INHIBITION EFFECT OF VASCULAR ENDOTHELIAL GROWTH FACTOR ANTISENSE OLIGODEOXYNUCLEOTIDES ON C6 GLIOMA

Objective: To explore the probability of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides as a developing new therapeutic strategy for glioma. Methods: VEGF protein expression was detected by S-P immunohistochemical technique. Tumor cell apoptosis was observed by TUNEL method. Results: Compared with control, VEGF protein expression was inhibited by antisense oligodeoxynucleotides in vitro. And the inhibitory effects increased with the increasing concentration. VEGF positive rate was 82.10% in control group, while in 2.5, 5, 10 mmol/L AODN groups, they were 70.00%, 57.85%, 53.20% respectively. No inhibition effect was found in the cell lines treated with missense and sense oligodeoxynucleotides. In vivo, antisense oligodeoxy-nucleotides therapy also inhibited VEGF protein expression and induced the increase of apoptotic tumor cells. However, it has no effect on tumor cell proliferation. Conclusion: It is hopeful that VEGF antisense oligodeoxynucleotides may be a new gene therapy method to glioma through its antiangiogenesis effect by inhibition of VEGF.

Effect of C-myc Antisense Oligodeoxynucleotides on Hypoxia-induced Proliferation of Pulmonary Vascular Pericytes

To study the effect of c myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin 11 dUTP labeled cDNA, 3H thymidine incorporation, immunocytochemical technique and image analysis methods were used to observe the effect of c myc antisense ODNs on expression of c myc gene and proliferating cell nuclear antigen (PCNA), and 3H thymidine incorporation of PC induced by hypoxia. The results showed that hypoxia could significantly enhance the expression of c myc and PCNA ( P <0.01), and elevate 3H thymidine incorporation of PC ( P <0.01), but antisense ODNs could significantly inhibit the expression of c myc and PCNA ( P <0.05), and 3H thymidine incorporation of PC ( P <0.01). It was suggested that hypoxia could promote the proliferation of PC by up regulating the expression of c myc gene, but c myc antisense ODNs could inhibit hypoxia induced proliferation of PC by downregulating the expression of c myc gene.

Inhibitory Effect of Tissue Transglutaminase (tTG) Antisense Oligodeoxynucleotides on tTG Expression in Cultured Bovine Trabecular Meshwork Cells

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides （tTG-ASDON） on tTG expression in cultured bovine trabecular meshwork cells （BTMCs） in vitro and explore a new treatment alternative for primary open angle glaucoma （POAG）, the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTCr-ASDON2 were significantly decreased as compared with that of the controls （P〈0.05）. On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.

Com bined effect of alphafetoprotein antisense oligodeoxynucleotidesand 5-fluorouracil on human hepato ma cell growth

Objective To investigate the effect of alpha fetoprotein (AFP) antisense phosphorothioate oligodeoxynucleotides in combination with 5 fluorouracil (5 FU) on the growth of BEL 7404 human hepatoma cells in vitro Methods Phosphorothioate oligodeoxynucleotides were synthesized by β cyanoethyl phosphoramidite chemistry AFP gene expression in human hepatoma cells was determined by avidin biotin peroxidase complex (ABC) immunocytochemical method Cell growth in the presence or absence of experimental agents was measured using 3 (4, 5 dimethylthiazol 2 yl) 2, 5 dipheny ltetrazolium (MTT) microculture tetrazolium assay Results AFP antisense oligomers markedly suppressed the growth of BEL 7404 human hepatoma cells in vitro by sequence specific blocking of the AFP gene expression in the cells (P<0 05) 5 FU also inhibited the hepatoma cell growth in a dose dependent manner when used alone (P<0 05) The combined treatment with AFP antisense oligomers and 5 FU showed significantly enhanced hepatoma cell growth inhibition than either AFP antisense or 5 FU treated cells alone (P<0 05) Conclusion Combined use of AFP antisense oligomers and 5 FU could more effectively inhibit the growth of BEL 7404 human hepatoma cells in vitro

