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1.
Chinese Journal of Immunology ; (12): 1141-1145, 2017.
Article in Chinese | WPRIM | ID: wpr-608834

ABSTRACT

Objective:To determine the effects of Foxp3-overexpressing lung cancer cells on activated CD4+T lymphocyte.Methods: Stable Foxp3-overexpressing lung cancer cells NCIH-1299,NCIH-hFoxp3,was generated by transfection of NCIH-1299 cells with plasmid pcDNA3-hFoxp3 mediated by Lipofectamine 2000 and by selection with G418,and validated by quantitative PCR and Western blot.The expression levels of IL-8 and IL-10 secreted by NCIH-hFoxp3 and NCIH-control were measured by ELISA.IL-2 secrection by activated human CD4+T lymphocyte which was tested after stimulation with 20% conditioned medium of NCIH-hFoxp3 and NCIH-control cells.The proliferation of activated human CD4+ T lymphocytes was assessed by MTT after coculture with NCIH-hFoxp3 cells.The adhesive ability of activated human CD4+ T lymphocytes was probed with NCIH-hFoxp3 cells by immunocytochemistry.Results: Compared with NCIH-control cells,NCIH-hFoxp3 secreted high level of IL-10 and low level of IL-8.NCIH-hFoxp3 with Foxp3 overexpression significantly suppressed the proliferation,adhesive potential and IL-2 expression by activated CD4+ T cells.Conclusion: Suppression of immune activities of activated CD4+ T cells by Foxp3 overexpression in lung cancer cells may correlate with cytokine IL-8 and IL-10,which can contribute lung cancer progression.

2.
Chinese Journal of Immunology ; (12): 1481-1484, 2016.
Article in Chinese | WPRIM | ID: wpr-504353

ABSTRACT

Objective:To determine the effects of enforced expression of Foxp3 in lung cancer cell with regards to proliferation and tumorgeneity. Methods: A stable subline NCIH-hFoxp3 was established by liopfectamin-mediated pcDNA plasmid transfection carrying exogenous hFoxp3. The growth curve and secrection of IL-8 and IL-10 of NCIH-hFoxp3 were evaluated using MTT and ELISA, respectively. The in vivo tumorigeneity was assessed as well by inoculation of NCIH-hFoxp3 subcutaneously in nude mice. Results:Lung cancer cell NCIH-hFoxp3 with enforced expression of Foxp3 proliferated slowly but exihited increased in vivo tumorgeneity compared with corresponding control subline. In addition,increased expression of hFoxp3 in NCIH-hFoxp3 augmented secretion and at-tenuated secretion of IL-8 and IL-10,respectively. Conclusion:Increased expression of Foxp3 may promote progression of lung cancer cell by change of cellular microenvironment and evasion of immune surveillance.

3.
Chinese Journal of Immunology ; (12): 832-837, 2016.
Article in Chinese | WPRIM | ID: wpr-490240

ABSTRACT

Objective:To express and purify the fusion protein of GST-FILIP-1L and prepare its polyclonal antibody. Methods:The constructed recombinant expression vectors pGEX-4T3-FILIP-1L were transformed into Escherichia Coli BL21. FILIP-1L fusion protein was induced by IPTG and purified by Glutathion Sepharse 4B . The rabbit was immunized by the purified fusion protein,and pro-duced serum with anti-FILIP-1L antibody. The titer of polyclonal antibody was detected by ELISA, the anti-FILIP-1L polyclonal antibody was purified by Active and its combining specificity with FILIP-1L protein was further identified by Western blot. Results:The GST-FILIP-1L fusion protein was highly expressed in E. coli, and its specific polyclonal antibody was obtained after the immunization. The polyclonal antibody purified by Active Ester Agrose was able to combine specially with FILIP-1L protein and transformed FILIP-1L protein in 293 cells and FILIP-1L protein of liver cancer cells, respectively. Conclusion: The GST-FILIP-1L fusion protein was expressed successfully,the anti-GST-FILIP-1L polyclonal antibodies with high titer and specificity are successfully prepared,these antibodies provide an useful experimental tool to research the biological function of the FILIP-1L protein.

