Objective To study the effects of glutaminase (GA) gene blocked by antisense nucleotide on apoptosis of transplanted gastric carcinoma cells in nude mice. Methods The plasmid containing antisense sequence of GA gene was trans-fected into gastric carcinoma cells , then the cells were injected to endermic tissue of nude mice to create animal models of gastric carcinoma. Apoptosis of tumor cells was detected by terminal deoxynucleotidyl transferase2mediated nick end labeling (TUNEL) method. The expression of GA mRNA in tumor tissue was measured by reverse transcription polymerase chain reaction (RT2PCR) technique. Results After the successful transfection of plasmid containing antisense sequence of GA gene into gastric carcinoma cells , the tumor’s growth speed decreased , apoptosis of tumor cells increased , and the expression of GA mRNA also decreased. Conclusion The antisense gene of GA could inhibit the expression of GA gene and significantly increase the apoptosis of gastric carcinoma cells.
Objective To evaluate the effect of vascular endothelial cell growth factor (VEGF) antisense oligodeoxynucleotides (ASODNs) on the expression of VEGF in rats with oxygen-induced retinopathy. Methods Thirty newborn Sprague-Dawley (SD) rats were randomly divided into 3 groups:normal control group, disposal group and non-disposed group, The animal models with oxygen-induced proliferative retinopathy were established by raising the rats in hyperoxic environment. Retrobulbar injection was performed with VEGF ASODNs or normal saline on the rats in 3 groups respectively. The intraocular tissues (all the tissues except the cornea, sclera, and lens) and serum were collected, and the expressions of VEGF were determined by using competitive enzyme immunoassay.Results The expressions of VEGF in intraocular tissues of rats in disposal group were significantly lower than those in non-disposed group (P＜0.05), and there was no statistical difference between the disposal and normal control group (P＞0.05). There was no significant difference of the expressions of VEGF in serum of rats between the disposal and non-disposed group (P＞0.05), which were both lower than those in the normal control group (P＜0.05). Conclusion VEGF ASODNs could significantly inhibit the expression of VEGF in intraocular tissues. (Chin J Ocul Fundus Dis，2003，19：172-174)
Objective To observe the effects of vascular endothelial growth factor antisense oligonucleotide (VEGF-ASODN) on expression of vascular endothelial growth factor (VEGF) and growth in gastric cancer cells. Methods The VEGF-ASODN was synthesized artificially with phosphorothioic acid. After transfecting with VEGF-ASODN in gastric cancer cells SGC-7901, the initial copy number of mRNA was detected by real-time RT-PCR, and the quantity of VEGF protein in both cell and supernatant were detected by ELISA. The levels of expression of survivin protein in cells were measured by Western blot. FCM and MTT method were used to detect cellular apoptosis and the activity of cells, respectively. The effect of transfection on the growth of cells was evaluated by growth curve. Results The copy number of VEGR mRNA, protein levels of VEGF in the cells and in culture fluid all decreased when the concentration of transfected VEGF-ASODN increased, as well as the levels of survivin protein （P＜0.05）. The ratio of apoptosis increased, the activity of cells also decreased as the concentration of transfected VEGF-ASODN increased （P＜0.05）. Conclusion Transfection with VEGF-ASODN in gastric cancer cells SGC-7901 can inhibit the expressions of VEGF and survivin remarkably. It can enhance cellular apoptosis and suppress growth of cells.
【Abstract】ObjectiveTo study the apoptosis of gallbladder carcinoma cell line GBCSD induced by antisense oligodeoxynucleotide (ASODN) targeting survivin. MethodsASODN targeting survivin was transfected into GBCSD cells mediated by lipofectin. Cultured cells were divided into 3 groups: control group,sense oligonucleotide (SODN) group and ASODN group. After transfected for 16 h, the cultured cells were harvested and the following texts were carried out. The expression of survivin mRNA was detected by RTPCR. Flow cytometer were used to detect apoptosis. Morphological changes were observed by electron microscopy. ResultsThe expression of survivin mRNA was decreased 47.83% in ASODN group while apoptosis was increased from (0.50±0.23)% to (26.28±3.91)%. Abnormal morphological changes of cells were observed in ASODN group and apoptosis bodies were found in some gallbladder carcinoma cells. ConclusionThe expression of survivin may be decreased in GBCSD cells after ASODN transfection.ASODN targeting survivin could induce gallbladder carcinoma cells apoptosis effectively.