EFFECT OF TWO NEW BCL-2 ANTISENSES ON DRUG-SENSITIVITY OF CELLS FROMN LEUKEMIA PATIENTS

Objective: To investigate the effect of two antisense oligonucleotides on cell surviving, bcl-2 expression and apoptosis of leukemia cells. Methods: The experimental assays were performed with cell culture, immunochemistry and flowcytometry. Results: The two antisense oligodeoxynucleotides, combined with Vp16 or Ara-c or DNR, were able to decline the survival rate of myeleukemic cells, downregulate bcl-2 gene expression and induce apoptosis of leukemic cells significantly, as compared with Vp16 or Ara-c or DNR alone. Conclusion: It is possible for the two new bcl-2 antisenses to be developed into clinical trials for leukemia and tumor with bcl-2 gene overexpression. Key words Leukemia - Antisense oligodeoxynucleotides - bcl-2 gene - drug-resistance CLC number R733.7 Foundation item: this work was supported by the Key Foundation of Science & Technology Program of Guangzhou (No. 2001-Z-037-01); and the Nature Science key Foundation of Guangdong Province(No. 021195).Biography: LEI Xiao-yong(1970–), male, associate professor, doctor of medicine, Institute of Pharmacy and Pharmacology, Nanhua University, majors in tumor pharmacology.

Inhibitory effects of antisense oligodeoxynu-cleotide on expression of vascular endothelia growth factor by human hepatocarcinoma cells

BACKGROUND: It is widely recognized that the growth of solid tumor depends on angiogenesis. Vascular endothelia growth factor (VEGF) is an endothelial cell-specific mito- gen that promotes angiogenesis in solid tumor. Inhibition of angiogenesis is considered a promising approach for cancer therapy, and treatments including administration of antisense drugs and RNA interference for the VEGF gene are geared to the suppression of tumor angiogenesis. METHODS: As a new approach for gene therapy of hepato- cellular carcinoma (HCC), four groups of antisense oli- godeoxynucleotide (ASODN) (A-Cap, A-AUG, A-UGA and A-Exon-3) were used to block the expression of VEGF, then VEGF mRNA and protein were detected by RT-PCR and Western blot. RESULTS: After treatment with ASODN, the relative VEGF mRNA levels of A-Cap, A-AUG, A-UGA, and A- Exon-3 were decreased significantly to (32±9)%, (63 ± 1)%, (86 ±3)%, and (70 ±5)%, respectively(F=64.18, P< 0.001). The relative VEGF protein levels of A-Cap, A- AUG, A-UGA and A-Exon-3 were decreased significantly to (41 ±5)%, (59 ±3)%, (88 ±7)%, and (79 ±9)% respec- tively (F =60.64, P<0.001). CONCLUSIONS: Among the four ASODNs, the ASODN for Cap structure showed the strongest inhibitory effect and that for A-UGA, the least (P <0.05 ). The inhibitory effect of ASODN on the expression of VEGF proteins was similar to that of VEGF mRNA expression.

Inhibition of telomerase with human telomerase reverse transcriptase antisense increases the sensitivity of tumor necrosis factor-α-induced apoptosis in prostate cancer cells

Aim： To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase （hTERT） antisense on tumor necrosis factor-α （TNF-α-induced apoptosis in prostate cancer cells （PC3）. Methods： Antisense phosphorothioate oligodeoxynucleotide （AS PS-ODN） was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol （TRAP） and polymerase chain reaction enzyme-linked immunoassay （PCR-ELISA）. hTERT mRNA was measured by reverse transcription PCR （RT-PCR） assay and gel-image system, hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-（4, 5-dimethylthiazol-2-yl）-2,5-Diphenyltetrazolium （MTT） assay. Cell apoptosis was observed by morphological method and determined by flow cytometry. Results： The telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide （S PS-ODN） and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-α treatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-α treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group. Conclusion： hTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-α- induced apoptosis of PC3 cells.