4.
Military Medical Sciences ; (12): 45-50, 2016.
Article in Chinese | WPRIM | ID: wpr-491795

ABSTRACT

Objective To examine whether Tim-3 plays a protective role in paraquat poisoning induced excessive immune response and tissue damage based on the critical roles of Tim-3 controlling inflammatory response.Methods A paraquat poisoning model was established in wild type and in Tim-3 transgenic C57BL/6 mice by intraperitoneal injection of paraquat (40 mg/kg) .In addition, C57BL/6 mice with paraquat poisoning were injected with Tim-3 soluble protein( sTim-3) or control protein to see the effect of Tim-3 blocking on the progression of paraquat poisoning.Samples were collected at 6 and 24 h after paraquat injection respectively and were examined for tissue damage, cytokine expression and paraquat metabolism.Results After paraquat poisoning, there was significantly attenuated tissue damage in the lungs and kidneys and decreased TNF-α,IL-6 and IL-1 beta expression in the PBMCs or in the serum from Tim-3 transgenic mice compared to wild type mice.The serum concentration of paraquat in Tim-3 transgenic mice was also significantly decreased.However, in sTim-3 treated paraquat poisoning mice, there was significantly increased cytokine expression and tissue damage compared to control protein treated mice.The in vitro data showed that Tim-3 signaling negatively regulated macrophages mediated inflammatory response.Conclusion Tim-3 plays a critical role in maintaining the homeostasis after paraquat poisoning. Further investigation on the regulatory roles of Tim-3 in inflammation will shed new light on the pathogenesis of paraquat poisoning and provide new therapeutic strategies.

5.
Military Medical Sciences ; (12): 974-977,1012, 2016.
Article in Chinese | WPRIM | ID: wpr-606208

ABSTRACT

Objective To clone and express the alkaline protease AprA , one important virulence factor secreted by Pseudomonas aeruginosa(PAE)in Escherichia coli, to clone and express the inhibitor of AprA (AprI) and its substrate flagellin , and to detect the function of AprA and the inhibitory function of AprI .Methods The genes encoding AprA ,AprI and flagellin gene were amplified respectively by PCR using PAE PAO 1 genome DNA as the template .The expression vec-tors (pET-28a-AprA, pET-28a-AprI and pET-28a-Flagellin) were constructed and transformed into E.coli BL21(DE3) respectively.The recombinant AprA protein was expressed by IPTG induction and purified via denaturing and renaturation. The recombinant AprI and flagellin were expressed and purified by Ni 2+affinity chromatography .The cleavage activities of AprA on flagellin were detected by SDS-PAGE.Results Recombinant AprA , AprI and flagellin protein were expressed and purified .It was demonstrated that AprA cleaved flagellin , which was blocked by AprI .Conclusion Recombinant AprA could cleave its substrates as an alkaline protease , and its inhibitor AprI inhibits the activities of AprA .This study will contribute to further investigations on the role of AprA in the pathogenesis of PAE .

6.
Military Medical Sciences ; (12): 321-324, 2015.
Article in Chinese | WPRIM | ID: wpr-463524

ABSTRACT

Objective To explore the signal transduction mechanism of inhibitor kappa B kinase α( IKKα) , one of the catalytic subunits of IKK complex , for regulating p53 transactivation in the cellular ultraviolet radiation ( UVB) repsonse. Methods The transactivation of p53 was determined by dual-luciferase reporter gene analysis system while the expression and activation of IKKα, IKKβ, p53 and p38K was detected by Western blotting assay .Results UVB exposure induced activation and transactivation of p 53 in the wild type mouse fibroblasts ,but the effect was blocked by IKKa deficiency and recovered by reconstitution of IKKαexpression.Under the same conditions , IKKαregulated p38K activation, while inhibi-ting p38K activation down-regulated p53 transactivation under UVB exposure .Conclusion IKKαregulates UVB-induced phosphorylation and activation of p 53 in a p38K-dependent manner .

