dendritic DC2.4 cells were co-cultured with Mycobacterium tuberculosis H37Rv. F-actin of DC2.4 cells were strained with phalloidin-TRITC, the microtubule was stained with anti-β-tubulin monoclonal antibody and FITC-conjugated AffiniPure anti-mouse IgG. The changes of cytoskeleton in DC2.4 cells induced by Mycobacterium tuberculosis H37Rv were determined by fluorescence microscopy and the rates of F-actin rearrangements were calculated. The expressions of PLC in cytoplasm and cytomembrane of DC2.4 cells were measured by ELISA. DC2.4 cells were pretreated with PLC inhibitor U73122, then F-actin rearrangements induced by invasion of Mycobacterium tuberculosis were observed.</p><p><b>RESULTS</b>Bacterial invasion was observed while DC2.4 cells were co-incubated with Mycobacterium tuberculosis H37Rv for 2 h. The rates of invasion were (26.1 ± 4.5)%, (39.9 ± 5.6)%, (51.2 ±5.9)%, (57.9 ± 6.1)% and (63.9 ± 6.8)% at 4, 6, 8, 10 and 12 h of co-culture, respectively; while those were (13.6 ± 3.1)%, (14.2 ± 3.9)%, (15.1 ± 4.3)%, (16.8 ± 4.0)% and (18.3 ± 5.2)% after blocked by PLC, respectively. The rates of the F-actin rearrangements at 2, 4, 6, 8, 10 and 12 h after DC2.4 cells were invaded by H37Rv were (26.9 ± 1.5)%, (59.3 ± 2.8)%, (72.7 ± 4.8)%, (78.2 ± 5.9)%, (63.3 ± 2.9)% and (43.2 ± 2.6)%, respectively; while those were (18.5 ± 1.2)%, (22.3 ± 1.7)%, (3.6 ± 2.5)%, (24.8 ± 2.3)%, (22.3 ± 1.3)% and (23.8 ± 1.8)% after blocked by PLC, respectively. There were no changes of the microtubule observed in DC2.4 cells at the same time points. The rates of the F-actin rearrangements before blocked by PLC were higher than those after PLC blockade at 4, 6, 8 and 10 h (P <0.05). The expressions of PLC in cytomembrane in DC2.4 cells increased after 2 h and reached its highest level at 8 h. The PLC inhibitor U73122 inhibited the expressions of PCL in cytomembrane of DC2.4 cells, but not in cytoplasm.</p><p><b>CONCLUSION</b>Mycobacterium tuberculosis can provoke to F-actin rearrangements through PLC molecule, which would further lead to Mycobacterium tuberculosis invasion of DC2.4 cells.</p>
Subject(s)
Animals , Mice , Actins , Metabolism , Cell Line , Coculture Techniques , Cytoskeleton , Metabolism , Dendritic Cells , Cell Biology , Microbiology , Microtubules , Metabolism , Mycobacterium tuberculosis , Virulence , Type C Phospholipases , MetabolismABSTRACT
Objective To explore how Vibrio vulnificus (Vv) invades dendritic cells (DC) and induces acute necrosis of DC via toll-like receptor (TLR) 2 and 4 pathways.Methods Vv 1.1758 strain and DC 2.4 mixed culture model was established,observed the infection rates of DC with optical microscope,the location of Vv and structural changes of DC by transmission electron microscope.The expression levels of TLR2 and TLR4 mRNA were determined by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and tumor necrosis factor-α (TNFα) protein titers were measured by enzyme-linked immunosorbent assay (ELISA).DNA ladder qualitative test was used to detect cell apoptosis,while flow cytometry was used to quantify cell apoptosis and necrosis rates.Statistical analysis was done by chi-square test and one-way ANOVE.Results The infection rates of DC after 0.5,1,2,4 and 6 h of mixed culture were (7.8±0.8) ％,(13.9± 1.1) ％,(34.6±4.9) ％,(77.8± 10.2)％ and (95.8 ± 13.1)％,respectively.Vv was generally located in the internal cell membrane of DC 2.4.After 2 h co-culture,nuclear chromatins of DC became active and intranuclear apoptosis bodies appeared.After 4 h,cytoplasmic vacuoles appeared,chromatin gathered,and cell membranes were seriously damaged.After 6 h,mitochondria was highly swelled and distorted,and cell apoptosis and necrosis occurred.TLR2 and TLR4 mRNA levels reached peak values after co-culture for 0.5 h; TNF-α level began to increase at 1 h (P＜0.05) and reached peak values at 2 h.DNA Ladder electrophoresis presented scouring necrosis after 2 h culture and apoptotic bands appeared between 720 bp and 900 bp after 4 to 5 h culture.Early apoptosis rates of DC after 2,4 and 6 h culture were (3.1±3.8)％,(7.8±4.7)％ and (12.7±8.2)％,and necrosis rates of DC were (16.7±12.5)％,(41.6±25.9)％ and (75.5±33.6)％,higher than that of control group (all P＜0.05).Conclusions Vv infects DC and induce DNA degradation through up-regulated expression of TLR2 and TLR4 and increasing of TNF-α inflammatory mediators.During cell degradation,apoptosis and necrosis coexist,while necrosis is predominant.