Antisense Oligodeoxynucleotide Inhibits Expression of Re-com binantPorcine Follicle-Stim ulating Horm one Receptor

To assess the role of follicle stimulating hormone receptor(FSHR) gene expression in regulating expression of FSHR protein in the plasma membrane, the effects of a porcine FSHR cDNA antisense oligodeoxynucleotide (ODN) on FSHR mRNA levels and 125 I FSH binding were determined in Chinese hamster ovary cells expression recombinant porcine FSHR (pFSHR CHO cells). An 18 mer phosphorothioate endcapped antisense ODN that corresponded to the region surrounding the translation initiation codon of the porcine FSHR cDNA was synthesized. An 18 mer nonsense sequence of identical nucleotide composition, which had little homology to known DNA sequences, was synthesized for use as a control. pFSHR CHO cells were cultured in 24 well plates (10 5 cells/well) in the absence or presence of 1 20 μmol/L antisense or nonsense ODN for 24 h and then assayed for porcine FSHR mRNA, using quantitative reverse transcription and competitive polymerase chain reaction, and for 125 I FSH binding activity. Treatment with 10 μmol/L antisense ODN caused a paradoxical increase in porcine FSHR mRNA from 0.89±0.06 to 1.64±0.08 ng/mg total RNA ( P <0.05). Transfection with lipofectamine and 0.33 μmol/L antisense ODN caused an increase in porcine mRNA from 0.95±0.08 to 1.53±0.07 ng/mg total RAN. This was probably due to upregulation of mRNA synthesis resulting from inhibition of porcine FSHR protein translation. The nonsense ODN had no effect on porcine FSHR mRNA. Antisense, but not nonsense, ODN (10 μmol/L) inhibited membrane binding of 125 I FSH by 13.6± 0.8 % ( P <0.05) in 24 h. Treatment of cells with antisense ODN (10 μmol/L) for 48 h resulted in a 76±1.5 % ( P <0.05) inhibition of 125 I FSH binding. In contrast, transfection with lipofectamine and 0.33 μmol/L antisense ODN at 0 h caused a 76.1±1.3 % ( P <0.05) reduction in binding within 24 h. Binding had returned to 52.3±2.3 % ( P < 0.05) of normal by 48 h. These results indicate that an antisense ODN corresponding to the region of the translation start site of the porcine FSHR cDNA is an effective specific inhibitor of porcine FSHR synthesis and that inhibition of receptor synthesis causes a decrease in functional membrane bound FSHR.

Effect of Antisense Oligodeoxynucleotide Directed to NF-κB-RelA on Bcl-x_L mRNA in Extended Drug Resistance Leukemia Cell Line HL- 60/E6

To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF κB RelA in HL 60/E6 cells. FCM analysis and RT PCR were used to detect the efficiency of liposome mediated ODN transfection and the change of bcl x L mRNA levels after 5 μmol/L phosphorothioate (PS) derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL 60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL 60/E6 cells,but in the cytoplasm of HL 60 cells, the efficiency of liposome mediated ODN transfection was significantly higher than that of null ODN ( P <0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL 60/E6 cells to 5 μmol/L AS PS ODN directed to RelA led to a maximal 40 % decline of bcl x L mRNA levels within 8 h. The inhibition rate of bcl x L mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF κB was involved in regulating bcl x transcription. It was suggested that NF κB was an important factor for drug resistance in leukemia cells.