7.
Article in Chinese | WPRIM | ID: wpr-464065

ABSTRACT

Objective To investigate the self-assembly and cellular localization patterns of fila-mentous temperature-sensitive protein Z (FtsZ) in Escherichia coli (E.coli) strains by using FtsZP74R, FtsZG77D and FtsZA81R mutants.Methods YFP or His labeled FtsZ proteins and the plasmids of FtsZ mu-tants were constructed by using molecular clone and site-directed mutagenesis methods.The targeted proteins were purified by affinity chromatography.FL37(△ftsZ-Cat) strains were constructed via linear DNA homol-ogous recombination.Living cell imaging was performed to observe the cellular localization patterns of FtsZ protein and its mutants in E.coli strains.The interactions between FtsZ-FtsZ/FtsZ mutants were examined by coi-mmunoprecipitation assay . The polymerization properties of FtsZ mutants were analyzed by light scattering.The activities of GTPase were monitored by using high performance liquid chromatography.Re-sults The P74, G77 and A81 amino acids were respectively replaced by different polar amino acids to change the amphipathicity of the helix within the domain of FtsZ ( 74-82 ) .The YFP-labeled FtsZP74R , FtsZG77D and FtsZA81R mutants failed to assemble into functional Z-ring structure in E.coli strains.The inter-actions between FtsZ protein and its mutants were weakened or completely disappeared.In addition, in vitro experiments showed that P74R, G77D and A81R mutations caused a decrease in the polymerization efficien-cy of FtsZ monomer.The activity of GTPase was significantly decreased in the FtsZA81R mutant. Conclusion The P74, G77 and A81 were critical amino acids in the function and assembly of FtsZ protein in E.coli strains.Moreover, A81 amino acid regulated the lateral interaction of FtsZ monomer and the activity of GTPase.

8.
Article in Chinese | WPRIM | ID: wpr-464571

ABSTRACT

PD-1/PD-L1 signaling pathway as a T cell immune response co-stimulatory signaling pathway plays an important role in adaptive immunity. PD-1 is a major co-receptor expressing on T cells, binding with its ligands(PD-L1 and PD-L2), PD-1 can inhibit T cell activation and protect the body against the attacks from its own immune system. In addition to adjusting and maintaining autoimmune tolerance, in tumor cells PD-L1 expression is up-regulated, while in the virus-infected T cells PD-L1 expression is also upregulated. PD-1 / PD-L1 are involved in the tumor and infectious pathogen immune evasion, thus blocking the PD-1 / PD-L1 signaling pathway has become a hot research of cancer and chronic diseases. Currently, there are several anti-PD-1 or PD-L1 monoclonal antibodies approved by the FDA to enter clinical studies, which have shown significant anti-cancer effect.

9.
Chinese Journal of Pathophysiology ; (12): 1699-1703, 2015.
Article in Chinese | WPRIM | ID: wpr-479281

ABSTRACT

AIM:To clarify the impact of heart shock factor 4b (Hsf4b) K65R mutation on the regulation of downstream protein expression .METHODS:Non-functional Lys mutant plasmid pWZL-blast-HA-Hsf4b/K65R was genera-ted by replacing single , homologous amino acids using KOD-Plus-Mutagenesis-Kit.Mouse lens epithelial mLEC stable cell lines expressing Hsf4b or Hsf4b/K65R were constructed by lentivirus infection .The expression of Hsf4b in the mutant and the wildtype mLEC cells was confirmed by Western blotting .The expression of Hsf4b downstream proteins such as heat shock protein ( Hsp)70, Hsp90, Hsp27 and CryAB was examined by Western blotting and real-time PCR.RESULTS:The results of PCR and DNA sequencing confirmed the successful construction of mLEC Hsf 4b/K65R mutant.The K65R mutation didn’t influence Hsf4b expression in the mLEC cells.After K65R mutation in Hsf4b, the expression levels of Hsp27 and CryAB were down-regulated and the expression of Hsp 70i and Hsp90a upregulated.CONCLUSION: pWZL-blast-HA-Hsf4b/K65R can be used to construct a stable cell line by infecting with lentivirus .Hsf4b/K65R mutation influ-ences the regulation of downstream heat shock proteins .

