Objective To investigate the effect on expression of c-myc and proliferating cell nuclear antigen （PCNA） of vein grafts transferred by c-myc antisense oligodeoxynucleotides（ODN） of soluble stent. Methods A rabbit model of common carotid arteries grafted by external jugular veins was constructed in 50 New Zealand rabbits and were randomly divided into five groups, 10 rabbits each group. Control group： no stents ; group 1： soluble stent ; group 2： soluble stent with sense-ODN; group 3： soluble stent with antisense-ODN; group 4.. soluble stent with mismatch-ODN. At 7 d, 28 d and 90 d after surgery, vein grafts were harvested. The expression of c-myc and PCNA were identified by immunochemistry methods. Results At 7d, 28d, 90d after surgery, the expression of c-myc and PCNA of the intima and media of vein grafts in control group, group 1, group 2, group 4 were higher significantly than that in group 3 （P〈0. 01）. At 28d, 90d after surgery, the expression of c-myc in five groups were higher than that in the same group at 7d after surgery （P〈0. 01）. Conclusion Soluble stent can transfer ODN effectively. C- myc antisense-ODN transferred by soluble stent can inhibit significantly the expression of c-myc and PCNA in the intima and media of vein grafts.
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.
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 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.
Objective To observe the inhibitory effects of local co-transfection of tissuetype plasminogen activator(tPA) gene and proliferating cell nuclear antigen antisense oligodeoxynucleotides(PCNA-ASODN) on the intima proliferation and restenosis of autograft artery in rabbits. Methods One hundred and twenty male Zelanian rabbits were randomly divided into four groups(n=30, in each group): control group, PCNA-ASODN group, tPA group and tPA+PCNAASODN group. The left and right external iliac arteries (length 1.0 cm) were transplanted reciprocally. The transplanted arteries were respectively soaked in lipofection, PCNAASODN, pBudCE4.1/tPA and pBudCE4.1/tPA+PCNA-ASODN solution about 15 minutes. The transplanted arteries were sutured with 9-0 sutures soaked in PCNA-ASODN and pBudCE4.1/tPA solution. Each group were divided into five subgroups(n=6, in each subgroup) according to the sacrifice time (3 d, 7 d, 14 d, 28 d and 56 d after operation). On every sacrifice time point, the vascular specimens were harvested. The thrombocyte assembling and thrombus forming lining vessel wall were observed by scanning electron microscope. The pathological morphology of transplanted arteries were observed under microscope(HE). The intimal areas and stenosis ratio(%) of transplanted arteries were calculate and analyzed statistically among groups by computer system. The mRNA expression of tPA gene in transplanted ressel wall was detected with vevere transcriptionPCR(RT-PCR). The number of PCNA positive cells in transplanted vessel wall was counted by SP immunochemisty.Results The mRNA expression of tPA gene in the transplanted vessel wall in tPA and tPA+PCNA-ASODN groups was higher than that of the other two groups(P＜0.01).The number of PCNA positive cells in the transplanted arteries in PCNAASODN, tPA and tPA+PCNAASODN groups were significantly lower than that of control group(P＜0.05，P＜0.01). The intimal areas and degrees of luminal stenosis of PCNAASODN, tPA and tPA+PCNAASODN groups were lower than those of control group(P＜0.05，P＜0.01), and those of tPA＋ PCNA-ASODN group were lower than those of PCNA-ASODN and tPA groups(P＜0.05). Scanning electron microscopy showed that there were a few thrombocytes lining the vessel wall of tPA group and tPA+PCNAASODN group and no thrombus, whereas there were abundant thrombocytes and thrombi lining the vessel wall of the control group. Conclusion Co-transfection of tPA gene and PCNA-ASODN can effectively inhibit the proliferation of VSMC, hyperplasia of intima and restenosis of transplanted artery.
Objective To investigate an inhibitive effect of the chitosan nanoparticles with the proliferation cell nuclear antigen （PCNA）-antisense oligo deoxy nucleotides (ASODN) on the intimal cell proliferation after the vein grafting.Methods Fiftyfour male SD rats, weighing 450-600g, were randomly divided in the experimental group and the control group of 27 rats each. In the experimental group, the chitosan nanoparticles with PCNAASODN were infused into the anastomosis segment of the right jugular artery and vein; then, the anastomosis segment was transplanted to the jugular artery on the same side. The rats in the control group were infused with normal saline by the same procedures. There were 24 rats in each group which used to experiment. The hemodynamic data were obtained from the Doppler ultrasound examinations at 1, 2, 3 and 4 weeks. The specimens were taken. Immunohistochemistry, Westernblot, and bloodvesselwall histopathology were performed at the different week points. Results There was no significant difference in the thrombogenesis rate between the experimental group and the control group (3/27 vs. 3/27,P＞0.05). During the 4 week observation, PCNA Westernblot showed that the PCNA level was lower in the grafted vein and the anastomosis segment in the experimental group than in the control group. The indexes of the PCNA postive proliferating cells in the intimal area (0.13%±0.11%，0.79%±0.28%，0.45%±0.29%, 0.43%±0.25%) and the medial area (1.90%± 0.84%，2.11%±0.98%，2.48%±0.77%，2.17%±0.36%) were significantlydecreased at 1,2,3 and 4 weeks in the experimental group when compared with those in the control group（P＜0.05）. The lumen areas in the grafted vein (88.71±16.96，95.98±21.44，88.48±32.81，97.86±34.11 μm 2) and the anastomosis segment (41.49±3.34，45.15±11.65，46.27±8.90，51.62±8.85 μm 2) were significantly greater in the experimental group than in the control group (P＜0.05). The ratios of the initmal area to the medial area in the grafted vein (22.73%±3.11%，32.40%±4.55%，45.14%±3.19%，45.70%±5.01%) and the anastomsis segment (41.49%±3.34%，45.15%±11.65%，46.27%±890%，51.62%±8.85%) were significantly smaller in the experimental group than in the control group（P＜0.05）. The maximum velocities (Vmax) of the blood flow inthe grafted vein and the anastomsis segment were almost the same in the two groups at 1 week, but had different changes at the next 3 weekpoints. In the control group, the Vmax of the blood flow gradually increased and at 3 weeks it reached the peak point; however, at 4 weeks it decreased. In the experimental group,the Vmax of the blood flow gradually decreased, and at 3 weeks it decreased to the lowest point; however, at 4 weeks it increased. So, at 4 weeks the Vmax of the blood flow in the grafted vein and the anastomsis segment was almost the samein the two groups. There was no significant difference in the Vmax of the bloodflow between the two groups (P＞0.05), but in the same group there wasa significant difference at the different time points. Conclusion The chitosan nanoparticles with PCNAASODN can effectively inhibit the intimal cell proliferation after the grafting of the blood vessel, so that the neointimal thickening can be prevented.