Transfection of Antisense Oligodeoxynucleotide Inhibits Heparanase Gene Expression and Invasive Ability of Human Pancreatic Cancer Cell in vitro

Extracellular matrix （ECM） degradation is an essential step that allows tumor cells to penetrate a tissue barrier and become metastatic. Heparanase （HPSE） is an endoglycosidase that specifically degrades heparin sulfate proteoglycans （HSPG）, a chief component of ECM, HPSE is not expressed in normal epithelial cells but can be detected in a variety of human carcinomas including pancreatic cancer. In the present study, human pancreatic cancer cell line Panc-1 was transfected with HPSE antisense oligodeoxynucleotide （AS-ODN） in vitro, then the inhibitory effect of ASODN on HPSE gene expression and invasive ability of Panc-1 cells in vitro was examined. The HPSE mRNA and protein expression of Panc-1 cells transfected with AS-ODN was significantly inhibited. However, there were no marked inhibitory effects in Panc-1 cells treated with nonsense oligodeoxynucleotide （NS-ODN）. Moreover, a modified Boyden chamber assay demonstrated that transfection with HPSE AS-ODN significantly inhibited invasive potential of Panc-1 cells in vitro after AS-ODN transfection. This suggests that HPSE AS-ODN may contribute to the inhibition of HPSE mRNA and protein expression, and results in a decrease of the invasive ability of Panc-1 in vitro.

EFFECTS OF CALM /AF10 ANTISENSES ON PRIMARY LEUKEMIC CELLS WITH CALM /AF10 FUSION TRANSCRIPTS IN VITRO

Objectives: To define the involvement of CALM and AF10 fusion transcripts in primary leukaemias with t(10; 11). Methods: The AF10 and CALM fusion in five t(10; 11) leukemia samples were checked by reverse transcriptase-polymerase chain reaction (RT-PCR), and effects of CALM/AF10 antisense phosphorothioate oligodeoxynucleotides (AS PS-ODNs) on chemotherapy sensitivity and apoptosis of leukemia cells in vitro were observed. Results: Five different-sized AF10-CALM products and four different-sized CALM/AF10 products were detected by RT-PCR. The chemotherapy sensitivity of leukemic cells with t(10; 11) to drugs in vitro was lower than that of leukemic cells without t(10; 11). AS PS-ODNs increased the chemotherapy sensitivity and apoptotic rate. There were 4 cases positive at 5 μmol/L concentration, all cases positive at 10 μmol/L and 20 μmol/L concentration, P<0.01 vs only chemotherapeutic drugs (3 cases positive), and chemotherapeutic drugs + S-PS-ODNs (10 μmol/L) (3 cases positive). After cells were treated with 10 μmol/L AS-PS-ODNs + chemotherapeutic drugs for 48 h, 72 h, 96 h, the apoptotic indexes were 14.22±2.86, 29.39±3.57, and 41.26±4.52, respectively. These were significantly higher than those of only chemotherapeutic drugs-treated cells and chemotherapeutic drugs + S-PS-ODNs-treated cells at corresponding time (P<0.01). There was no difference between only drugs group and S-PS-ODNs group at corresponding time (P>0.05). Conclusion: The CALM and AF10 fusion transcripts are involved in the pathogenesis of haematological malignancies with t(10, 11), and is associated with a poor prognosis. AS-PS-ODNs might be useful in therapy of t(10, 11) leukemia. Key words AF10 - CALM - Fusion transcript - Primary leukemia cell - In vitro sensitivity - Antisense oligodeoxynucleotide CLC number R733.7 Biography: LIU Ge-xiu (1968–), male, associate professor, Institute of Hematology, Medical College, Jinan University, majors in hematology.

Effect of MUC2 Antisense Oligodeoxynucleotide on Cell Proliferation,Adhesion,and Proteolytic Enzyme in Human Gastric Carcinoma in vitro

