By using decoy-oligodeoxynucleotides (decoy-ODNS) technique, the effects of Stathmin gene on the proliferation and differentiation of in vitro cultured precartilainous stem cells (PSCs) were investigated. The Stathmin decoy-ODNs were transfected into PSCs in rats by using gene transfection technique. Under the induction of cortisol (1 micromol/L), electrophoretic mobility shift assay was used the inhibitory effects of decoy-ODNS on Stathmin gene. MTT and cytometry were used to test the cell proliferation. The expression of collagen II and V and Stathmin protein was detected by using Western blot. The results showed that Stathmin decoy-ODNs inhibited the Stathmin activity in a dose-dependent manner. When the concentration of decoy-ODNs was 10 times of standard concentration, the proliferation of PSCs was obviously suppressed and the differentiation happened. Compared to the control group, the difference was significant (P<0.05). It was concluded that decoy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.
Cartilage/*cytology ; Cell Differentiation/*drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Oligodeoxyribonucleotides/genetics ; Oligodeoxyribonucleotides/*pharmacology ; Rats, Sprague-Dawley ; Stathmin/*genetics ; Stathmin/pharmacology ; Stem Cells/*cytology

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

OBJECTIVE: To study the effects of oligodeoxynucleotides complementary Stat3 on apoptosis in laryngeal carcinoma Hep-2 cell. METHOD: Oligodeoxynucleotides complementary Stat3 was designed, which was transferred into laryngeal carcinoma Hep-2 cell by lipofection. Expression of Stat3 and p-Stat3 were detected by Western blot and PCR. MTT was used to observe the growth-inhibiting ratio. DNA ladder, AO/EB and FCM were used to observe the apoptosis of laryngeal carcinoma cell Hep-2 in vitro. RESULT: Western blot and PCR results demonstrated that oligodeoxynucleotides complementary Stat3 could significantly inhibit the expression of Stat3 and p-Stat3 in Hep-2 cell. MTT results showed that it could significantly suppress the growth of Hep-2 cell. The DNA ladder, AO/EB and FCM results showed it could inhibit the expression of Stat3 and induce the apoptosis of Hep-2 cell in a concentration-dependent manner. CONCLUSION Oligodeoxynucleotides complementary Stat3 could induce the apoptosis and suppress cell proliferation in laryngeal carcinoma Hep-2 cell.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Oligodeoxyribonucleotides ; genetics ; pharmacology ; STAT3 Transcription Factor ; genetics ; Transfection

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

OBJECTIVETo investigate the effect of antisense cDNA of cyclin D1 on the cyclin D1 gene expression and cell proliferation of human hepatocarcinoma HepG2 cells in vitro.

METHODSPlasmids containing cyclin D1 antisense cDNA were constructed and transfected into HepG2 cells. Their effects on cell proliferation were examined by MTT method, RT-PCR, immunohistochemical means, and flow cytometry.

RESULTSCyclin D1 antisense cDNA significantly inhibited the growth of HepG2 cells. The inhibition peaked at 48 hour after transfection by MTT method. RT-PCR analysis showed that cyclin D1 antisense cDNA down-regulated cyclin D1 at the mRNA levels. Expression level of cyclin D1 protein was also decreased as shown by immunohistochemical studies. Cell-cycle analysis by flow cytometry showed that transfected HepG2 cells were arrested at the G1 phase of the cell cycle.

CONCLUSIONSOur data suggest that cyclin D1 antisense cDNA could specifically inhibit the expression of cyclin D1 mRNA and protein and regulate cell cycle and cell proliferation of HepG2 cells. Cyclin D1 antisense cDNA may serve as a potential antitumor strategy in regulating cell-cyclin treating advanced HCCs.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cyclin D1 ; genetics ; Genes, bcl-1 ; genetics ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Oligodeoxyribonucleotides, Antisense ; 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 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

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

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 observe the ability of triple helix-forming oligonucleotides (TFO) modified with manganese porphyrin to combine with and cleave HBV DNA fractions.

METHODSThe ends of TFO were modified with manganese porphyrin and acridine; At 37 degrees C and pH 7.4 condition in vitro, TFO modified with manganese porphyrin and acridine were bound with 32P labeled HBV DNA fragments, the affinity and specificity binding to target sequence were tested by electrophoretic mobility shift and DNase 1 footprinting assays, the ability to cleave HBV DNA fractions was observed with cleavage experiments.

RESULTSTFO modified with manganese porphyrin and acridine could bind to target sequence in a sequence-dependent manner with Kd values of 3.5 x 10(-7) mol/L and a relative affinity of 0.008. In the presence of KHSO5, TFO modified with manganese porphyrin and acridine could cleave target sequence in the region forming triple DNA.

CONCLUSIONIn the presence of KHSO5, TFO modified with manganese porphyrin and acridine could cleave target HBV-DNA in sequence-dependent manner.

Binding, Competitive ; DNA Fingerprinting ; Deoxyribonuclease I ; metabolism ; Electrophoretic Mobility Shift Assay ; Hepatitis B virus ; genetics ; Manganese ; chemistry ; Metalloporphyrins ; chemistry ; Nucleic Acid Conformation ; Oligodeoxyribonucleotides ; chemistry ; genetics ; metabolism