Objective To investigate the reversal effect of antisense phosphorothioate oligonucleotide （ASOND) on human hepatoma resistant cells. Methods Human hepatoma resistant cells SMMC-7721 was transfected with synthetic antisense phosphorothioate oligonucleotide complementary to the 5′ region flanking the AUG initiation codon mediated by lipofectamine. In vitro drug sensitivity was measured by MTT assay. The expression of P-170 was determined by flow cytometry and mRNA was assessed by RT-PCR. Results ASOND inhibited the expression of mRNA and p-170 in SMMC-7721, enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug. The best inhibitory effect was achived by the dose of 0.5μmol/L. Conclusion ASOND enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug and reversed the multidrug resistance of SMMC-7721 partially.
To investigate the inhibitory effect of Col I A1 antisense ol igodeoxyneucleotide (ASODN) transfection mediated by cationic l iposome on Col I A1 expression in human hypertrophic scar fibroblasts. Methods Scar tissue was obtained from volunteer donor. Human hypertrophic scar fibroblasts were cultured by tissue block method. The cells at passage 4 were seeded in a 6 well cell culture plate at 32.25 × 104 cells/well, and then divided into 4 groups: group A, l iposomeand Col I A1 ASODN; group B, Col I A1 ASODN; group C, l iposome; group D, blank control. At 8 hours, 1, 2, 3 and 4 days after transfection, total RNA of the cells were extracted, the expression level of Col I A1 mRNA was detected by RT-PCR, the Col I A1 protein in ECM was extracted by pepsin-digestion method, its concentration was detected by ELISA method. Results Agarose gel electrophoresis detection of ampl ified products showed clear bands without occurrence of indistinct band, obvious primer dimmer and tailing phenomenon. Relative expression level of Col I A1 mRNA: at 8 hours after transfection, group A was less than groups B, C and D (P lt; 0.05), and groups B and C were less than group D (P lt; 0.05), and no significant difference was evident between group B and group C (Pgt; 0.05); at 1 day after transfection, groups A and B were less than groups C and D (P lt; 0.05), and there was no significant difference between group A and group B, and between group C and group D (P gt; 0.05 ); at 2 days after transfection, there were significant differences among four groups (P lt; 0.05); at 3 and 4 days after transfection, group A was less than groups B, C and D (P lt; 0.05), group B was less than groups C and D (P lt; 0.05), and no significant difference was evident between group C and group D (P gt; 0.05). Concentration of Col I protein: at 8 hours after transfection, group A was less than groups B, C and D (P lt; 0.05), groups B and C were less than group D (P lt; 0.05), and no significant difference was evident between group B and group C (P gt; 0.05); at 1 day after transfection, significant differences were evident among four groups (P lt; 0.05); at 2, 3 and 4 days after tranfection, groups A and B were less than groups C and D (P lt; 0.05), and no significant difference was evident between group A and group B (P gt; 0.05). Conclusion Col I A1 ASODN can inhibit mRNA and protein expression level of Col I A1. Cationic l iposome, as the carrier, can enhance the inhibition by facil itating the entry of ASODN into cells and introducing ASODN into cell nucleus.
【Abstract】Objective To construct a recombinant adenoviral vector carrying antisense matrix metalloproteinase2 (MMP2) for use in the gene therapy to inhibit the invasiveness and migratory capacity of hepatocellular carcinoma (HCC) cell line HepG2 in vitro and in vivo models. Methods Total RNA was extracted from HCC, and then a 500 bp fragment at the 5′ end of human MMP2 cDNA was synthesized by polymerase chain reaction (PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV,with the resultant plasmid and the backbone plasmid pAdEasy-1,the homologous recombination took place in the E.coli BJ5183 and the recombinant adenoviral plasmid carrying the antisense MMP2 gene was constructed and generated. The adenoviruses(Ad-MMP2AS) were packaged and amplified in the HEK 293 cells.Then the viral titer was checked by GFP. Results The recombinant adenovirus vector carrying antisense MMP2 was constructed successfully, the b green fluorescence was observed in HEK 293 cells under a fluorescence microscopy. The viral titer was 1×108/ml. Conclusion The recombinant adenovirus Ad-MMP2AS constructed by us could introduce the antisense MMP2 into HepG2 effectively,which would provide experimental basis for reversing the overexpression of MMP2 in HCC and for inhibiting the invasiveness and migratory capacity of HepG2 in vitro and in vivo models.