Objective：To investigate the effect of MUC2 antisense oligodeoxynucleotide （ASODN） on cell proliferation, adhesion and proteolytic enzyme inhuman gastric carcinoma cell line （SGC7901）. Methods： Phosphorothioate MUC2 ASODN was synthesized and packaged by lipofectin, and then transfected to SGC7901 cells. The expression of MUC2 mRNA and protein after transfection was detected by reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemical method respectively, and the effect of MUC2 ASODN on cell proliferation, adhesion and proteolytic enzyme was determined by flow cytometry（FCM）, MTT method, Rose Bengal and immunohistochemical method. Results： Compared with the blank control group, ASODN efficiently downregulated the expression of MUC2 mRNA and protein in SGC7901 cells 48h after transfection（P〈0.01）. Various concentrations of ASODN could significantly inhibit the growth of SGC7901, and the inhibition peaked at the 48th hour after transfection（P〈0.05）. The apoptosis rate of the experimental group was about 4.38%, and the percentage of S-phase cells rose while G0/G1-phase cells fell because most of them were blocked at S-phase. In addition, cells treated with MUC2 ASODN showed lower adhesion ability with matrix and endothelial cells than control cells in vitro（P〈0.01）. By immunohistochemical method, the upregulation of E-cadherin proteins and the downregulation of MMP2 and cathepsinD proteins were also observed（P〈0.05）. Conclusion： MUC2 ASODN could efficiently inhibit SGC7901 cell proliferation, reduce cell adhesion ability and downregulate the expression levels of proteolytic enzyme in vitro.

Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Cell Proliferation and Apoptosis in Cells Derived from Human Ovarian Mucinous Cystadenocarcinoma in Vitro

The human ovarian mucinous cystadenocarcinoma （hOMC） cells were co-cultured with antisense oligodeoxynucleotide （antisense ODN）, nonsense ODN, and follicle-stimulating hormone （FSH） at different concentrations for the purpose of observing the effects of antisense ODN to FSH receptor （FSHR） on the proliferation and apoptosis of cultured hOMC cells in vitro. The inhibitory rates of growth were measured by using MTT method on the 2nd, 4th, 6th, 8th and 10th days after the interference of antisense ODN, nonsense ODN, and FSH, respectively. The apoptotic rates and the cell cycles were determined by means of flow cytometry, the apoptosis indexes were detected by using TUNEL, and the expression of caspase-3 was measured by using SP immunohistochemistry. Compared with that in the control group, the proliferative activity of hOMC cells was increased obviously in FSH groups （P〈0.05 or P〈0.01）, decreased distinctly in antisense ODN groups （P〈0.05 or P〈0.01）, and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could significantly antagonize the FSH-promoted cell proliferative activity （P〈0.01）. Compared with those in the control group, the apoptotic rates and the expression of caspase-3 were dramatically increased in the mid- and high-dose antisense ODN groups （P〈0.05 or P〈0.01）, while the number of cells in G1/G0 phase was significantly decreased and that in S phase distinctly increased （P〈0.01）, There was no change in nonsense ODN groups （P〉0.05）, It was suggested that FSH may improve the development of hOMC cells, However, antisense ODN could inhibit proliferative activity and the FSH-promoted proliferative activity in hOMC cells, at the same time, antisense ODN could inhibit hOMC cell growth by inducing apoptosis.

Local Delivery of C-myc Antisense Oligodeoxynucleotide by Gelatin Coated Platinium-Iridium Stent to Prevent Restenosis in a Normal Rabbit Carotid Artery

Objectives To investigate the feasibility and effect of local deliveryof c-myc antisense oligodeoxynucleotide (ASODN) by gelatin coated Platinium-Iridium stent to prevent restenosis in a normal rabbit carotid artery. Methods Gelatin coated Platinium-Iridium stent were implanted in the right carotid arteries of 32 rabbits under vision. Animals were randomized to the control group and the treated group receiving c-myc ASODN (n=16 respectively).7, 14, 30,90 days following the stenting procedure, morphometry for caculation of neointimal area and mean neointimal thickness were performed.The expression of c-myc protein was detected by immunohistochemical methods. Results 32 stents were successfully implanted into the right carotid arteries in 32 animals.Morphometric analysis showed that neointimal area and mean neointimal thickness siginificantly increased continuously up to 12 weeks after stent implantation,and at each time point , neointimal area and mean neointimal thickness were siginificantly smaller in the treated group than control group. (P<0.001,respectively).c-myc protein expression was weak positive or negative in treated group and positive in control group. Conclusions Gelatin coated Platinium-Iridium stent mediated local delivery of c- myc ASODN is feasibility , and it can inhibit neointimal hyperplasia to prevent restenosis in a normal rabbit carotid artery.