10.
Chinese Journal of Immunology ; (12): 1601-1605, 2014.
Article in Chinese | WPRIM | ID: wpr-457510

ABSTRACT

Objective:To explore the regulatory effect of Hsf 1 on PLC/PRF5 hepatoma cells proliferation.Methods: By shRNA gene silencing technology ,constructed PLC/PRF5 hepatoma cell line of Hsf 1 gene silencing.To detect the expression of Hsf 1, p53 and Rb proteins in PLC/PRF5 hepatoma cells by Western blot.The proliferation of PLC/PRF5 cell line was observed by methylthiazolyl tetrazolium assay ( MTT ) , plate clone formation assay ( PCFA ) and cell cycle assay.Results: shRNA-Hsf1 could significantly inhibit the expression of Hsf 1 in PLC/PRF5 cells.It could induce PLC/PRF5 cells stopping at G1 phase of cell cycle , inhibit cell proliferation and colonal formation;silencing Hsf1 caused up-regulation of p53 and Rb proteins expression in PLC/PRF5 cells.Conclusion: Silencing Hsf1 is involved in up-regulation of p53 and Rb proteins expression , which results in inhibiting proliferation of PLC/PRF5 hepatoma cells.

11.
Chinese Journal of Immunology ; (12): 1374-1377,1392, 2014.
Article in Chinese | WPRIM | ID: wpr-600255

ABSTRACT

Objective:In order to get recombinant protein OCILRP 2-Fc and anti-OCILRP2 antibody for further study of OCILRP2.Methods:Eukaryotic expression vector pIg-CD5-OCILRP2 which fused with extracellular domain of OCILRP 2 and human IgG1 Fc fragment was constructed.G418 was used for stable expression cell strain after pIg-CD5-OCILRP2 transfected into CHO cells.Recombinant protein OCILRP 2-Fc purified from CHO cell supernatant was used to immunize rabbit and anti-OCILRP2 polyclonal antibody was purified from rabbit serum by using protein G column.Results: ELISA data showed that we got a high-titer anti-serum and anti-OCILRP2 antibody purified from the rabbit serum.Western blot indicated this antibody could specifically bind to OCILRP 2-Fc and OCILRP2 in NIH/3T3 lysate.OCILRP2 expression in murine bone marrow derived dendritic cells ( DC) was detected by this polyclonal antibody ,too.Compared to immature DC ,OCILRP2 expression was elevated in LPS induced-mature DC.Conclusion: This study has offered an available tool and provided a clue for further study of the roles of OCILRP 2 in immune response.

12.
Chinese Journal of Immunology ; (12): 1330-1333, 2014.
Article in Chinese | WPRIM | ID: wpr-475309

ABSTRACT

Objective:To determine the correlation of BCG concentration in Freund complete adjuvant ( FCA) and severity of arthritis during arthritis establishment in DBA 1/j male mice induced by bovine type Ⅱcollagen.Methods:CIA was induced by the im-munization of DBA1/j mice with bovine type Ⅱcollagen and BCG of various concentrations dissolved in FCA.To ascertain the effects administering the collagen booster,CIA-related features(including body weight,clinical scoring of arthritis),TNF-αin serum and the histopathological changes in the spleen and the joints regions were measured.Results:4 mg/ml BCG induced more serious arthritis than 1 mg/ml in DBA1/j mice after collagen exposure.Conclusion:There is a positive correlation of arthritis severity with BCG.Which indicates that selection of adjuvant with suitable BCG concentration could determine pathological outcome in CIA mouse model .