ABSTRACT
Objective To observe the process of Vibrio vulnificus inducing dendritic cell strain apoptosis.Methods We established the mixed culture model of mouse dendritic cell ( DC 2.4 strain) and Vibrio vulnificus( Vv1.1758 strain ),analyzed morphological characteristics of cell apoptosis by DAPI fluorescence staining,detect DNA fragmentation level of apoptosis cells by DNA Ladder assay,analyze DC2.4 apoptosis rate by Annexin V FITC/PI staining,determine activities of caspase-3 and caspase-8 by means of spectrophotometric method and detect changes of mitochondrial transmembrane potential ( △ Ψm ) by JC-1 fluorescence labeling.Results After Vv1.1758 strain and DC2.4 cell were mixed and cultured for 4 h,DAPI fluorescence staining showed typical apoptosis characteristics-chromatin condensation and marginalization; DNA agarose gel electrophoresis showed apoptosis band; apoptosis rates at 2,4 and 6 h were respectively (37.8±9.8) ％,(54.3 ± 12.7 ) ％ and ( 68.2± 14.6 ) ％ ; Mitochondrial transmembrane potentials (△Ψm) at 1 h,2 h and 4 h reduced by 7.1％,16.1％ and 46.7％ respectively; caspase-8 activity increased at 1.5 h and reached the peak at 2 h [ (2.48±0.19) U/μg],while caspase-3 activity started to increase at 3h and reached the peak at 4 h [ ( 1.91 ±0.16) U/μg ].Conclusion Vibrio vulnificus could induce dendritic cells by two pathways: reducing mitochondrial transmembrane potential and activating caspase-8 promoter and finally activate effector caspase-3 to promote apoptosis.
ABSTRACT
Objective To investigate the invasion,internalization and the organelles damage of the cultured dendritic cells ( DC2.4 strain) during Vibrio vulnificus (Vv) infection.Methods The study model was the cultured DCs infected by Vv 1.1758 strain.Electron microscopy was used to observe the localization of bacteria in different time point of infection,cell morphology and the process of organelles changes.The cytoskeleton structure including the microfilaments and the microtubules rearrangement was examined by the fluorescence microscope.Results The Vv were pinocytosed into the DC cells through double-sides,and localized at 1-2 μm of the inner side membrane.It cost 1.27,1.87,3.43 hours reaching the infection ratio of 25％,50％,75％,respectively.Using electron microscopy,the DCs had been observed the phagosome formation within 1h,chromatin activation within 2 h,chromatin aggregation 4 h,and the significant cytoskeleton structure disruption within 6 h.Endoplasmic reticulum,mitochondria and lysosomes became swollen.In DCs,the protruding filaments gradually reduced,and their shape changed from the point-like to the linearlike aggregation at the inner side of the plasma membrane,extended microtubules disappeared,the microtubules at the outside nuclear membrane striking rearranged.Conclusion After DC was infected by Vv,the bacteria were pinocytosed into the inner side of DC membrane,and the microfilaments were observed to move from the cytoplasm to cell membrane.In addition,the microtubules moved from the synapse and the cell membrane to the nuclear membrane.The high lethality of Vv could provoke to the DCs cytoskeleton rearrangements.
