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Objective To investigate the effect of cyclin-dependent kinase 2 (cdk2) recombinant lentivirus in rats with lipopolysaccharide (LPS)-induced acute lung injury (ALI).Methods Thirty-six adult SD rats were divided into control group, LPS model group and gene intervention group according to the random number table, with 12 rats per group.Rats with LPS-induced ALI were established by intratracheal injection of LPS.Saline solution (60 μL/kg) was injected in control group at the time point of 0, 24, 48 h respectively.Control-lentivirus (60 μL/kg) and cdk2 recombinant lentivirus (60 μL/kg) were injected respectively in LPS model group and gene intervention group at the time point of 0 h and 24 h.After 48 h, LPS (60 μL/kg) with isotonic saline solution were injected in both LPS model group and gene intervention group.Lung tissue samples from right-lower areas were collected at 24 h postinjury to evaluate the pathological changes with HE staining.Expressions of cdk2, clara cell secretory protein (CCSP), phospholipase A2(PLA2) and p-C/EBP β protein were detected by Western blot.Inflammatory factors of tumor necrosis factor-α(TNF-α), interleukin (IL)-1β, IL-6 and IL-10 in serum were measured with ELISA method.Results Inflammatory infiltration and damage to the alveolar structure were serious in LPS model group than control group, while inflammatory infiltration decreased significantly and alveolar structure tended to be normal in gene-intervention group.Expression of Cdk2 in control group (1.00±0.21) and LPS model group (0.93±0.17) were similar, but both were lower than that in gene intervention group (4.29±0.73) (P<0.05).Expression of CCSP in gene intervention group (3.19±0.38) was significantly higher than that in control group (1.00±0.20) and LPS model group (0.32±0.19) (P<0.05).Expression of PLA2 in LPS model group (4.49±0.51) was higher than that in control group (1.00±0.13) and gene intervention group (1.76±0.26) (P<0.05).Meanwhile, the variation of p-C/EBPβ concentration among the groups was similar to CCSP.Expression of TNF-α in LPS model group[(196.34±30.17)pg/ml] was higher than that in control group [(71.24±5.13)pg/ml] and gene intervention group[(86.32±11.02)pg/ml](P<0.05).Changes in IL-1β, IL-6 and IL-10 among the groups were similar to TNF-α.Conclusions Over-expression of Cdk2 plays a protective role for LPS-induced ALIby up-regulating CCSP and down-regulating inflammatory factors such as PLA2, TNF-α, IL-1β, IL-6 and IL-10, as may relate to the phosphorylation of C/EBPβ.
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Objectives To analyze the relationship between phosphorylation of serine-3 of cofilin1 and Taxol resistance of ovarian cancer and to evaluate the prognostic value of cofilin1 phosphorylation in estimating ovarian cancer survival using clinical samples.Methods Wild type (WT) plasmids with over-expression of wild cofilin1,S3L plasmids with over-expression of cofilin1 and inhibited phosphorylation at serine-3,and siRNA-C plasmids with down-regulation of cofilin1 were constructed using molecular biology techniques.After the SKOV3 and SK-TR30 cell-lines were transfected with these plasmids,changes in biological characteristics and drug resistance after instant transfection were compared.Fifty-one elderly female patients with microscopically confirmed ovarian cancer,who had received chemotherapy with Taxol and cis-platinum (TC)after surgery,including 30 chemo-sensitive and 21 chemo-resistant cases,were recruited in this study.Immunohistochemical methods were used to detect the expression of cofilinl and phosphorylated cofilin in tissue sections from the two groups.Progression free survival(PFS)was analyzed.Results Compared with the control group,the proportion of cells increased at the G0/G1 phase(P=0.034)and decreased at the S phase (P=0.031),and the apoptosis rate decreased(P=0.020),in SKOV3 cells with high expression of cofilin1.However,these characteristics disappeared when the wild type was replaced with the inactive mutant S3L.When cofilin1 expression was down-regulated in SK-TR30 cells,the proportion of cells at the G0/G1 phase decreased(P=0.020).The expression of cofilin1 was detected in 56.7 ％ (17/30)and 57.1％ (12/21),respectively,sections of ovarian tumor tissues of the chemo-sensitive and chemoresistant groups,and there was no statistic difference (x2=0.332,P =0.943)between the two groups.The expression of phosphorylated cofilin was much higher in the chemo-resistant group (85.7％ or 18/21)than in the chemo-sensitive group (53.3％ or 16/30),and the difference was statistically significant(x2=5.829,P=0.016).The higher expression of phosphorylated cofilin was also correlated with shorter PFS(x2 =21.440,P＜0.01,95％ CI:0.068-0.883),especially in the chemo-sensitive group.Conclusions Serine-3 phosphorylation status of cofilin 1 is associated with paclitaxel resistance in ovarian cancer,but the underlying mechanisms of regulation need further investigation.
