OBJECTIVETo investigate the interference and the mechanisms of Bcl-2 and HER-2 genes antisense oligodeoxynucleotides combined transfection in the human tongue carcinoma Tca8113 cell lines.

METHODSThere were 6 groups in our study, normal control group, Bcl-2 sense experimental group, HER-2 sense experimental group, Bcl-2 antisense experimental group, HER-2 antisense experimental group, Bcl-2 and HER-2 genes antisense oligodeoxynucleotides combined transfection experimental group. In the different times after liposome-mediated transfection, the cell apoptosis, Bcl-2 and HER-2 expressing level were observed by RT-PCR and electronic microscope.

RESULTSAccording to the results of combined transfection experimental group, the apoptosis body and apoptosis cells were observed. The expression of genes were decreased statistically in both Bcl-2 and HER-2, respectively. Bcl-2 and HER-2 combined ASODN was superior to single ASODN in the intervention of tongue carcinoma.

CONCLUSIONBcl-2 and HER-2 ASODN can effectively interfere the expression of HER-2 and Bcl-2 genes. The expression of HER-2 and Bcl-2 can be reduced, and the apoptosis of cells can be enhanced.

Apoptosis ; Female ; Genes, erbB-2 ; Humans ; Oligodeoxyribonucleotides ; Oligodeoxyribonucleotides, Antisense ; Transfection

To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide (AS-ODNs) by insonated gas-filled lipid microbubbles, SF6-filled microbubbles were prepared by sonication-lyophilization method. An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles. Three levels of mixing speed, different durations of mixing and various delay time before ultrasound were examined, separately. Transfection efficiency was detected by fluorescence microscopy. Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared. From the results, there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells. AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration, but there is a negative relationship with delay time before ultrasound. The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r x min(-1) for 30-60 s with less than 60 s delay before ultrasound. For a successful transfection, long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids, because these vectors depend on endocytosis and membrane fusion to realize transfection. Unlike liposomes and cationic lipid-polymer hybrids, gas-filled lipid microbubbles depend on sonorporation effect to realize transfection. Therefore, the incubation of gene and microbubbles before mixing cells may not be necessary. Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.
Cell Line, Tumor ; Green Fluorescent Proteins ; Humans ; Microbubbles ; Oligodeoxyribonucleotides, Antisense ; genetics ; Sulfur Hexafluoride ; Transfection ; methods ; Ultrasonics

To study the effect of VEGF fully phosporothioated antisense oligodeoxynucleotide (VEGF-ASODN) on VEGF expression in acute monocyte leukemic cell line U937 in vitro, U937 cells were incubated with VEGF-ASODN at concentrations of 10, 20 and 30 micro mol/L or scrambled sequence as compared with negative control. The expression of VEGF mRNA was measured by semi-quantitative RT-PCR. The expression of VEGF protein was measured by Western blot. The result showed that VEGF-ASODN had obviously inhibitive effect on expression of VEGF in U937 cell, as compared with scrambled sequence and negative control (P < 0.05). Scrambled sequence group had no significant difference compared with negative control group (P > 0.05). It is concluded that the expressions of VEGF mRNA and protein in leukemic cell line U937 are down-regulated by VEGF-ASODN.
Humans ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; U937 Cells ; Vascular Endothelial Growth Factor A ; analysis ; antagonists & inhibitors ; genetics

Carcinoma, Hepatocellular ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Humans ; Liver Neoplasms ; genetics ; metabolism ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Tumor Cells, Cultured ; beta Catenin ; biosynthesis ; genetics

AIMTo investigate factors affecting the properties of antisense oligodeoxy nucleotides (ASON)-liposomes complex and their cellular uptake.

METHODSThree types of blank liposomes were prepared by reverse-phase evaporation vesicles, and the complex were obtained through physical absorption. The light microscope was used to observe morphology characteristics of the complex. Drug loading capacity was analyzed by agarose gel electrophoresis. The transfected cell percentage and means fluorescence intensity were determined by flow cytometric analysis using M3 myeloma cell as a model.