ABSTRACT
<p><b>OBJECTIVE</b>To establish the method of promoting human peripheral blood mononuclear cell proliferation by polysaccharide of snakegourd root and identify the effects of polysaccharide of snakegourd root on lymphocyte proliferation, T lymphocyte subsets and the different levels of TNF-alpha and IL-6.</p><p><b>METHOD</b>The polysaccharide of snakegourd root preparations were purified with dialysis and ethanol precipitation. The healthy human PBMC were used as the target cells for screening potency of the drugs. MTT colorimetry was established to examine the levels of lymphocyte proliferation on human PBMC by polysaccharide of snakegourd root in vitro. The percents of lymphocyte subsets (CD3+, CD4+, CD8+ T lymphocyte) and the different levels of TNF-a and IL-6 in PBMC were analysed by FCM and ELISA, respectively.</p><p><b>RESULT</b>1.0-50.0 mmol x L(-1) of polysaccharides of snakegourd root showed the significant effects of promoting proliferation of human PBMC (P < 0.05). The percents of CD3+, CD4+, CD8+ T lymphocytes in PBMC treated with 5.0 and 10.0 mmol x L(-1) of polysaccharides of snakegourd root were significantly higher than those of the control group (P < 0.05). The levels of TNF-alpha and IL-6 were significantly higher than those of the control group after 1.0, 5.0, 10.0 mmol x L(-1) of polysaccharides of snakegourd root stimulation on the human PBMC at 8 hours (P < 0.05).</p><p><b>CONCLUSION</b>The significant effects on promoting lymphocyte proliferation and activation of the polysaccharide of snakegourd root are confirmed in this study. The percents of lymphocyte subsets are increased in different degrees by the polysaccharide of snakegourd root. The high levels of TNF-alpha and IL-6 are secreted after the polysaccharides of snakegourd root stimulation on the human PBMC, which lays a foundation for further elucidating the immunocompetence effects and mechanism of the polysaccharide of snakegourd root.</p>
Subject(s)
Humans , Dietary Carbohydrates , Immunocompetence , PolysaccharidesABSTRACT
Objective To investigate the influence of intermittent catheterization methods on the urinarytract infection in patients with spinal cord injury. Methods Thirty-one cases of spinal cord injury and urinarytract infection were recruited. By use of intermittent catheterization, the bladder function was trained. Bacteria cul-ture and identification of in urine from the patients were conducted before the training (indwelling catheterization)and after 15 or 30 days of intermittent catheterization. Results The rates of urinary tract infection (colony count≥1?10~5cfu/ml) after 15 or 30 days of intermittent catheterization were 38. 7% and 35. 5%, respectively, whichwere significantly lower than those of indwelling catheterization (100%), P
ABSTRACT
@#ObjectiveTo study the effect of heuristic teaching to the medical students in interest, initiative spirit, and comprehensive ability. Methods110 students majoring in rehabilitation medicine were divided into two groups, heuristic teaching group and rote teaching group. The teaching contents included ROM(range of movement), ADL(activity of daily living) and the techniques of movement and transfer. Eight items were assessed. ResultsThere are no difference between the two groups in application of basic knowledge and ability of comprehension (P>0.05), obvious difference in the interest in study, the initiative spirit, and the ability of self study and analyzing (P<0.05).Conclusions In medical rehabilitation teaching, heuristic teaching is more advantageous than rote teaching in the interest in study, the initiative spirit, and the ability of self study and analyzing.
ABSTRACT
AIM:To construct a prokaryotic expression system of staphylococcal enterotoxin A(SEA)gene and determine the effects of the recombinant expression product rSEA in promoting lymphocyte proliferation and inhibiting tumor cell growth.METHODS:PCR was used to amplify entire SEA gene of S.aureus strain ATCC13565.The cloned SEA gene was sequenced after T-A cloning.SDS-PAGE was applied to measure the output of rSEA expressed by pET32a-SEA-E.coli BL21DE3.Ni-NTA affinity chromatography was performed to extract rSEA.Cytotoxicity of rSEA to Vero cells was detected using TCID_ 50 titration method and then the value of TCIC_ 50 was determined.MTT colorimetry was established to examine the effects of rSEA at different dosages on proliferation of mouse splenocytes and human peripheral blood mononuclear cells(PBMC)as well as on growth of HepG2 cells and HeLa cells in vitro.RESULTS:In comparison with the published corresponding sequences,similarities of the nucleotide and putative amino acid sequences of the cloned SEA gene were 100%.The output of rSEA was approximate 25% of the total bacterial proteins.rSEA had a cytotoxicity with TCIC_ 50 of 3.14 ?g to Vero cells.1.0-20.0 mg/L rSEA showed the significant effects of promoting proliferation of mouse splenocytes and human PBMC(P