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Objective To analyze the construction of mouse liver phosphoproteome and phosphorylated kinases to provide useful information for integrating mouse kinase phosphorylation regulatory networks.Methods A new method was established to identify phosphoproteome from the mouse liver.First of all, liver protein was digested with trypsin before the resulting peptides were subjected to a two-step phosphopeptide enrichment and separation procedure consisting of TiO2 chro-maphy enrichment combined with high pHHPLC separation.Samples were injected onto aNanolC-Ultra-2Dplus system cou-pled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument.Then data analysis was performed to provide information of new identified phosphorylation sites of kinase.Results and Conclusion Using our efficient and high-throughput platform, we reported the identification of 5386 phosophorylation sites and 4553 phosphopeptides from 1533 proteins of the mouse liver.126 new phosphorylation sites were identified from 116 kinases, which provides valuable infor-mation for phosphorylation networks in the mouse liver.
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Objective To investigate the effects of narrow-band ultraviolet B (NB-UVB) on the expression of keratin 17 (K17) in the human keratinocyte cell line HaCaT.Methods Cultured HaCaT cells were irradiated with different doses (0,50,100,200,400 mJ/cm2) of NB-UVB followed by additional culture for 6,12 and 24 hours respectively.To explore the mechanisms underlying the effects of NB-UVB on K17 expression,some HaCaT cells were pretreated with PD98059 (an inhibitor of mitogen-activated protein kinase kinase) followed by UVB radiation and additional culture as described above.Cell counting kit-8 (CCK 8) was used to evaluate the proliferation of,real time PCR to measure the mRNA expressions of K17 in,and Western blot to determine the protein expression of K17,Erk1/2 and phosphorylated Erk1/2 in,HaCaT ceils.Results Both the proliferation of and K17 expression in HaCaT cells were promoted by NB-UVB radiation at low doses (50,100 mJ/cm2),but inhibited by NB-UVB radiation at high doses (200,400 mJ/cm2).Significant differences were observed for the proliferative activity between HaCaT cells irradiated with NB-UVB at 100 or 400 mJ/cm2 and unirradiated HaCaT ceils at 12 and 24 hours (P ＜ 0.01 or 0.05).The phosphorylation of Erk1/2 was upregulated by NB-UVB radiation at 100 mJ/cm2,but downregulated by that at 400 mJ/cm2,and the upregulation induced by low dose NB-UVB could be suppressed by blocking the Erk 1/2 pathway.Conclusion The effects of NB-UVB radiation on K17 expression may be modulated by the Erk 1/2 pathway.
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Objective To investigate expressions of CDev6 and p-ERK1/2 in AML patients and its clinical significance.Methods Expressions of CD44v6 and p-ERK1/2 on bone marrow blasts in 152 AML patients and 22 normal controls were determined by flow cytometry.Meanwhile,the effects of CD44v6 expression(CD+44v6 and CD-44v6) on relapse rate and survival time were analyzed by following up of 129 AML patients.Results Double positive expressions of CD44v6 and p-ERK1/2 were observed in 4.5%(1/22) of the normal bone marrow blasts while single positive expression of CD44v6 and p-ERK1/2 was observed in 36.2%(55/162) and 64.5%(97/162) of AML patients,respectively.The MFI of p-ERK1/2 expression on blast cells in AML patients was [14(7 -58)],which was higher than that in normal controls [8(6 - 10),U =4.2,P＜0.01].Furthermore,MFI of p-ERK1/2 expression on blast cells in CD+44v6 AML patients was [28(15-61)] ,which was significantly higher than that in CD-44v6 AML patients [18(6- 37),U =6.7,P＜0.01].A strong correlation was obtained between CD44v6 and p-ERK1/2 expression(rs = 0.7,P＜ 0.01).Among 129 patients followed up for 4 to 65 months,the data also revealed that the relapse rate of CD-44v6 AML patients was 32.1%(26/81) ,which was much lower than that in CD44v6 AML patients [81.3%(39/48),x2 = 29.13,P＜ 0.01).And the overall survival in CD44v6 AML patients was(28.5 ± 1.8)months,which was significantly worse than that of CD44v6 AML patients [(51.2±2.0) months,x2 =48.2,P＜ 0.01).Conclusion CD44v6 expression was associated with a poor survival in AML patients,and CD44v6 might promote the expansion of leukemic blast cells by up-regulating p-ERK1/2.
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Objective To investigate the role of CRKL activity in leukemia cells with muhidrug resistance and find new factor related to multidrug resistance. Methods By flow cytometry, CRKL activity was compared in K562, HL-60 cells and its resistance cells. The change of CRKL activity was observed in sensitive cells treated with and withdrawal daunorubicin. Results With the comparison of K562, HL-60 sensitive cells, in K562, HL-60 resistant cell lines, the level of CRKL phosphorylation in K562, HL-60 resistance cells treated with daunombicin 72 hours increased markedly. The level of CRKL phosphorylation was time-dependent with chemotherapy drugs, not change of CRKL activity was found in Jurkat ceils.Conclusion The level of CRKL activity is new factor related to muhidrug resistance in leukemia cells.