Objective To investigate the reversal of the multidrug resistant gene mdr1 in vivo by antisense oligodeoxynucleotide （ASODN） on the basis of study in vitro. Methods The cultured drug-resistant human hepatocellular carcinoma cells were injected under the skin of axilla to establish the tumor model of nude mice. mdr1 ASODN accompanied by Lipofectamine were injected locally and ADM was injected intraperitoneally. Control 1 and control 2 were locally injected by Lipofectamine and normal saline separately, and ADM was also injected intraperitoneally. Results As time went on the tumor size increased and from the 5th day on alterations were marked, tumor size in different time phase showed marked difference to the prior time phase with significant difference (P＜0.05）. Tumor size in group ASODN was marked smaller than that of other 3 groups after the 5th day (P＜0.05)，while tumor size of group control 1,2 and group SODN in different phase showed no significant difference (Pgt;0.05). The results suggested that SODN and Lipofectamine showed no marked effect on tumor growth of nude mice and ASODN had marked inhibition effect on tumor growth. Conclusion mdr1 ASODN can also reverse multidrug resistance of drug-resistant human hepatocellular carcinoma cells in vivo. After the treatment the tumor’s growth in nude mice will slow down in a range of time.
Objective To study the effects on MCF-7 breast cancer cells with combination of tamoxifen（TAM） and antisense oligonucleotide （ASODN） targeting survivin mRNA. Methods MCF-7 breast cancer cells were treated with a 20 mer ASODN targeting survivin mRNA and TAM, which were divided into three groups: TAM group (treated by TAM only), ASODN group (by ASODN only), and TAM+ASODN combined group (by TAM+ASODN combination). The growth inhibition of MCF-7 cells, the changes of cell cycles and apoptotic rate, the positive rate of survivin mRNA expression, and the activity of caspase-3 were tested by MTT, flow cytometry, hybridization in situ, and spectrophotometric method, respectively.Results The rate of growth inhibition of MCF-7 cells in the TAM+ASODN combined group was (62.26±3.92)%, which was significantly higher than that in the TAM group 〔(42.30±6.63)%〕 or ASODN group 〔(54.77±9.99)%〕, Plt;0.05. The apoptotic rate of MCF-7 cells was (28.08±4.32)% in the TAM+ASODN combined group, which was significantly higher than that in the TAM group 〔(18.94±4.01)%〕 or ASODN group 〔(21.12±3.95)%〕, Plt;0.01. The effect of arresting MCF-7 cells in G0/G1 phase in the TAM+ASODN combined group was ber than that in the TAM or ASODN group (Plt;0.05, Plt;0.01). The positive rate of survivin mRNA in the TAM+ASODN combined group was (13.38±3.45)%, which was significantly lower than that in the TAM group 〔(39.67±7.42)%〕 or ASODN group 〔(27.50±5.80)%〕, Plt;0.01. The activity of caspase-3 in the TAM+ASODN combined group （0.93±0.13） was significantly higher than that in the TAM group (0.50±0.09) or ASODN group (0.64±0.08), Plt;0.01. Conclusion The ASODN targeting survivin mRNA can promote the apoptosis of MCF-7 breast cancer cells, and make MCF-7 cells more sensitive to tamoxifen.
The pathogenesis of diabetic retinopathy (DR) is complex. Antisense non-coding RNA (ANRIL) in the INK4 locus in long-chain non-coding RNA (lncRNA) is closely related to cell proliferation, differentiation, and individual development. It plays an important role in the dysplasia of retinal vascular endothelial cells and is a new field in the study of the pathogenesis of DR. According to the researches at present, ANRIL may plays its role in the occurrence and development of DR through the signal pathway of nuclear factor-κB and ROS/polyadenylation diphosphate ribose polymerase, and interact with p300, miR-200b, and EZH2 to regulating the expression and function of VEGF. Specific blocking ANRIL and its related pathways may become a new target in the treatment of DR.