Objective: Hepatocellular carcinoma(HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples.Methods: Lectin affinity chromatography(LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography(LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins.Results: A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain(FGG), FOS-like antigen 2(FOSL2), and α-1, 6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B(MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples.Conclusion: A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC.
Objective:To develop a safe and efficient non-viral gene delivery vector. Methods:A new polymer,Tween 85-polye...
Jisheng XiaoXiaopin DuanQi YinHaijun YuYaping Li Center of PharmaceuticsShanghai Institute of Materia MedicaChinese Academy of SciencesShanghai 201203China
Objective To investigate the effects of microvesicles(MVs) derived from hypoxia/reoxygenation(H/R)-treated human umbilical vein endothelial cells(HUVECs) on endothelium-dependent relaxation of rat thoracic aortic rings.Methods H/R injury model was established to induce HUVECs to release H/R-EMVs.H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium.H/R-EMVs were characterized using 1 urn latex beads and anti-PE-CD144 by flow cytometry.Thoracic aortic rings of rats were incubated with 2.5,5,10,20 μg/ml H/R-EMVs derived from H/R-treated HUVECs for 4 hours,and their endothelium-dependent relaxation in response to acetylcholine(ACh) or endothelium-independent relaxation in response to sodium nitroprusside(SNP) was recorded in vitro.The nitric oxide(NO) production of ACh-treated thoracic aortic rings of rats was measured using Griess reagent.The expression of endothelial NO synthase(eNOS) and phosphorylated eNOS(p-eNOS,Ser-1177) in the thoracic aortic rings of rats was detected by Western blotting.Furthermore,the levels of SOD and MDA in H/R-EMVs-treated thoracic aortic rings of rats were measured using SOD and MDA kit.Results H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation.The membrane vesicles(< 1 urn) induced by H/R were CD144 positive.ACh-induced relaxation and NO production of rat thoracic aortic rings were impaired by H/R-EMVs treatment in a concentration-dependent manner(P<0.05,P<0.01).The expression of total eNOS(t-eNOS)was not affected by H/R-EMVs.However,the expression of p-eNOS decreased after treated with H/R-EMVs.The activity of SOD decreased and the level of MDA increased in H/R-EMVs treated rat thoracic aortic rings(P<0.01).Conclusion ACh induced endothelium-dependent relaxation of thoracic aortic rings of rats was impaired by H/R-EMVs in a concentration-dependent manner.The mechanisms included a decrease in NO production,p-eNOS expression and an increase in oxidative stress.
Objective To investigate the effects of endothelial microvesicles(EMVs) induced by calcium ionophore A23187 on H9c2 cardiomyocytes. Methods Human umbilical vein endothelial cells(HUVECs) were treated with 10 μmol/L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. EMVs were characterized using 1 and 2 μm latex beads and antiPE-CD144 antibody by flow cytometry. For functional research, EMVs at different concentrations were cocultured with H9c2 cardiomyocytes for 6 h. Cell viability of H9c2 cells and the activity of LDH leaked from H9c2 cells were tested by colorimetry. Moreover, apoptosis of H9c2 cells was observed through Hoechst 33258 staining and tested by FITC-Annexin V/PI double staining. Results EMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. We identified the membrane vesicles(< 1 μm) induced by A23187 were CD144 positive. In addition, the EMVs could significantly reduce the viability of H9c2 cells, and increase LDH leakage from H9c2 cells in a dose dependent manner(P<0.05). Condensed nuclei could be observed with the increasing concentrations of EMVs through Hoechst 33258 staining. Furthermore, increased apoptosis rates of H9c2 cells could be assessed through FITC-Annexin V/PI double staining by flow cytometry. Conclusion Microvesicles could be released from HUVECs after induced by A23187 through calcium influx, and these EMVs exerted a pro-apoptotic effect on H9c2 cells by induction of apoptosis.
Man SHANGQi ZHANGMeng-xiao ZHANGYao WANGYan CHENYan-na WUJun-qiuSONGMing-lin LIUYan-xia LIU
