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

AIMTo investigate the feasibility of transfer antisense oligodeoxynucleotides (AS-ODNs) by the phospholipids-based gas-filled microbubbles (PGM) under ultrasound activation.

METHODSAn antisense oligodeoxynucleotides sequence ZL combined with luciferase reporter plasmid was used. A breast cancer cell line SK-BR-3 was exposed to different conditions to investigate the effects of such factors as ZL concentration, PGM concentration, mechanical index (MI) and ultrasound exposure duration on transfection efficiency and cell viability. The transfection efficiency and cell viability by other lipid vectors such as lipofectamine and liposome were also tested, whose results were comparied with that of PGM. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by PI (propidium iodide) assay.

RESULTSAmong the factors tested, ultrasound exposure duration, MI and PGM concentration had obvious impacts on transfection efficiency and cell viability. The results showed that the optimal ultrasound condition was the exposure to ultrasound at MI 1.0 for 30 s with 2% PGM concentration, which gave an overall transfection efficiency of 78% +/- 10%, increased nearly 18 folds over the transfection by PGM (4.0%) or lipofectamine (4.3%) without ultrasound. Under same ultrasound conditions, different vectors showed significant difference in transfection efficiency while there are similar results in cell viability.

CONCLUSIONUnder proper ultrasound conditions, PGM can markedly enhance AS-ODNs transfection efficiency.

Breast Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Cell Survival ; Drug Carriers ; Female ; Humans ; Lipids ; Liposomes ; Luciferases ; genetics ; metabolism ; Microbubbles ; Microscopy, Fluorescence ; Oligodeoxyribonucleotides, Antisense ; genetics ; Phospholipids ; Transfection ; methods ; Ultrasonics

To explore the effect of antisense phosphorothioate oligodeoxynucleotide (ASODN) of human telomerase reverse transcriptase (hTERT) gene on telomerase activity in CEM cells, PCR enzyme-linked immunoassay was used to determine telomerase activity. The expression levels of hTERT mRNA and protein were assayed by RT-PCR and immunofluorescence assay using fluoresce isothiocyanate label respectively. The results showed that the expression levels of hTERT mRNA and protein in CEM cells decreased with time after hTERT ASODN treatment. There was no difference in hTERT mRNA and protein levels between control and sense oligodeoxynucleotide-treated cells. Telomerase activity decreased when CEM cells were treated with ASODN for 48 hours. Telomerase activity of CEM cells was significantly inhibited when treated with ASODN for 72 hours. There was no difference in telomerase activity levels between control and hTERT sense oligodeoxynucleotide-treated cells. These results suggested that hTERT ASODN inhibited telomerase activity of CEM cells.
Cell Division ; drug effects ; Cell Line ; DNA-Binding Proteins ; Flow Cytometry ; Humans ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; Telomerase ; analysis ; genetics ; metabolism

The present study was carried out to investigate the effect of antisense c-myb oligodeoxynucleotides (ODN) on hCG-induced testosterone secretion in isolated rat Leydig cells. The effects of cAMP, Ca(2+) and cycloheximide (CYX) on c-Myb protein expression and testosterone secretion were also observed. The results showed that antisense c-myb ODN inhibited hCG-induced testosterone secretion of isolated rat Leydig cells in a dose-dependent manner. At the same time, integral optical density immunostaining of Myb in Leydig cells was also remarkably reduced. Nonsense tat ODN had no effect on Leydig cells. Further experiments showed that dbcAMP (100 micromol/L) obviously increased hCG-induced testosterone secretion and integral optical density (IOD) immunostaining of Myb in Leydig cells. Verapamil (10 micromol/L), a Ca(2+) channel blocker, and cycloheximide (50 microg/ml), a protein synthesis inhibitor, reduced the immunostaining of c-Myb, and also lowered hCG-induced testosterone secretion in isolated rat Leydig cells. The results indicate that c-myb closely correlates with hCG-induced testosterone secretion, and that cAMP and Ca(2+)-dependent pathway participates in the expression of protooncogene.
Animals ; Cell Separation ; Cells, Cultured ; Chorionic Gonadotropin ; pharmacology ; Leydig Cells ; secretion ; Male ; Oligodeoxyribonucleotides, Antisense ; physiology ; Proto-Oncogene Proteins c-myb ; physiology ; Rats ; Rats, Sprague-Dawley ; Testosterone ; secretion

OBJECTIVETo investigate the effect of hTERT antisense oligodeoxynucleotide on telomerase activity in bladder cancer cells.

METHODSAntisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by telomerase PCR ELISA kit. hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR), and hTERT protein by immunohistochemistry and flow cytometry.

RESULTSTelomerase activity was decreased in T24 cells 48 h after treatment with AS PS-ODN, and was significantly inhibited at 72 h.

CONCLUSIONAS PS-ODN can significantly inhibit telomerase activity by down-regulating hTERT mRNA and protein expression in bladder cancer cells.

Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Telomerase ; genetics ; metabolism ; Urinary Bladder Neoplasms ; enzymology ; pathology

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

OBJECTIVESTo study the inhibitory effects of mouse telomerase RNA (mTR) antisense oligodeoxynucleotide(ASODN) on telomerase activity in rat spermatogonia.

METHODS9-mer phosphorothioate mTR-ASODN was encapsulated by Lipofect AMINE 2000 (LF 2000) and transfected to type A spermatogonia in Snrague Dawley (SD) rat. Telomerase activity was detected by aid of TRAP-SYBR-Green staining and Bioluminescence technique in type A spermatogonia treated or untreated with ASODN.

RESULTSmTR-ASODN conjugated with LF 2000 could significantly inhibit telomerase activity of spermatogonia(P < 0.01). mTR mRNA level also decreased while the spermatogonia were treated with ASODN for 24 h. No change of telomerase activity and apoptosis were observed when SODN, RODN or single LF 2000 was used.

CONCLUSIONSAntisense oligodeoxynucleotide of mTR conjugated with LF 2000 could significantly inhibit telomerase activity of spermatogonia. mTR-ASODN might inhibit telomerase activity of spermatogonia at transcription level.

Animals ; Male ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA ; antagonists & inhibitors ; genetics ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Spermatogonia ; cytology ; enzymology ; ultrastructure ; Telomerase ; antagonists & inhibitors ; genetics ; 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