Expression of heparanase mRNA in anti-sense oligonucleotide-transfected human esophageal cancer EC9706 cells

AIM： To investigate the effed3 of anti-sense oligonucleotides （ASODNs） on mRNA expression of heparanase in human esophageal cancer EC9706 cells. METHODS： One non-sense oligonucleotide （N-ODN） and five ASODNs against different heparanase mRNA sites were transfected into EC9706 cells, then the expression of heparanase mRNA in EC9706 cells was studied by in situ hybridization. RESULTS： The expression of heparanase mRNA could be inhibited by ASODNs.There was no significant difference among five ASODNs （P〉0.05）, but there was a significant difference between ASODNs and N-ODN or non-transfected group （ASODNI： 2.25±0.25, ASODN2： 2.21±0.23, ASODN3： 2.23±0.23, ASODN4：2.25±0.24 vs N-ODN： 3.47±2.80 or non- transfected group： 3.51±2.93 respectively, P〈0.05）. CONCLUSION： The expression of heparanase mRNA in EC9706 cells can be inhibited by ASODNs in vivo, and heparanase ASODNs can inhibit metastasis of esophageal squamous cell carcinoma or other tumors by inhibiting the expression of heparanase.

Anti-sense oligonucleotide labeled with technetium-99m using hydrazinonictinamide derivative and N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycline: A comparison of radiochemical behaviors and biological properties

AIM:To explore and compare the radiochemical behavior and biological property of anti-sense oligonuc-leotide (ASON) labeled with technetium-99m using N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycl ine (NHS-MAG3) and hydrazinonictinamide derivative (HYNIC). METHODS:After HYNIC and NHS-MAG3 were synthesized, ASON was labeled with technetium-99m using HYNIC and NHS-MAG3 as a bifunctional chelator. The in vivo and in vitro stability, binding rates of labeled compounds to serum albumen, biodistribution of 99mTc-MAG3-ASON and 99mTc-HYNIC-ASON in BALB/C mouse and its HT29 tumor cellular uptake were compared. RESULTS:The labeling efficiency and stability of 99mTc-MAG3-ASON were significantly higher than those of 99mTc-HYNIC-ASON (P = 0.02, and P = 0.03, respectively). 99mTc-MAG3-ASON had a significantly lower rate of binding to serum albumen than 99mTc-HYNIC-ASON (P < 0.05). In contrast to 99mTc-HYNIC-ASON, the biodistribution of 99mTc-MAG3-ASON was significantly lower in blood, heart, liver and stomach (P < 0.05), slightly lower in intestines and spleen (P > 0.05) and significantly higher in lung and kidney (P < 0.05). The HT29 tumor cellular uptake rate of 99mTc-MAG3-ASON was significantly higher than that of 99mTc-HYNIC-ASON (P < 0.05). CONCLUSION:99mTc-MAG3-ASON shows superior radiochemical behaviors and biological properties than 99mTc-HYNIC-ASON. 99mTc-MAG3-ASON is a potential radiopharmaceutical agent for in vivo application.

Binding tendency with oligonucleotides and cell toxicity of cetyltrimethyl ammonium bromide-coated single-walled carbon nanotubes

Functionalized carbon nanotubes （CNTs） were made for the delivery of genes and drugs and CNT-based biosensors. The basis of CNTs is for binding with biomolecules in biomedical applications. The binding tendency with small interfering RNA oligonucleotides and cytotoxicity of cetyltrimethyl ammonium bromide （CTAB）-coated single-walled carbon nanotubes （SWNTs） were studied. The field emission scanning electron microscopy and transmission electron microscopy results show that a SWNT suspension in CTAB solution was well-dispersed and stable. CTAB is the cross-linker between SWNTs and oligonucleotides. The CTAB-coated SWNTs have less cytotoxicity to human umbilical vein endothelial cells than single SWNTs and the cytotoxicity of CTAB-coated SWNTs depended on the concentration of CTAB-coated SWNTs.