13.
Article in Chinese | WPRIM | ID: wpr-454446

ABSTRACT

Objecitv e To investigate the effects of a recombinant protein osteoclast inhibitory lectin related protein 2( OCILRP2)-Fc on LPS-induced endotoxemia by blocking OCILRP 2 signaling pathway and to in-vestigate the roles of OCILRP2 during inflammation.Methods Real-time PCR was used to detect OCILRP2 ex-pression at mRNA level in RAW264.7cells before and after in vitro stimulation with LPS.A mouse model of en-dotoxemia was established by intraperitoneal injection of BALB /c mice with a median lethal dose of LPS .Two hours prior to LPS treatment, mice were intraperitoneally injected with OCILRP2-Fc, human IgG or PBS, re-spectively .Several parameters including the survival rate of BALB/c mice with and without LPS treatment , spleen weight for arterial hyperemia analyzing , histopathological changes of lung and liver by HE staining , serum levels of inflammatory cytokines (IL-6, IL-12, TNF-αand IFN-γ)by ELISA , NF-κB activity by Western blot, were analyzed .Results Real-time PCR showed that LPS elevated in vitro OCILRP2 expression at mRNA level in macrophages (P<0.05).Upon the treatment of OCILRP2-Fc, BALB/c mice suffered from endotoxemia showed obviously increased survival rate , decreased spleen hyperemia , attenuated pathological injury of lung and liver, reduced levels of IL-6, IL-12, TNF-αand IFN-γin serum samples (P <0.05) as compared with mice treated with human IgG and PBS .LPS induced NF-κB p65 phosphorylation and IκB degradation were inhibited by OCILRP2-Fc treatment.Conclusion OCILRP2-Fc protects mice from endotoxemia by blocking OCILRP 2 signaling, which suggests that OCILRP2 plays an important role in LPS induced inflammation.

14.
Acta Anatomica Sinica ; (6): 493-499, 2014.
Article in Chinese | WPRIM | ID: wpr-455028

ABSTRACT

Objective To identify differentially expressed N-linked glycoproteins between hepatocellular carcinoma ( HCC) and adjacent non-tumorous liver tissues .Methods N-linked glycoproteome was extracted by multi-lectin affinity chromatography comprising concanavalin A (ConA), lentil lectin (LCH), and snowdrop lectin (GNA) and subsequently subjected to two-dimensional electrophoresis ( 2DE ) and mass spectrometry ( MS ) for identification of differential glycoproteins between 10 pairs of HCC and adjacent non-cancer tissue .Western blotting was used to verify different expression of human liver carboxylesterase 1 (hCE1), haptoglobin (HP)and cathepsin D (CD).Invasion potential in vitro was examined after si-RNA mediated CD gene scilencing .Results LC-ESI-MS/MS identified a total of 28 differentially expressed glycoproteins (14 up-regulation and 14 down-regulated).Western blotting detected consistent down-regulation of hCE1 and HP, and up-regulation of pro-cathepsin D (pCD) in HCC.Up-regulation of ConA-binding CD (ConA-CD), however , was verified in HCC only after ConA-CD enrichment by ConA chromatography .Down-regulation of CD expression mediated by CD-siRNA markedly inhibited the in vitro invasive potential of SNU449 and SNU473.Conclusion Dysregulation of HP , hCE1 expression and alteration of glycans linked to CD may play crucial roles in pathogenesis of HCC.

15.
Article in Chinese | WPRIM | ID: wpr-432400

ABSTRACT

Objective To investigate the expression of TIPE2 in splenic lymphocytes derived from the mouse model of cattle collagen Ⅱ-induced rheumatoid arthritis (CIA) and from peripheral blood of patients with rheumatoid arthritis (RA) for its role in the pathogenesis of RA.Methods The CIA mouse model was established by immunization of DBA-1/J mice with cattle collagen Ⅱ.Peripheral blood lymphocytes were collected from RA patients and healthy donors.The TIPE2 mRNA and protein expression in splenocytes of CIA and control mice were measured by semi-quantitative RT-PCR and Western blot,respectively.In addition,fluorescence quantitative RT-PCR and Western blot were used to determine the TIPE2 mRNA and protein expression in peripheral blood lymphocytes derived from patients with RA and healthy donors.Results Both mRNA and protein expression of TIPE2 were higher in splenocytes of CIA mice and lymphocytes of peripheral blood from RA patients compared with corresponding controls.Furthermore,the mRNA and protein expression of TIPE2 increased significantly in patients with active RA.TIPE2 expression were positively correlated with DAS28 scores (P<0.001).Conclusion Our results suggest that TIPE2 plays a role in the RA pathogenesis.The level of TIPE2 may also indicate the severity of rheumatoid arthritis.

