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.
Objective To analyze the expression of apoptosis-related genes of retinal blood vessel in early diabetic rats by gene chip technology. Methods To make diabetic rat model by intraperitoneal injection of streptozotocin (STZ). On the 6th week after blood pressure increased, 10 rats were executed in Diabetic group and normal control group respectively. 20 retinal blood vessels were extracted and the RNA was isolated. The probe was made of alpha;-32 P-deoxyadenosine triphosphate (dATP)-labeled sample which hybridized 1176 nylon chips, and then analyzed by software. Three different expression genes were selected to verify by reverse transcription polymerase chain reaction (RT-PCR). Results On the 6th week, 136 （11.5％） genes were differentially expressed ［up-regulated genes were 90(7.6%), down-regulated genes were 46(3.9%)］in diabetic group. These genes involved into different groups according to their function. Especially in 72 apoptosis-related genes, 15 genes were differentially expressed. The up-regulated genes were some TNF receptor family members such as TNFRSF12, TRAIL, TNFRSF9, FADD；Bcl-2 family members such as bcl-w, bax, bak1 and AKT. The down-regulated genes were FAF1 which related to fas. Conclusions The expression of retinal vascular gene in early diabetic rats has been changed complicatedly. In particular, the multiple apoptosis-related genes have been changed in early diabetic, and most of them are at the upstream of apoptosis pathway. These findings indicate that the development of diabetic retinopathy is associated with multiple signaling pathways leading to apoptosis, while the alterations on the level of molecular biochemistry are still limited in apoptosis induction period. （Chin J Ocul Fundus Dis，2008，24：244-248）
Objective To investigate the effect of topical treatment with antisense oligonucleotides（ASON）targeting tumor necrosis factor-alpha;（TNF-alpha;）on the pathological process of experimental herpes simplex virus type-Ⅰ（HSV-Ⅰ）induced chorioretinitis in mouse eye. Methods Fifty BALB/c mice were randomly divided into experimental and control group（twenty five mice in each group）.HSV chorioretinitis model was induced in each mouse by inoculating 1times;105 plaque-forming units （pfu） of HSV-Ⅰ（KOS strain）into anterior chamber of the right eye.In experimental group，Fluorescein isothiocyanate （FITC）-labeled ASON targeting TNF-alpha; 2 mu;l were injected sub-conjunctiva in the left eye1day before and 1 and 4 days after the infection;while phosphate buffer solution was injected in the same way in control group.The inflammation changes of the eyes in the 2 groups were observed and the clinical grades were assessed according to the extends of anterior-chamber inflammation，vasodilatation of cornea and iris，formation of cataract，and vitreous opacity. All of the mice were executed 10 days after the infection and were observed histologically. The contents of TNF-alpha; in retina and choroid were measured by enzyme-linked immunobsorbent assay（ELISA）. Results After the infection，acute inflammation appeared in the right eyes in both groups. The inflammation of the left eyes in experimental group was significantly milder than which in the control group.Twelve left eyes had necrotic chorioretinitis in different degrees in the control group while 2 left eyes had mild chorioretinitis in the experimental group. The difference of the number of inflammatory cells between the 2 groups was statistically significant in retina，choroid，and ciliary body（P＜0.05）and was not obvious in anterior chamber，vitreous cavity，and iris（P＞0.05）.The content of TNF-alpha; in choroid and retina was（60plusmn;1.25）pg in the experimental group and（305plusmn;1.03）pg in the control group（P＜0.05）. Conclusions TNF-alpha; ASON treating HSV-Ⅰinduced chorioretinitis may reduce the content of TNF-alpha; in affected mice eyes and decrease the inflammatory reaction. (Chin J Ocul Fundus Dis, 2006, 22: 245-248)
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 investigate the role of anti apoptosis gene bcl-2 in the survival of cultured uveal melanoma UM cells. Methods Antisense oligodeoxynucleotides (AS-ODN) bcl-2 were delivered with cationic lipid to primary cultured UM cells. The inhibitory effect of AS-ODN bcl-2 on proliferation of UM cells was examined by 3,-4,5 Dimethyliazol-2,5 diphenyl tetrazolium bromide (MTT) method. Using DNA ladder to determine if the UM cells had been apoptotic. Bcl-2 expression was detected by RT-PCR and Westernblot technics. Results The effect of AS-ODN bcl-2 in inhibiting the proliferation of cultured UM cells had opposite relation to dosage. It down regulated the mRNA and protein level of bcl-2 gene, and the sense ODN didn′t have this effect. Conclusion AS-ODN bcl-2 can down regulate bcl-2 expression, inhibits UM cells proliferation and induces apoptosis. (Chin J Ocul Fundus Dis, 2002, 18: 38-41)
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 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.