Effect of Dexamethasone and Aquaporin-1 Antisense Oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.

Enhanced therapeutic effects of combined chemotherapeutic drugs and midkine antisense oligonucleotides for hepatocellular carcinoma

AIM: To evaluate the effect of combined antisense oligonucleotides targeting midkine (MK-AS) and chemotherapeutic drugs [cisplatin(DDP), 5-fluorouracil (5-FU) and adriamycin (ADM)] on inhibition of HepG2 cell proliferation, and to analyze the efficacy of MK-AS used in combined ADM in in situ human hepatocellular carcinoma (HCC) model. METHODS: HepG2 cells were treated with MK-AS and/or chemotherapeutic drugs mediated by Lipofectin, and cell growth activity was determined by MTS assay. An in situ HCC model was used in this experiment. MK- AS, ADM and MK-AS + ADM were given intravenously for 20 d, respectively. The animal body weight and their tumor weight were measured to assess the effect of the combined therapy in vivo. RESULTS: Combined treatment with MK-AS reduced the IC50 of DDP, 5-FU and ADM in HepG2 cells. MK-AS significantly increased the inhibition rate of DDP, 5-FU and ADM. Additionally, synergism (Q 1.15) occurred at a lower concentration of ADM, 5-FU and DDP with combined MK-AS. Combined treatment with MK-AS and ADM resulted in the more growth inhibition on in situ human HCC model compared with treatment with chemotherapeutic drugs alone. CONCLUSION: MK-AS increases the chemosensitivity in HepG2 cells and in situ human HCC model, and thecombination of MK-AS and ADM has a much better in vitro and in vivo synergism.

Inhibitory Effects of Anti-sense PTTG on Malignant Phenotype of Human Ovarian Carcinoma Cell Line SK-OV-3

To construct eukaryotic expression vector expressing full length anti-sense pituitary tumor transforming gene (PTTG) mRNA and observe its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3. PCR primers containing designed enzyme cut sites were used for cloning full-length PTTG gene fragment, and the resulting PCR product was inserted into the eukaryotic vector pcDNA3.1 in the antisense direction. The recombinant vector was then transfected into SK-OV-3 by Lipofectamine. The positive cell clone was screened by G418, PTTG and bFGF at protein level expression were detected by Western blot. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay. Our results showed that SK-OV-3 clones stably expressing full-length recombinant pcDNA3.1-PTTGas were obtained. The expressions of PTTG and bFGF protein in transfected cells were decreased by 61.5 % and 52.3%, respectively as compared with non-transfected ones. The number of colony formation was reduced significantly in transfected cells as compared with empty vector transfected and non-transfected cells. It is concluded that the recombinant vector pcDNA3.1-PTTGas is a novel tool and provides an alternative anti-sense gene therapy targeted at PTTG in human carcinoma.

Combination of telomerase antisense oligonucleotides simultaneously targeting hTR and hTERT produces synergism of inhibition of telomerase activity and growth in human colon cancer cell line

