Objective To study the effects of survivin antisense RNA on SGC7901 cell’s apoptosis and chemosensitivity to taxotere, and to investigate its effect on the expression of multi-drug resistance gene-1 (MDR-1). Methods Survivin antisense eukaryotic vector anti-pcDNA3-svv was transfected into SGC7901 cell lines by lipofectamine and positive clones were screened out then. Survivin protein and MDR-1 mRNA were measured by western blot and RT-PCR, respectively. Apoptosis that was induced by anti-pcDNA3-svv was observed by electronic microscope, and the sensitivity of SGC7901 cell to taxotere was examined by MTT. Results The expressions of survivin protein and MDR-1 mRNA in transfected SGC7901 cells both decreased more significantly than that of non-transfected cells (P＜0.05， P＜0.01), and the indices of MDR of transfection group and non-transfection group were 0.196±0.013 and 3.126±0.019, respectively, at the late phase of apoptosis, which had a significant difference between each other (P＜0.01), IC50 of the transfected cells to taxotere was （16.7±1.98） ng/ml and that of the non-transfected cells was (55.7±1.89) ng/ml, which also had a significant difference (P＜0.01). Conclusion Surivivin antisense RNA could induce the apoptosis of SGC7901 cancer cell line and could increase the cells’ sensitivity to taxotere, which may help to reverse drug resistance.
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 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.
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 the effect s of T lymphoma invasion and metastasis inducing factor 1 ( Tiam 1) antisense oligonucleotides (ASODN) on morphological remodeling of gast ric cancer cells. Methods The high-invasive and metastastic subgroup (MH ) was separated f rom human gast ric cancer cell line MKN245 (M0 ) by laminin adhesion method in vi t ro. And they were divided into four group s according to different further t reatment s : no t ransfection group (cont rol group ) , liposome t ransfection group , sense oligonucleotides2liposome t ransfection group ( SODN t ransfection with liposome group ) and antisense oligonucleotides2liposome t ransfection group (ASODN t ransfection with liposome group) . Then the expressions of Tiam 1 mRNA and protein were detected by RT-PCR and flowcytomet ry , respectively. The morphology changes between Tima 1 ASODN t ransfected MH cells and no t ransfected cells were observed by using HE stain , cytoskeletal protein stain and scanning elect ronic microscope (SEM) . Results Compared with the other group s , the expressions of Tiam 1 mRNA and protein in MH cells were significantly decreased af ter the cells were t ransfected with 0. 43 μmol/ L Tiam 1 ASODN ( P lt; 0. 01) . Additionally , it was observed that the t ransfected MH cells had less membrane surface projections , fewer or shortener pseudopodia , less irregular cytoskeletal network and less spotted-like actin bodys than no t ransfected MH cells did. Conclusion ASODN t ransfection could effectively suppress the expression of Tiam 1 and the remodeling in gast ric cancer cells , which may play an important role in the invasion and metastasis of gast ric cancer cells.
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.
【Abstract】ObjectiveTo study the effect of down-regulation of E-cadherin on the invasion ability of tumor cells. MethodsHuman pancreatic carcinoma cell line JHP-1 was treated with E-cadherin antisense oligodeoxynucleotied (ASODN). The immunocytochemistry, Western blot were used to detect the expression and the contents of E-cadherin in the tumor cells, and the invasive ability of tumor cells were evaluated by invasive-MTT assay. Results Treated with E-cadherin ASODN, the expression of E-cadherin on JHP-1 cells were reduced, and the protein contents were decreased as well compared with control groups and ODN group. The invasive ability of JHP-1 cells to the basement membrane was increased (P＜0.001) compared with ODN group and control group. ConclusionE-cadherin was related to the invasive ability of tumor cells.
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.
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.