RESULTSThe neutral liposome showed no aggregation while the cationic liposomes appeared some different extent aggregation in different medium when associated antisense oligodeoxynucleotides. The drug loading capacity depended on the ratio of +/- and the cationic charge density on the lipid membrane. The two kinds of cationic liposomes appeared different principles of loading ASON. As far as cellular uptake, The neutral liposomes showed no improvement of cellular uptake of ASON. However, the cationic liposomes were shown to enhance the cellular uptake of ASON if the appropriate +/- charge ratio was used. The optimal cellular uptake was achieved when +/- charge ratio was at 0.5:1 and 1:1 for SA-I liposome and SA-II liposomes, respectively.

CONCLUSIONThe cationic liposomes improved the loading capacity and cell uptake of antisense oligodeoxynucleotides, which was determined by +/- charge ratio and charge density.

Amines ; metabolism ; Biological Transport ; Drug Carriers ; Drug Delivery Systems ; Humans ; Liposomes ; pharmacokinetics ; Multiple Myeloma ; Oligodeoxyribonucleotides, Antisense ; administration & dosage ; pharmacokinetics ; Random Allocation ; Tumor Cells, Cultured ; metabolism

OBJECTIVETo investigate the effect of S-phase kinase-associated protein 2 antisense oligodeoxynucleotide (Skp2 ASODN) on the growth and proliferation of gastric carcinoma SGC-7901 cells and its mechanism.

METHODSThe Skp2 oligodeoxynucleotides (ODNs) were embedded in cationic liposome Lipofectamine 2000 reagent and transfected into SGC-7901 cells. The cell growth and proliferation were observed with light microscopy and MTT assay. Cell cycle was measured by flow cytometry. The expression levels of Skp2 and p27 mRNA were detected by reverse transcription-polymerase chain reaction. The expression levels of Skp2 protein and its substrate p27 protein were detected by Western blot.

RESULTAfter treatment with Skp2 ASODN, the growth and proliferation of SGC-7901 cells were inhibited in a dose-dependent manner with a peak value at 48 h. The inhibition rate of 200 nmol/L group at 48 h was 42.4 % (P<0.01). In cell cycle study the percentage of S phase cells in 200 nmol/L group was significantly higher than that in normal control group (P<0.05). Both Skp2 mRNA and its protein levels in 200 nmol/L group were significantly lower than those in control group and in Skp2 nonsense oligodeoxynucleotide (Skp2 NSODN) group (P<0.05). However, p27 mRNA level remained unchanged although its protein level was significantly higher than that in control group and NSODN group (P<0.05).

CONCLUSIONSkp2 ASODN can inhibit the growth and proliferation of SGC-7901 cells, which may be mediated by interfering with ubiquitin-proteosome pathway and cell cycle regulation.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Humans ; Oligodeoxyribonucleotides, Antisense ; genetics ; pharmacology ; S-Phase Kinase-Associated Proteins ; genetics ; pharmacology ; Stomach Neoplasms ; pathology ; Transfection

To explore the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynucleotide (ASODN) on telomerase activity in primary acute myeloid leukemia (AML) cells and chronic myeloid leukemia (CML) cells, polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) was used to determine telomerase activity. The expression levels of hTERT protein were assayed by flow cytometry using immunofluorescence labeling. Immunofluorescence assay showed that the expression levels of hTERT protein in AML and CML cells decreased with time after hTERT ASODN treatment. There was no difference in hTERT protein levels between control and sense oligodeoxynucleotide (SODN)-treated cells. Telomerase activity decreased when AML and CML cells were treated with ASODN for 48 h. Telomerase activity of AML and CML cells was significantly inhibited when the cells treated with ASODN for 72 h. There was no difference in telomerase activity levels between control and hTERT SODN-treated cells. It was concluded that hTERT ASODN could inhibit hTERT protein expression level, thus decreasing the telomerase activity in primary AML and CML cells.
Adolescent ; Adult ; DNA-Binding Proteins ; Female ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; enzymology ; Leukemia, Myeloid, Acute ; enzymology ; Male ; Middle Aged ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Telomerase ; antagonists & inhibitors ; metabolism

OBJECTIVETo explore the effect of homeobox B2 (HOXB2) anti sense oligodeoxynucleotides (asodn) on the proliferation and expression of primary human umbilical vein endothelial cells (HUVECs).