AIM: To investigate synergism of inhibition of telomerase activity and proliferation of human colon cancer cells by combination of telomerase antisense oligonucleotides (ASODNs) simultaneously targeting human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in vitro. METHODS: ASODN of hTR and ASODN of hTERT were transfected into human colon cancer SW480 cells by liposomal transfection reagents. Telomerase activity of SW480 cells was examined using telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (PCR-ELISA). Proliferation activity of SW480 cells was tested by methyl thiazolyl tetrazolium assay. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: The telomerase activity and cell survival rate in SW480 cells transfected with 0.2 μmol/L of ASODN of hTR or ASODN of hTERT for 24-72 h were significantly decreased in a time-dependent manner compared with those after treatment with sense oligonucleotides and untreated (telomerase activity: 24 h, 73%, 74% vs99%, 98%; 48 h, 61%, 55% vs98%, 99%; 72 h, 41%, 37% vs 99%, 97%; P<0.01; cell survival rate: 24 h, 88%, 86% vs594%, 98%; 48 h, 49%, 47% vs94%, 97%; 72 h, 44%, 42% vs92%, 96%; P<0.01). Moreover, the telomerase activity and the cell survival rate in SW480 cells treated by the combination of telomerase anti-hTR and anti-hTERT were more significantly suppressed than single anti-hTR or anti-hTERT (telomerase activity: 24 h, 59% vs 73%, 74%; 48 h, 43% vs61%, 55%; 72 h, 18% vs41%, 37%; P<0.01; cell survival rate: 24 h, 64% vs88%, 86%; 48 h, 37% vs49%, 47%; 72 h, 25% vs44%, 42%; P<0.01). Meanwhile, the apoptosis rates in the combination group were markedly increased compared with those in the single group (24 h, 18.0% vs7.2%, 7.4%; 48 h, 23.0% vs13.0%, 14.0%; 72 h, 28.6% vs 13.2%, 13.75; P<0.01). Cells in combination group were arrested at G0/G1 phase. CONCLUSION: Telomerase anti-hRT and anti-hTERT suppress telomerase activity, and inhibit growth of human colon cancer cells probably via induction of apoptosis and retardation of cell cycle. Additionally, combined use of telomerase ASODNs targeting both hTR and hTERT yields synergistic action selective for human colon cancer.

Inhibition of PCNA Antisense Oligonucleotides Mediated by Liposome on mRNA Expression and Proliferation of h-RPE Cells

Summary： The proliferating cell nuclear antigen （PCNA） gene expression was blocked and retinal pigment epithelium （RPE） proliferation was inhibited by using antisense oligonucleotides （AS-ODN） mediated by liposome, to find a new genetic therapy of proliferative vitreoretinopathy （PVR）. RPE cells cultured in vitro were transfected with synthetic fluorescence labled AS-ODN mediated by liposome-Lipofectamine, and the intracellular distribution and persistence time of AS-ODN were dynamically observed. AS-ODN （0.07, 0.28 and 1.12 μ mol/L and sense oligonucleotides （S-ODN with the same concentrations as AS-ODN） mediated by liposome were delivered to the RPE cells cultured in vitro, and CPM values were measured by ^3H-TdR incorporation assay and analyzed statistically by variance by comparison with blank control group. Expression ofPCNA mRNA in RPE cells was detected by in situ hybridization after the treatment of different concentrations of PCNA AS-ODN and S-ODN, and the average optic density （AOD） was measured by image analysis system and was subjected to q-test and correlation analysis with CPM. Our results showed that AS-ODN mediated by liposome could quickly aggregate in cellular plasma and nuclei in 30 min and 6 h, and stayed for as long as 6 days. AS-ODN （0.28 and 1.12 μmol/L） markedly suppressed proliferation of RPE cells in a dose-dependent manner with the difference being statistically significant （P〈0.05 and P〈0.01, repectively） as compared with blank control group. AOD was well correlated with CPM （r=0.975）. It is concluded that liposome could increase transfection efficiency of AS-ODN in RPE cells, and AS-ODN could sequence-specifically suppress PCNA mRNA expression and proliferation of human RPE cells.

Obstructive Effects of Ultrasonic Microbubble Intensifier on CHG-5 Cell with Survivin Antisense Oligonucleotides Transfection

Objective： To study the effects on human glioma cell line CHG-5 by ultrasonic microbubble intensifier with survivin antisense oligonucleotides （ASODN） transfection. Methods： Antisense oligonucleotides targeting survivin mRNA was designed and synthesized. Four regimen groups were designed, group A： survivin antisense oligonucleotides transfected with ultrasonic microbubble intensifier combined with ultrasound irradiation, group B： survivin antisense oligonucleotides transfected with lipofectamine combined with ultrasound irradiation, group C： survivin antisense oligonucelotides with lipofectamine transfection, group D： blank control. The expression changes of surviving protein were measured by immunohistochemical staining and Western blotting, and MTT assay was used to measure the changes of proliferation. Results： Survivin protein expression in group A was decreased significantly in human glioma cell line CHG-5 than other groups（P〈0.05）, and the proliferating rate of CHG-5 in group A was also significantly inhibited（P〈0.05）. Conclusion： Ultrasonic microbubble intensifier transfection combined with ultrasound irradiation is a promising method in gene transfection effectively and noninvasively.

