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 permeability and stability of the transfection of antisense oligonucleotide (ASODN) hybridized epidermal growth factor receptor (EGFR) to retinal glial cells (RG).Methods Phosphorothioate and unmodified EGFR ASODN conjugated with 5′-isothioc yanate (5′-FITC) were encapsulated with or without lipofectin, and then added into human retinal glial cells culture media. The cellular permeability and stability of the transfection were observed by fluorescence microscopy in fixed cells.Results In the absence of lipofectin, phosphorothioate and unmodified EGFR ASODN were found in a few RG cells at 30 minutes, and in about 50% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in RG cells for 3-4 hours and disappeared at about 8 hours. In the presence of lipofectin, phosphoro thioate and unmodified EGFR ASODN were found in a few RG cells at 15 minutes and about 70%-80% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in cells for 10-12 hours, and phosphorothioate and unmodified EGFR ASODN were disapp eared at about 14 hours and 4 hours respectively.Conclusion 5′-FITC EGFR ASODN encapsulated with lipofectin could enter RG cells and express stably in RG cells. (Chin J Ocul Fundus Dis，2003，19：52-54)
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 proliferating cell nuclear antigen (PCNA) gene expression in retinal pigment epithelium (RPE) cells and inhibition of antisense oligonucleotides(AS-OND) encoding PCNA mRNA to gene expression and proliferation of RPE cells, so as to search for new genetic therapy way for pro1iferative vitreoretinopathy (PVR). Methods (1) Rabbit RPE cells cultured in vitro were detected for PCNA expression by streptoavidin-biotin-enzyme complex (SABC) immunohistochemistry at several times. (2) The liposome-mediated synthetic antisense oligodeoxynucleotides (AS-ODN) and sense oligodeoxynucleotides (S-ODN) encoding PCNA were delivered to the RPE cells at different concentrations, then PCNA expresstion were detected by immunohistochemistry. (3) Exposed to different concentrations of AS-ODN and S-ODN, growth activity and suppressive rate of RPE cells were measured by methyl thiazolyl tetrazolium (MTT) methods. Results (1) PCNA were expressed in RPE cells, culmination in 48 hours of culture. (2) PCNA expression were markedly suppressed in the RPE cells treated with 0.28 and 1.12 μmol/L PCNA AS-ODN. (3) 0.28 μmol/L and 1.12 μmol/L PCNA AS-ODN significantly inhibited proliferative activity of RPE cells in a dose-dependent manner, the arrest rates of cellular growth reached 53% and 81% respectively. Conclusion AS-ODN complementary to PCNA mRNA at some concentration can sequence-specifically suppress PCNA expression in RPE cells and cellular proliferative activity, and show potential application to further experimental study for PVR genetic medication. (Chin J Ocul Fundus Dis, 2002, 18: 231-233)
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.
【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 inhibitory effect of proliferation cell nuclear antigen (PCNA) antisense oligonucleotides mediated by liposome transfection on hepatocellular carcinoma cell proliferation. MethodsThe antisense oligonucleotides were complementary to 18mer sequences next to the start codon of PCNA mRNA sequences. The human hepatocellular carcinoma cell line Bel7404 was treated with antisense oligonucleotides. The inhibition of proliferation was estimated by MTT method. We compared the deference between the liposome mediated transfection technique and direct transfection technique. ResultsThe cell proliferation was inhibited effectively by antisense oligonucleotides. A sense sequence oligomer showed no effect.Liposome mediated transfection could enhance the inhibitory effect. Conclusion Liposome mediated transfection could enhance the inhibitory effect of PCNA antisense oligonucleotides on hepatocellular carcinoma cell proliferation.
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 investigate the effect of suppression of ischemia-induced retinal neovascularization by VEGF antisense oligodeoxyribonucleotides. Methods Mouse models of hyperoxia-induced ischemic retinopathy were established. Retrobulbar injections were performed with VEGF antisense oligodeoxyribonucleotides or NS in 4 groups:normal control and various doses respectively. The nuclei of new vessel buds extending from the retina into the vitreous in differ ent groups were counted and compared under the light microscope. Results There were plenty of new vessel buds in the eyes of mice in hyperoxic condition., while the number of the nuclei of new vessel buds is less in the murine eyes with retrobulbar injection of VEGF antisense oligodeoxyribonucleotides,especially the nuclei were redused with 59.3% in eyes with large dose. Conclusion The proliferation of retinal new vessel may be suppressed by using the retrobulbar injection of VEGF antisense oligodeoxyribonucleotides. (Chin J Ocul Fundus Dis, 2001,17:141-143)
Objective To investigate the inhibitory effect of survivin antisense oligonucleotides (ASODN) on proliferation of pancreatic cancer cells PANC-1. Methods The ASODN and sense oligodeoxynucleotides (SODN) were complementary to survivin sequences. FAM-marked ASODN was transfected into PANC-1 cells mediated by positive ion liposome as ASODN group. Blank control group (normal cells), negative control group (normal medium)， and SODN group were established for comparison. The transfection efficiency was detected by flow cytometry (FCM) after transfection; MTT assay was used to detect cytotoxicity; Cell morphological changes were examined by transmission electron microscopy; The cell cycle and apoptotic rate were analyzed by FCM; Immunohistochemical staining techniques were used, and the expressions of survivin were observed under light microscopy, examined and analysed by computer image. Results ①The transfection efficiency was 31.9%, 37.4%, 41.4%, 52.6%, 24.2%, 11.4%, 16.1%, and 15.5% when the transfecting concentration of ASODN was 50, 100, 150, 200, 250, 400, 600, and 800 nmol/L, respectively; The transfection efficiency was 12.0%, 50.8%, and 11.2% when the inoculated cells was 2×104/well, 2×105/well, and 2×106/well, respectively; The transfection efficiency was 58.8%, 34.0%, and 23.6% when 2 μl, 3 μl, and 4 μl liposome was used during transfection, respectively. ②Cell gap was oversize, morphous was round, adherent cells were less after transfection under fluorescence microscope. ③The inhibition rate in the ASODN group was higher than that in each control group (Plt;0.05) on 24, 36, 48 h after treating by survivin ASODN, which increased as time prolonged (Plt;0.05). ④The apoptosis showed a ladder-shaped line in the ASODN group. ⑤Apoptotic morphology was demonstrated in the ASODN group, such as apoptotic cells with nuclear chromatin highly concentrated, crescent nuclear staining aggregated by the side nuclear membrane, nucleolus disappeared by AO and EB stains. ⑥The apoptotic rate 〔（38.1±3.4）%〕 in the ASODN group was higher than that in the SODN group 〔（4.16±1.7）%〕, Plt;0.05. ⑦G2/M cell cycle arrested in the ASODN group. ⑧After transfection, the expression of survivin protein in the ASODN group was significantly lower than that of each control group (Plt;0.05). Conclusions The optimal transfection conditions are as following: the cell count of 2×105/well, concentration of ASODN 200 nmol/L, and cationic liposome oligofectamine 2 μl, respectively. Survivin ASODN can inhibit the proliferation of pancreatic cancer cells and induce their apoptosis.