16.
Article in Chinese | WPRIM | ID: wpr-426290

ABSTRACT

ObjectiveTo investigate the expression and clinical significance of heat shock transcription factor 1 (HSF1) protein in human hepatocellular carcinoma (HCC) tissues,and deduce the probable molecular mechanism of HSF1 in the development and advancement of HCC.MethodsSixty-seven samples of HCC tissue and 21 samples of normal liver tissue were obtained from March 2006 to March 2007 at the Xijing Hospital.The expressions of HSF1 protein and heat shock protein 70 (HSP70) were detected by using immunohistochemistry.The probable molecular mechanism of HSF1 in the development and advancement of HCC was deduced according to the relationship between the expressions of HSF1 protein and HSP70.Positive rates of HSF1 protein in different tissues and the relationship between HSF1 protein expression in the HCC tissues and clinical pathological factors were analyzed by the chi-square test and by calculating Fisher exact probability,respectively.The correlation between the expressions of HSF1 protein and HSP70 in the HCC tissue was analyzed by the Spearman correlation coefficient.The survival curve was drawn by the Kaplan-Meier method,and the survival rate was analyzed by the Log-rank test.ResultsThe positive rates of HSF1 protein expression was 69% (46/67) in the HCC tissue,which was significantly higher than 29% (6/21) in the normal liver tissue ( x2 =10.628,P < 0.05 ),The positive rates of HSP70 expression in the HCC tissue was 57% (38/67),which was significantly higher than 24% (5/21) in the normal liver tissue ( x2 =6.929,P < 0.05 ).The expression of HSF1 protein in the HCC tissue was positively correlated with that of HSP70 (r=0.319,P <0.05).The high expression of HSF1 protein was correlated with the integrity of capsule of HCC,tumor differentiation and TNM stage (x2 =5.935,9.762,5.159,11.267,P<0.05 ),while the high expression of HSF1 protein was not correlated with the gender,age,levels of hepatitis B surface antigen and alpha fetoprotein,and portal vein tumor thrombus ( x2 =0.822,0.172,2.059,P >0.05 ).The survival time was (21.4 ± 1.9 )months for patients with positive HSF1 protein expression and (29.8 ± 2.7 ) months for patients with negative HSF1 protein expression.There was a significant difference in the survival time between patients with positive and negative HSF1 protein expression ( x2 =4.276,P < 0.05 ).Conclusions HSF1 is correlated with the development,advancement,invasion,metastasis and malignant prognosis of HCC.HSF1 takes effects by regulating the expression of HSP70,and it has a good perspective of clinical application for the diagnosis and treatment of HCC.

17.
Acta Pharmaceutica Sinica ; (12): 409-16, 2012.
Article in Chinese | WPRIM | ID: wpr-415071

ABSTRACT

Multidrug resistance (MDR) is one of the main causes leading to the failure in cancer treatment. Differential proteins between esophageal squamous cell carcinoma (ESCC) cell line EC9706 and its cisdiamminedichloroplatinum (CDDP)-resistant subline EC9706/CDDP revealed by quantitative analysis may provide deeper insights into the molecular mechanisms of MDR implicated in ESCC. EC9706/CDDP was generated by exposure of its parental sensitive EC9706 to a step-wise increase of CDDP concentration during EC9706 cultivation. The stable isotope labeling with amino acids in cell culture (SILAC) was used to label EC9706 and EC9706/CDDP with heavy and light medium, separately. Mixed peptides derived from EC9706 and EC9706/CDDP were analyzed by high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS/MS) and subsequently subjected to bioinformatics analysis to identify differential proteins between EC9706 and EC9706/CDDP. Compared to parental EC9706, EC9706/CDDP manifested phenotypes of slow proliferation, cell pleomorphology, atypia and increased resistant-index 3.23. Seventy-four differential proteins identified in the present study belongs to various families with multiple functions, such as cytoskeleton (20%), energy metabolism (11%), transcription regulation and DNA repair (11%), redox homeostasis (9.5%), protein biosynthesis and mRNA processing (12%), ribosome constituent (8.1%), molecular chaperone (8.1%), immunity/inflammation (5.4%), intracellular transport (5.4%) and nucleosome assembly (2.7%), which indicated that development of MDR is a complicated process involving dysregulation of multiple molecules and pathways. The data is of great value for in-depth elucidation of molecular mechanisms of the MDR implicated in ESCC and may represent potential molecular targets for future therapeutic development.