Following Inhibition of BCL-2 by Antisense Oligonucleotides Compensatory Suppression of Apoptosis Involves the Direct Signal Transduction Pathway of LNCaP Cells

Previously we have shown that when LNCaP cells are treated with antisense oligonucleotides (oligos) directed against BCL-2, compensatory changes in non-targeted genes take place in attempts to restore apoptosis and promote tumor aggressiveness. In addition to the inhibition of BCL-2, we find that the apoptosis promoter caspase-3 activity is suppressed, the transcription activity of STAT-3 is enhanced, while other regulators (bax, clusterin, AKT-1) associated with mitochondrial regulated apoptosis and caspase cascade are either unchanged or undetectable. We now evaluate proteins associated with the second pathway of apoptosis activation mediated by direct signal transduction involving fas, fas-ligand (a tumor necrosis factor-like cell surface receptor aka CD95), as well as the similar programmed death cell surface receptor (PD-1) and its respective ligand (PD-1L). This study evaluates the growth inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against BCL-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)];and employing RT-PCR. The expression of these four proteins was evaluated. Expression of fas-ligand, PD-1 and PD-L1 were all significantly enhanced, whereas fas itself was undetectable. This suggests that in addition to pathways associated with the mitochondrial pathway of apoptosis, compensatory changes occur in the direct signal transduction pathway of this process. In addition to alterations in androgen sensitivity, growth factor expression and oncogene expression, these data suggest that suppressive BCL-2 therapy involves multiple pathways, including those involved with immune targeting and cytotoxicity and must be taken into account to make gene therapy more efficacious.

Combined transfection of Bcl-2 siRNA and miR-15a oligonucleotides enhanced methotrexate-induced apoptosis in Raji cells

Objective: B-cell lymphoma 2 (Bcl-2) is an important member of the Bcl-2 family of proteins that regulate the induction of apoptosis. This study aims to investigate whether Bcl-2 small interfering RNA (siRNA) combined with miR-15a oligonucleotides (ODN) could enhance methotrexate (MTX)-induced apoptosis in Raji cells. Methods: Chemically synthesized miR-15a ODN and Bcl-2 siRNA were transfected in Raji cells by using a HiPerFect Transfection Reagent and then combined with MTX. Expression levels of Bcl-2 protein were detected by Western blot. Cell proliferation was determined by CCK8 assay. The rate of cell apoptosis was determined by Annexin V/PI double staining. The morphology of apoptotic cells was observed by Hoechst-33 258 staining. Results: After the cells were transfected with miR-15a ODN combined with Bcl-2 siRNA, Bcl-2 protein levels were evidently decreased. CCK8 assay showed that cell proliferation was significantly decreased and was significantly lower in miR-15a ODN combined with Bcl-2 siRNA plus MTX group than in miR-15a ODN with methotrexate group, Bcl- 2 siRNA with MTX group, and single MTX group (P<0.05). Hoechst 33258 staining revealed numerous apoptotic cells. AnnexinV/PI double staining showed that the apoptotic rates were (13.13±1.60)%, (34.47±2.96)%, (32.87±3.48)%, and (45.47±2.16)% in MTX, Bcl-2 siRNA plus MTX, miR-15a ODN plus MTX, and miR-15a ODN combined with Bcl- 2 siRNA plus MTX groups, respectively. Among these groups, the apoptotic rate of miR-15a ODN combined with Bcl-2 siRNA plus MTX group was the highest; this apoptotic rate was also significantly different from that of miR-15a ODN or Bcl-2 siRNA plus MTX (P<0.05). Conclusions: Bcl-2 siRNA combined with miR-15a ODN could enhance MTX-induced apoptosis in Raji cells. Bcl-2 siRNA and miR-15a combined with MTX may be a useful approach to improve the treatment effects on lymphoma.

Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis

AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.

SELECTION AND THEIR ANTITUMOR ACTIVITY OF ANTISENSE OLIGONUCLEOTIDES TARGETING MESSENGER RNA OF VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2

Objective： To select the antisense oligonucleotides （asONs） which hybridize with the mRNA of vascular endothelial growth factor receptor2 （VEGFR2, also named as kinase insert domain-containing receptor：KDR） in an effective and specific way, and to investigate their antitumor activity in MCF-7 cells. Methods： The effective antisense oligonucleotides were chosen by computer prediction combined with oligonucleotide microarrays. The inhibition effect on MCF-7 cells proliferation was measured by MTT; and VEGFR2 expression was surveyed by Western-blotting and RT- PCR. Results： Using predicting secondary structure of VEGFR2 mRNA with RNA folding program, computer prediction designed 30 antisense oligonucleotide probes that were directed to local loose regions of RNA structure. In 30 probes, 4（4/30, 13.33%） antisense oligonucleotides showed strong hybridization intensities in oligonucleotide microarrays test and were selected. All these antisense oligonucleotides targeting 4 different sites of VEGFR2 mRNA lowered the level of VEGFR2 mRNA and protein present in MCF-7 cells. Proliferation of MCF-7 cells was reduced by 4 antisense oligonucleotides, respectively, in which asON1 was the most effective, with the inhibitory rates being 53.06% at 0.8 I.tmol/L. Conclusion： Combination of computer prediction with oligonucleotide microarrays is an effective way in selecting optimal antisense oligonucleotides. The antisense oligonucleotides showed good correlation between their antitumor activity and the hybridization intensities. The antisense oligonucleotides targeting VEGFR2 mRNA demonstrated prominent antitumor role in vitro.

P53 Regulation of Leukemia Cells with the Blockage of MDM2 by Antisense Oligonucleotides

Summary： The changes of expression and function of MDM2 and P53 by MDM2 specific antisense oligonucleotides were investigated in HL60 cells. Cells were divided into control group, AS group （MDM2 specific antisense oligonucleotides group）, cisplatin group, and combined treatment group. FCM analysis and Western blot and RT-PCR were used to estimate apoptosis and the expression of MDM2 and P53. Our results showed that the transfection of MDM2 specific antisense oligonucleotides obviously inhibited MDM2 expression （P〈0.01） and increased the expression of P53 （P〈0.05）. Apoptosis rate were reduced by MDM2 specific antisense oligonucletides and cisplatin （P〈0.01）. It is concluded that MDM2 specific antisense oligonucletides can inhibit the expression of MDM2, induce the expression of P53 and increase the apoptosis of leukemia cells after chemotherapy.

Effects of human papillomavirus type-specific antisense oligonucleotides on the proliferation of cervical cancer cells containing papillomavirus type 16

Objective: To investigate the effects of antisense phosphorothioate oligonucleotides (S--ODNs ) on theproliferation of cervical cancer cell line SiHa, which harbor HPV16. Methods: Three different antisenseoligodeoxynucleotides were used in this study. Two of them, S--ODN1 (AE6 ) and S--ODN2 (AE7 ), arecomplementary to sequences flanking the start codons of HPV16 E6 and E7 open reading frames,respectively. SODN3 is a nonsense sequence. SiHa cells were treated with various concentrations of oligos. Growth assay and 3HTdR incorporation were used. Results: Both AE6 and AE7 markedly inhibited the proliferation and DNA synthesisof SiHa cells which harbor HPV16 but had little effects on HeLa cells that do not. S--ODN3 had none of theseeffects. Conclusion: Antisense oligodeoxynucleotides can inhibit the proliferation and DNA synthesis of HPV16positive human cervical cancer cells.