METHODSVarious concentrations of HOXB2 asodn modified by thiophosphate transfected the induction of liposome into HUVECs. MTT a nd RT-PCR methods were employed to determine the effect of different conc ent rations of asodn on the endothelial proliferation and the expression level of HOXB2 mRNA.

RESULTSAfter the transfection of HOXB2 asodn, the endothelial proliferation was inhibited in a dose-dependent fashion. Simultaneously, the expression of HOXB2 mRNA decreased significantly.

CONCLUSIONSHOXB2 plays an important role in the proliferation of endothelia.

Cell Division ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Gene Expression ; Homeodomain Proteins ; genetics ; Humans ; Oligodeoxyribonucleotides, Antisense ; RNA, Messenger ; Transcription Factors ; genetics ; Umbilical Veins ; cytology

OBJECTIVETo study the effect of interleukin (IL)-18 ASPODN on regeneration of allogeneic partial liver graft in rats.

METHODSNinety donor SD rats and ninety recipient LEW rats were randomly divided into 3 groups: 50% partial liver transplantation group (PLT group); PLT+IL-18 antisense phosphorothioate oligodeoxynucleotide (ASPODN) treatment group (IL-18 ASPODN group) and PLT+IL-18 SPODN treatment group (IL-18 SPODN group) in which liposomes encapsulated IL-18 ASPODN or IL-18 SPODN were intravenous injection every day after PLT. BrdU labeling of hepatocytes, expression of IL-18 protein and IFN-gamma mRNA in liver graft, and serum level of IFN-gamma were measured with immunohistochemistry analysis, Western blotting, semi-quantification RT-PCR, and ELISA, respectively.

RESULTSAlthough regeneration of liver graft from each group peaked 72 hour after transplantation, BrdU labeling of hepatocytes in IL-18 ASPODN group (58.3%+/-7.5%) were significantly higher than those of PLT group (31.6%+/-6.7%) (t=6.503, P<0.001) and IL-18 SPODN group (33.4%+/-5.5%) (t=6.558, P<0.001). Expression of IL-18 protein and IFN-gamma mRNA in liver graft, and serum level of IFN-gamma in IL-18 ASPODN group from 48 hour, 72 hour and 96 hour after transplantation were significantly suppressed compared with PLT group (IL-18protein: t=2.950, t=5.916, t=7.947, P<0.05, P<0.001; INF-gamma mRNA: t=2.558, t=6.292, t=8.925, P<0.05, P<0.001; IFN-gamma level: t=16.998, t=15.483, t=54.723, P<0.001) and IL-18 SPODN group (IL-18 protein: t=2.845, t=6.062, t=6.973, P<0.05, P<0.001; INF-gamma mRNA: t=3.117, t=6.154, t=8.738, P<0.05, P<0.001; IFN-gamma level: t=14.531, t=18.139, t=46.924, P<0.001).

CONCLUSIONIL-18 ASPODN could promote hepatocyte regeneration of allogeneic partial liver graft by the suppression of IL-18 and IFN-gamma production.

Animals ; Hepatocytes ; physiology ; Interferon-gamma ; biosynthesis ; Interleukin-18 ; antagonists & inhibitors ; genetics ; Liver Regeneration ; Male ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Rats ; Rats, Inbred Lew ; Rats, Sprague-Dawley ; Transplantation, Homologous

Animals ; Drug Carriers ; Drug Delivery Systems ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; Maleates ; metabolism ; Molecular Weight ; Oligodeoxyribonucleotides, Antisense ; metabolism ; Polyvinyls ; metabolism ; Prodrugs ; metabolism ; Proteins ; metabolism