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.
【Abstract】ObjectiveTo study the effect of transfection with antisense DNMT3b gene eukaryotic expression vector on the expression of DNMT3b gene in human cholangiocarcinoma cell line QBC-939. MethodsThe constructed antisense DNMT3b gene eukaryotic expression vector was transfected into the human cholangiocarcinoma cell line QBC-939 by using lipofectamine transfection reagents, and positive cell clones were obtained by using G418 selection after transfection. Whether the constructed recombinant vector was transfected into QBC-939 cells successfully was confirmed by amplifying the exogenous neoR gene with PCR method. The expression of DNMT3b gene mRNA and protein were detected by semi-quantitative RT-PCR and FCM methods respectively. ResultsFollowing the transfection of antisense DNMT3b gene eukaryotic expression vector, the mRNA level of DNMT3b gene in QBC-939 cells of human cholangiocarcinoma decreased from 0.956±0.053 to 0.209±0.023, and the protein level of DNMT3b gene also decreased from (75.38±3.22)% to (29.87±3.46)%. There were very significant differences on the expression levels of DNMT3b gene between non-tranfections group and the antisense DNMT3b gene eukaryotic expression vector transfection group (P＜0.01). ConclusionTransfection with antisense DNMT3b gene eukaryotic expression vector significantly reduces the expression level of DNMT3b gene in human cholangiocarcinoma cell line QBC-939, and this study may provide a valid tool and method to investigate the function of DNMT3b gene and its role in cholangiocarcinoma.
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.
Objective Col I A1 antisense oligodeoxyneucleotide (ASODN) has inhibitory effect on collagen synthesis in cultured human hypertrophic scar fibroblasts. To investigate the effects of intralesional injection of Col I A1 ASODN on collagen synthesis in human hypertrophic scar transplanted nude mouse model. Methods The animal model of humanhypertrophic scar transplantation was established in the 60 BALB/c-nunu nude mice (specific pathogen free grade, weighing about 20 g, and aged 6-8 weeks) by transplanting hypertrophic scar without epidermis donated by the patients into the interscapular subcutaneous region on the back, with 1 piece each mouse. Fifty-eight succeed models mice were randomly divided into 3 groups in accordance with the contents of injection. In group A (n=20): 5 μL Col I A1 ASODN (3 mmol/L), 3 μL l iposome, and 92 μL Opti-MEM I; in group B (n=20): 3 μL l iposome and 97 μL Opti-MEM I; in group C (n=18): only 100 μL Opti-MEM I. The injection was every day in the first 2 weeks and once every other day thereafter. The scar specimens were harvested at 2, 4, and 6 weeks after injection, respectively and the hardness of the scar tissue was measured. The collagens type I and III in the scar were observed under polarized l ight microscope after sirius red staining. The ultrastructures of the scar tissues were also observed under transmission electronic microscope (TEM). Additionally, the Col I A1 mRNAs expression was determined by RT-PCR and the concentrations of Col I A1 protein were measured with ELISA method. Results Seventeen mice died after intralesional injection. Totally 40 specimens out of 41 mice were suitable for nucleic acid and protein study, including 14 in group A, 13 in group B, and 14 in group C. The hardness of scars showed no significant difference (P gt; 0.05) among 3 groups at 2 weeks after injection, whereas the hardness of scars in group A was significantly lower than those in groups B and C at 4 and 6 weeks (P lt; 0.05), and there was no significant difference between groups B and C (P gt; 0.05). The collagen staining showed the increase of collagentype III in all groups, especially in group A with a regular arrangement of collagen type I fibers. TEM observation indicated that there was degeneration of fibroblasts and better organization of collagen fibers in group A, and the structures of collagen fibers in all groups became orderly with time. The relative expressions of Col I A1 mRNA and the concentrations of Col I A1 protein at 2 and 4 weeks after injection were significant difference among 3 groups (P lt; 0.05), and they were significantly lower in group A than in groups B and C (P lt; 0.05) at 6 weeks after injection, but no significant difference was found between groups B and C (P gt; 0.05). Conclusion Intralesional injection of Col I A1 ASODN in the nude mice model with human hypertrophic scars can inhibit the expression of Col I A1 mRNA and collagen type I, which enhances the mature and softening of the scar tissue. In this process, l iposome shows some assistant effect.