18.
Article in Chinese | WPRIM | ID: wpr-404158

ABSTRACT

Objective:To investigate mitochondrial-dependent molecular mechanisms of a novel agonistic anti-human death receptor 5 (DR5) monoclonal antibody(mDRA-6) inducing apoptosis in Jurkat cell.Methods:The dose-dependent and time-dependent cell growth suppression of mDRA-6 in Jurkat cells was determined by MTT assay.The measurement of the mitochondrial transmembrane potential(ΔΨm) of Jurkat cells was detected by flow cytometry with JC-1 single staining.Caspase-8,9 as well as Bid,Bax,Bcl-2 and Cyto c of apoptotic Jurkat cells were analyzed by Western blot after mDRA-6 treatment.Results:The mDRA-6 induced cell growth suppression and cytotoxicity in dose-dependent manner and time-dependent manner.After mDRA-6 treatment at 2.0 μg/ml for15 min,30 min,60 min and 120 min,the change in ΔΨm were 20.14 %,19.34 %,21.11% and 30.90% respectively by JC-1 single staining.Western blot revealed that the level of active fragments of Caspase-8,9 and Bid,Bax,Bcl-2 and Cyto c respectively,and the amount of Cyto c was increased in cytosol concomitant with the related attenuation of Cyto c in mitochondria.Conclusion: Apoptotic pathway of Jurkat cells induced by mDRA-6 is initiated upon DR5 ligatian to mDRA-6 and exogenic Caspase-dependent cell apoptotic cascades is activated,and endogenic mitochondrial-dependent cell apoptosis pathway is activated.mDRA-6 may be a useful agent in investigating human leukemia therapy by using TRAIL/DR5.

19.
Article in Chinese | WPRIM | ID: wpr-381236

ABSTRACT

Objective To explore the effect of TNF related apoptosis inducing ligand (TRAIL) in apoptosis induced by LPS. Methods After LPS injected mice blocking TRAIL with soluble death receptor 5 (sDRS), detecting ALT, AST and LDH of mice serum at different times, apoptotic effects of LPS to mice hepatocyte were detected by HE and flow eytometry (FCM) with Annexin V-FITC/PI staining. The expres-sion of DR5 in mice hepatocyte was assayed with immunohistochemistry and FCM. Results Apoptotic effect was promoted by up-regulated DR5 expression on hepatocyte. Blocking TRAIL with sDR5 markedly amelio-rated the hepatocyte damage and reduced apoptosis. Conclusion These results establish a critical role for TRAIL in apoptosis during disease process of LPS.

20.
Tumor ; (12): 611-615, 2009.
Article in Chinese | WPRIM | ID: wpr-434186

ABSTRACT

Objective:To identify differentially expressed proteins related with malignant transformation of esophageal squamous cell carcinoma (ESCC) using proteomic analysis. Methods:Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization timE-of flight mass spectrometry (MALDI-TOF-MS) in combination with protein database searching were used to determine and identify differentially expressed proteins in esophageal cancer cell lines (EC1, EC18, and EC109) and immortal cell line (NECA-E6E7-hTERT). Western blotting and immunocytochemistry were used to verify the differential expression of annexin 2 in esophageal cancer cell lines and immortal cell line (NECA-E6E7-hTERT). Real-time fluorogentic quantitative PCR(RFQ-PCR) was performed to analyze the expression level of annexin A2 mRNA.Results: A total of 15 differentially expressed proteins were identified with more than 5 folds difference. Among them three proteins were down-regulated and 12 proteins were up-regulated. Western blotting and immunocytochemical analysis verified the down-regulation of annexin A2 protein in ESCC cell lines. However, differential expression pattern of annexin A2 mRNA was not consistant with its protein expression in ESCC cell lines and immortal cell line (NECA-E6E7-hTERT). Conclusion:The findings provide important clues for identifying the candidate biomarkers for high-risk population screening and early diagnosis of ESCC. Post-translative regulation/modification contributes to the down-regulation of annexin A2 protein.

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