Identification and functional analysis of arabinogalactan protein expressed in pear pollen tubes

Arabinogalactan proteins(AGPs)are widely distributed in the plant kingdom and play a vital role during the process of plant sexual reproduction.In this study,we performed a comprehensive identification of the PbrAGPs expressed in pear pollen and further explored their influences on pollen tube growth.Among the 187 PbrAGPs that were found to be expressed in pear pollen tubes,38 PbrAGPs were specifically expressed in pollen according to the RNA-seq data.The PbrAGPs were divided into two groups of highly expressed and specifically expressed in pear pollen.We further tested their expression patterns using RT-PCR and RT-qPCR.Most of the PbrAGPs were expressed in multiple tissues and their expression levels were consistent with reads per kilobase per million map reads(RPKM)values during pollen tube growth,implying that PbrAGPs might be involved in the regulation of pear pollen tube growth.We also constructed phylogenetic trees to identify the functional genes in pear pollen tube growth.Therefore,19 PbrAGPs(PbrAGP1 to PbrAGP19)were selected to test their influences on pollen tube growth.Recombinant proteins of the 19 PbrAGP-His were purified and used to treat pear pollen,and 11 of the PbrAGP-His recombinant proteins could promote pear pollen tube growth.Additionally,pollen tube growth was inhibited when the expression levels of PbrAGP1 and PbrAGP5 were knocked down using an antisense oligonucleotide assay.PbrAGP1 and PbrAGP5 were localized in the plasma membrane and might not alter the distribution of pectin in the pollen tube.In summary,this study identified the PbrAGPs expressed in pear pollen and lays the foundation for further exploring their functions in pollen tube growth.

INHIBITION OF APOPTOSIS BY bcr-abl FUSION GENE IN K562 CELLS

Objective: To investigate the effect of bcr-abl fusion gene on CML cell apoptosis. Methods: Apoptosis of ex-vivo cultured K562 cells were observed after exposure to synthetic 18 mer antisense oligodeoxynucleotide complementary to the bcr-abl junction (b3a2). Results: Apoptosis of K562 cells was significantly increased associated with inhibition of bcr-abl expression. Conclusion: bcr-abl fusion gene formation due to chromosome translocation may be the major mechanism of CML via inhibition of apoptosis.

Inhibition of the VEGF Expression and Cell Growth in Hepatocellular Carcinoma by Blocking HIF-1α and Smad3 Binding Site in VEGF Promoter

In order to investigate the inhibitory effects on the vascular endothelial growth factor （VEGF） expression and cell growth in hapatocellular carcinoma （HCC） by blocking HIF-1α and Smad3 binding site in the VEGF promoter, antisense oligodeoxynucleotides （ASODN） were designed to block HIF-1α and Smad3 binding site in the VEGF promoter. Different concentrations of ASODN and ODN were transfected into HCC cells respectively. The expression of VEGF mRNA and protein was detected by SABC, Western blot and RT-PCR techniques and the inhibitory effects on the expression of VEGF and cell growth of the HCC cells stimulated by the supernatants were determined by using MTT method. Immunohistochestry revealed that after co-inoculation of hepatocellular carcinoma cells with different concentrations of ODN and ASODN for 48 h, there was no significant difference in the expression of VEGF protein between ODN group and control group （P 〈0. 05）, but there was significant difference between ASODN group and control group （P〈 0.05）. At a concentration of 10 μmol/L ASODN, the difference was very significant （P〈0.01）. Western blot and RT-PCR revealed that, after treatment for 48 h at a concentration of 10 μmol/L, the integral gray levels and RNA odds were 59743.2±10412.5 and 0. 783±0. 032 in ODN group, and 38694.5±10925.1 and 0.468±0. 015 in ASODN group, respectively, with the difference being very significant （P〈0. 01）. Antisense ODN could inhibit the growth of HCC cells in a concentration-dependent manner. It was concluded that anti-gene technique of aiming at HIF-1α action site in the VEGF promoter could suppress the VEGF expression and inhibit HCC cell growth, and it is promising that anti-gene technique works as a new gene therapeutic tool for anti-angiogenesis of HCC.

