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

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 effects of antisense epidermal growth factor receptor (EGF-R) oligodeoxynucleotides on ultraviolet-induced c-jun activity of keratinocytes after EGF-R oligodeoxynucleotides transfect to HaCaT in vitro.

METHODSc-jun DNA binding activity after ultraviolet-B (UVB) irradiation and EGF-R oligodeoxynucleotides transfection were determined with a highly sensitive and specific colorimetric method. After EGF-R oligodeoxynucleotides transfection, the mRNA level of EGF-R was detected by reverse transcription polymerase chain reaction method.

RESULTSCompared with control groups, c-jun activity increased significantly in UVB (10, 20, 30 mJ/cm2) irradiation groups (P < 0.05). EGF-R mRNA and c-jun activities induced by UVB were inhibited after the keratinocytes were transfected with EGF-R antisense oligodeoxynucleotides at 2, 4 and 8 microg/ml concentrations (P < 0.01).

CONCLUSIONThe ultraviolet-induced c-jun activity of keratinocytes can be mediated by EGF-R and inhibited by EGF-R antisense oligodeoxynucleotides, which is transfected to keratinocytes and mediated by lipofectamine.

Cell Line ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Keratinocytes ; drug effects ; metabolism ; radiation effects ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; Transfection ; Ultraviolet Rays

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

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

OBJECTIVETo investigate the effect of hTERT antisense oligodeoxynucleotide (ASODN) on the oncogenicity and the inductive apoptosis of HL-60 cells.

METHODSApoptosis of HL-60 cells was detected by flow cytometry (FCM) and agarose gel electrophoresis. Both treated and untreated HL-60 cells were collected and transplanted into 5 BALB/c nude mice respectively, the formation of transplanted neoplasm and its morphologic change were observed. After the transplanted neoplasms were uniform with the ameliorated method in another 10 BALB/c nude mice, they were divided into 2 groups and injected ASODN and PBS into the neoplasm respectively. Seven days later, the tumor were measured, its morphology were observed, and the apoptotic cells were detected with a TUNEL kit.

RESULTSAfter 72 h treatment there were DNA ladders and early apoptosis peak in hTERT ASODN treated HL-60 cells but was none in SODN treated and blank control cells. In tumor formation experiment, neoplasms were formed in ASODN treated group at 16-17 d and untreated group at 12-13 d. Neoplasm was formed in 2 of 5 ASODN treated mice and 4 of 5 untreated mice respectively. In untreated mice tumor tissues were rich in blood vasa and stromal tissue compared with that in ASODN treated mice. In tumor therapy experiment, before treatment, there was no difference in the average neoplasm physical volume between ASODN treated group [(100.9 +/- 24.6) mm3] and PBS treated group [(98.4 +/- 23.1) mm3] (P > 0.05). After treatment, the neoplasm volume in ASODN treated group [(422.7 +/- 326.4) mm3] was smaller than that in PBS treated group [(786.4 +/- 357.6) mm3] (P < 0.05). Histologically, there were many apoptosis cells in ASODN treated group, but was seldom seen in PBS treated group. The TUNEL positive cells in ASODN treated group were much more than that in PBS treated group (P < 0.05).

CONCLUSIONThe hTERT ASODN induces apoptosis of HL-60 cells in vitro, reduces the tumor formation in BALB/c nude mice and inhibits the growth of the transplanted neoplasm.

Animals ; Apoptosis ; drug effects ; HL-60 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Telomerase ; genetics ; Transfection ; Xenograft Model Antitumor Assays

OBJECTIVETo observe the role of corticotropin releasing factor receptor 2 antisense oligodeoxynucleotide (CRFR2ASO) of hypothalamus in hypermetabolism in rats with severe burn.

METHODSStainless-steel cannula were implanted into the 3rd ventricle. According to different medicine delivered into the 3rd ventricle, 30 SD rats with 30% TBSA full-thickness burn were divided randomly into burn control group (BC, with injection of 3 microL saline), CRFR1ODN group (with injection of CRFR1ODN 10 microg), CRFR1ASO group (with injection of CRFR1ASO 10 microg), CRFR2ODN group(with injection of CRFR2ODN 10 microg), CRFR2ASO group (with injection of CRFR2ASO 10 microg), with 6 rats in each group. Another 6 rats served as normal control (NC) and they received isotonic saline 3 microL instead. Different medicines were respectively delivered into respective group on 5, 6 post injury day (PID), then 3 microL gentian violet was introduced on 7 PID. Resting energy expenditure (REE) value and the expression level of hypothalamus CRFR2mRNA and CRFR2 protein were determined.

RESULTSREE value in BC, CRFR1ODN, CRFR1ASO, CRFR2ODN, CRFR2ASO groups was 11 840 +/- 987, 11 133 +/- 1100, 10 733 +/- 1338, 11 123 +/- 1321, 7563 +/- 890 kJx(m2)(-1)xd(-1), respectively, which were obviously higher than that in BC group [6641 +/- 526 kJx(m2)(-1)xd(-1), P < 0.05]. REE value in CRFR2ASO group was obviously lower than that in CRFR2ODN group (P < 0.01). The expression level of hypothalamus CRFR2 mRNA and its protein in BC group were increased after burn, which were obviously lower in CRFR2ASO group than NC group (P < 0.01).

CONCLUSIONCentral application of CRFR2ASO can downregulate the expression level of hypothalamus CRFR2 mRNA and its protein, and reduce hypermetabolism. Hypothalamus CRFR2 may mediate hypermetabolism in burn rats.

Animals ; Burns ; metabolism ; Hypothalamus ; metabolism ; Male ; Oligodeoxyribonucleotides, Antisense ; metabolism ; RNA, Messenger ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Corticotropin-Releasing Hormone ; genetics ; metabolism