A Novel Substrate for in situ Synthesis of Oligonucleotides: Hydrolyzed Microporous Polyamide-6 Membrane

A novel substrate for in situ synthesis of oligonucleotide was prepared by hydrolyzing microporous polyamide-6 membranes in a 0.01mol/L NaOH/(H2O-CH3OH) mixture medium. The formation of amines (NH2) on the surface was proved by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The treated membrane was applied for in situ synthesis of oligonucleotide and a single step coupling efficiency determined by ultraviolet (UV) spectra was above 98 %.

In situ Synthesis of Oligonucleotides on Plasma Treated Polypropylene Microporous Membrane

Polypropylene microporous membranes were treated with plasma in a mixture of N2 and H2 (1:2 in volume). Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FT1R), X-ray photoelectron spectroscopy (XPS) and ultra-violet (UV) spectra demonstrated the success of grafting amino groups. The density of the polar amino groups on the membrane surface is about 0.59 μmol/cm2. The as-treated membranes were successively applied to the in situ synthesis of oligonucleotides and an average coupling yield was more than 98%. The surface feature of the treated membrane is suggested to be responsible for its advantage over a glass slide.

Application of Artificially Induced Double-strand Breaks (DSB) and Triplex-forming Oligonucleotides (TFO) in the Improvement of Gene Targeting Efficiency

Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB） and triplex forming oligonucleotide （TFO） are currently developed methods to improve the targeting efficiency. This paper summarized the basic principles, design ideas and application in gene targeting efficiency improvement of these two methods, analyzed and com- pared their characteristics, and finally proposed prospects for their future development.

Effects of connective tissue growth factor antisense oligonucleotides on the proliferation and collagen synthesis of the cultured human keloid fibroblasts in vitro

Objective: To explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid. Methods: CTGF antisense oligonucleotides (ASODN) conjugated with isothiocyananate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblasts (HKFs) culture media. The intracellular distribution of CTGF ASODN was observed by fluorescence microscopy in the fixed HKFs. The proliferation of HKFs was measured by MTT test. The collagen synthesis of HKFs was measured by 3H-proline incorporation method. Results: Compared with control group, the CTGF ASODN can inhibit the proliferation and collagen synthesis of the HKFs (P<0.01). Conclusion: CTGF ASODN has anti-fibrotic effects on keloid in vitro, and CTGF play an important role in promoting the fibrosis of keloid.

Depressive Effect of the Antisense Oligonucleotides of C-myc and PCNA on the Proliferation of VSMC

Objectives To study the depressive effect of the antisense oligonuceotides （ASODN） of c-myc and proliferating cell nuclear antigen （PCNA） on the proliferation of VSMC. Methods Taking the VSMC obtained from rat aorta tho- racalis cultured 4 - 8 generation as research object. The objects were divided into three groups to carry out control study： control group, PCNA ASODN group and c-myc ASODN group. The ASODNs＇ working concentration all were 1 ： 50. The depressive effect of ASODN on VSMC proliferation was investigated by cell counting, MTT and ^3H-TdR incorporation assay; PCNA and c-myc expression were detected by immunohistochemical method after transferring PCNA and c-myc ASODN into VSMC. Results ① PCNA and c-myc ASODN could inhibit the proliferation of VSMC significantly, compared with control group （ P 〈 0. 05）. ②Transferring PCNA and c-myc ASODN into VSMC obtained successfully ; the corresponding gene was inhibited obviously ; compared with control group （ P 〈 0. 05 ）. Conclusions PCNA and c-myc might play a considerable role in the VSMC proliferation process. The corresponding gene could be depressed successfully after transferring PCNA and c-myc ASODN into VSMC, and then the proliferation of VSMC was slowed down. This study presented a beneficial proposal and theoretical fundament for atherosclerotic treatment.