Role of Connective Tissue Growth Factor in Extracellular Matrix Degradation in Renal Tubular Epithelial Cells

In order to investigate the effects of connective tissue growth factor （CTGF） antisense oligodeoxynucleotide （ODN） on plasminogen activator inhibitor-1 （PAI-1） expression in renal tubular cells induced by transforming growth factor β1 （TGF-β1） and to explore the role of CTGF in the degradation of renal extracellular matrix （ECM）, a human proximal tubular epithelial cell line （HKC） was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 （5 μg/L）, the mRNA level of PAI-1 was detected by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhibited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may he a novel way in preventing renal fibrosis.

Enhanced lysosome escape mediated by 1,2-dicarboxylic-cyclohexene anhydride-modified poly-L-lysine dendrimer as a gene delivery system

Antisense oligodeoxynucleotide(ASODN)can directly interfere a series of biological events of the target RNA derived from tumor cells through Watson-Crick base pairing,in turn,plays antitumor therapeutic roles.In the study,a novel HIF-1αASODN-loaded nanocomposite was formulated to efficiently deliver gene to the target RNA.The physicochemical properties of nanocomposite were characterized using TEM,FTIR,DLS and zeta potentials.The mean diameter of resulting GEL-DGL-FA-ASODN-DCA nanocomposite was about 170–192 nm,and according to the agarose gel retardation assay,the loading amount of ASODN accounted for 166.7 mg/g.The results of cellular uptake showed that the nanocomposite could specifically target to HepG2 and Hela cells.The cytotoxicity assay demonstrated that the toxicity of vectors was greatly reduced by using DCA to reversibly block the cationic DGL.The subcellular distribution images clearly displayed the lysosomal escape ability of the DCA-modified nanocomposite.In vitro exploration of molecular mechanism indicated that the nanocomposite could inhibit m RNA expression and HIF-1αprotein translation at different levels.In vivo optical images and quantitative assay testified that the formulation accumulated preferentially in the tumor tissue.In vivo antitumor efficacy research confirmed that this nanocomposite had significant antitumor activity and the tumor inhibitory rate was 77.99%.These results manifested that the GEL-DGL-FA-ASODNDCA nanocomposite was promising in gene therapeutics for antitumor by interacting directly with target RNA.

Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Expression of Proliferating Cell Nuclear Antigen and Vascular Endothelial Growth Factor in Primary Culture Cells Derived from Human Ovarian Mucinous Cystadenocarcinoma

The effects of antisense oligodeoxynucleotide （antisense ODN） to follicle-stimulating hormone receptor （FSHR） and follicle-stimulating hormone （FSH） on the expression of proliferating cell nuclear antigen （PCNA） and vascular endothelial growth factor （VEGF） were studied in primary culture cells derived from human ovarian mucinous cystadenocarcinoma （OMC）. The primary （）MC cells were cultured with the enzyme digestion method, and the expression of pan Keratin protein and FSHR mRNA was detected for identification of the cells. OMC cells were co-cultured with antisense ODN, nonsense ODN and FSH with different concentrations for 48 h and 72 h. The expression of PCNA and VEGF was detected by using SP immunohistochemistry. Compared with that in the control group, the PCNA and VEGF expression was increased obviously in FSH groups （P〈0.05 or P〈 0.01）, while decreased significantly in antisense ODN groups （P〈0.05 or P〈 0.01） and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could antagonize the increased expression of PCNA and VEGF caused by FSH significantly （P〈0.01）. It was suggested that FSH might promote the development of OMC to some extent. Antisense ODN could inhibit the proliferative activity of OMC cells and the promoting proliferative activity enhanced by FSH.