OBJECTIVE:To establish two-dimensional electrophoresis (2-DE) pattern for proteome analysis on K562 cells before and after treatment with 6-gingerol, and mass-spectrometry was applied to identify and analyze the differentially expressed proteins.METHODS:K562 cells were treated with 6-gingerol. Cell proliferation was analyzed by CCK-8 assay. Total protein of K562 cells was extracted by 2D-DIGE and then imaged by SDS gel scanning. The differentially expressed proteins were identified using Imagine Master 2D Platinum 6.0 software and functionally classified by MALDI and MALDI-TOF MS.RESULTS:6-gingerol could significantly inhibit K562 cells proliferation and the efficacy was concentration and dose-dependent. After being treated for 24, 48, 72 h, IC50 was 22.86, 15.75, 11.18 microg/mL, respectively. 42 differentially expressed proteins were identified, including 19 up-regulated expressed proteins and 10 down-regulated expressed proteins.CONCLUSION:Proteomic technique can be used to screen multiple proteins associate with the anti-leukemia effect of 6-gingerol, involving some important proteins related to oxidative stress, cell cycle regulation, apoptosis signal transduction, biosynthesis and glycometabolism.

OBJECTIVE:To observe the effects of 6-gingerol on reactive oxygen species (ROS) and mitochondrial membrane potential(deltapsim) of chronic myeloid leukemia K562 cells and human acute T lymphoblastic leukemia MOLT4 cells, to investigate the role of mitochondrial pathway in the signal transduction of leukemia cell.METHODS:With different concentrations of 6-gingerol treatment, using 2,7-dichloro fluoresceinciactate (DCFH-DA) as ROS probe, rhodamine-123 as deltapsim probe, the levels of ROS and deltapsim of K562 cells and MOLT4 cells were tested by flow cytomentry.RESULTS:After treated with 6-gingerol, the ROS levels of K562 cells were significantly higher than control group (P < 0.01), while the deltapsim were significantly lower than control group (P < 0.01), and the ROS levels of MOLT4 cells were significantly higher than control group (P < 0.05).CONCLUSIONS:6-gingerol can significantly increase ROS levels of K562 cells and MOLT4 cells, decrease deltapsim of K562 cells,induce apoptosis of leukemia cells by mitochondrial pathway.

目的:研究生姜活性成分姜酚及其衍生物姜酚肟对慢性粒细胞系K562细胞的增殖活性、细胞周期及凋亡的影响.方法:采用CCK-8法检测姜酚和姜酚肟对K562细胞增殖活性的影响,PI单染检测其对K562细胞周期的影响,Annexin V-PI双染检测早期细胞凋亡,Hochest 33258染色观察细胞形态学的变化.结果:(1)姜酚作用于K562细胞48、72 h后,显著抑制K562细胞增殖,48 h的IC_(50)值为15.75 μg/mL,且具有时间及剂量依赖性.姜酚肟的抑制作用亦有时间及剂量依赖性,但较姜酚抑制作用弱.(2)姜酚15 μg/mL组细胞周期阻滞于S期,姜酚肟作用组细胞周期与对照组比较无明显差异(P＞0.05).(3)姜酚和姜酚肟作用48 h后出现早期凋亡群,姜酚各浓度组间的早期凋亡率差异显著(P＞0.05),随着药物浓度的增加凋亡率升高.(4)荧光显微间观察出现少量的细胞胞核固缩,核边集,出现凋亡小体.结论:姜酚和姜酚肟对K562细胞有显著的抑制作用,姜酚较姜酚肟抑制作用强,姜酚的抑制机制可能与影响细胞周期及诱导细胞凋亡有关,姜酚肟的抑制机制可能与诱导细胞凋亡有关.

Aim:Examining the expression patterns folate receptor in hematology malignant cell lines and synthesis a new potential targeted gene carrier vector folate-polyethylenimine. Methods: ①Real-time fluorescence quantitative PCR was used to detect the expression patterns of folate receptor in three kinds of hematology tumor cell lines.② The activated folate(N-hydroxysuccinimide ester of folic acid) was conjuated to the surface of PEI via the branched amino groups.Then the FA-PEI copolymer was purified with Sephadex G-25 column and completely separated from unreacted folate.Folate-polyethylenimine was determined by ultraviolet(UV) spectrophotometric analysis and hydrogen nuclear magnetic resonance spectroscopy(1H NMR).Results: ①Three patterns of folate receptor α、β、γ were expressed in hematology tumor cell lines K562、K562/A02、U266 and the expression levels of α-FR was significantly higher than those of β-FR and γ-FR.Thus the expression of α-FR was dominant.② Ultraviolet(UV) spectrophotometric analysis found that the isolated sample displays the characteristic absorption peak of folate in 365 nm.③1HNMR test showed that the characteristic chemical shifts of methylene proton was in 2.5～3.2 and the characteristic aromatic hydrogen proton signal of folic acid was in 6.5～9.0.Conclusions:① the expression levels of α-FR was higher than those of β-FR and γ-FR in the three hematology tumor cell lines.②The successful coupling of FA-PEI can be used as a potential targeted gene delivery vector and providing evidence in the treatment of hematology tumor.

BACKGROUND: CpG oligodeoxynucleotide (ODN) is a type of highly effective immune adjuvant with low toxicity, which has an extensive application in gene therapy for many diseases. However, the specificity for species and cells leading to low uptake by cells and degradation by nuclease blocks its clinical application. OBJECTIVE: To explore the specific delivery and its immunologic efficacy of CpG ODN targeting B lymphocytes of umbilical cord blood by CD40 ligand-receptor-mediated carrier system. DESIGN, TIME AND SETTING: An observation and control experiment was performed at the Department of Hematology, and Department of Pediatric, the First Affiliated Hospital of Jinan University from April 2004 to October 2007. MATERIALS: Fresh umbilical cord blood with heparin was obtained from healthy, natal infant. Informed consent was obtained from his parents, and the experiment was approved by the hospital Ethics Committee. METHODS: CD40 ligand (CD40L)-EDC-PLL-CpG ODN conjugated complex was prepared. Mononuclear cells (MNCs) from umbilical cord blood were co-cultured with conjugated complexes. Uptake rate, mean fluorescence intensity of FAM marked CpG ODN, expressions of MNCs, proliferations of lymphocytes and the IgG levels of culture supematants were detected by flow cytometry, fluorescence techniques, MTT assay and ELISA, respectively. MAIN OUTCOME MEASURES: The uptake rate, the mean fluorescence intensity of CpG ODN by MNCs, subgroups and proliferations of lymphocytes, and IgG levels of culture supematants. RESULTS: Compared to the pure CpG ODN group, the uptake rate of the conjugated complexes group was higher (98%), the peak level of up-taking occurred earlier, and intracellular fluorescence intensity maintained much more stable. Expressions of CD19+, CD22+, and CD20+ was increased, A value and IgG levels in supematants were all higher than that of the control group. CONCLUSION: CD40 tigand-receptor-mediated carrier system is helpful for CpG ODN delivery targeting to B lymphocyte, enhancing its immunological efficiency.

BACKGROUND: CpG oligodeoxynucleotide (ODN) is a type of highly effective immune adjuvant with low toxicity,which has an extensive application in gene therapy for many diseases. However,the specificity for species and cells leading to low uptake by cells and degradation by nuclease blocks its clinical application. OBJECTIVE: To explore the specific delivery and its immunologic efficacy of CpG ODN targeting B lymphocytes of umbilical cord blood by CD40 ligand-receptor-mediated carrier system. DESIGN,TIME AND SETTING: An observation and control experiment was performed at the Department of Hematology,and Department of Pediatric,the First Affiliated Hospital of Jinan University from April 2004 to October 2007. MATERIALS: Fresh umbilical cord blood with heparin was obtained from healthy,natal infant. Informed consent was obtained from his parents,and the experiment was approved by the hospital Ethics Committee. METHODS: CD40 ligand (CD40L)-EDC-PLL-CpG ODN conjugated complex was prepared. Mononuclear cells (MNCs) from umbilical cord blood were co-cultured with conjugated complexes. Uptake rate,mean fluorescence intensity of FAM marked CpG ODN,expressions of MNCs,proliferations of lymphocytes and the IgG levels of culture supernatants were detected by flow cytometry,fluorescence techniques,MTT assay and ELISA,respectively. MAIN OUTCOME MEASURES: The uptake rate,the mean fluorescence intensity of CpG ODN by MNCs,subgroups and proliferations of lymphocytes,and IgG levels of culture supernatants. RESULTS: Compared to the pure CpG ODN group,the uptake rate of the conjugated complexes group was higher (>98%),the peak level of up-taking occurred earlier,and intracellular fluorescence intensity maintained much more stable. Expressions of CD19+,CD22+,and CD20+ was increased,A value and IgG levels in supernatants were all higher than that of the control group. CONCLUSION: CD40 ligand-receptor-mediated carrier system is helpful for CpG ODN delivery targeting to B lymphocyte,enhancing its immunological efficiency.