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AIM: To observe the inhibitory effect of antisense eukaryotic expression vectors for c-myc on rat airway smooth muscle cells. METHODS: Antisense and sense eukaryotic expression vectors for c-myc pcDNA3-myc-antisense and pcDNA3-myc-sense were constructed. Lipofectin was used to introduce antisense and sense eukaryotic expression vectors for c-myc into rat. The inhibitory effect was assayed by MTT cell proliferation assay. Cell cycles were detected by flow cytometry technology. The expression of c-Myc was detected by immunohistochemistry. RESULTS: The results showed that antisense eukaryotic expression vector for c-myc inhibited rat airway smooth muscle cells proliferation. Rat airway smooth muscle cells were prohibited in S phase and the expression of c-Myc was decreased after antisense eukaryotic expression vectors for c-myc were transfected into cells. CONCLUSION: Antisense eukaryotic expression vectors for c-myc inhibit rat airway smooth muscle cell proliferation. 相似文献
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AIM: To study the induction of apoptosis by c-myc antisense oligonucleotide in osteosarcoma cell (MG-63).METHODS: The designed c-myc antisense oligonucleotide fragment was transfected into human osteosarcoma MG-63 cells. The cell growth and apoptosis were measured by the methods of MTT, FCM, HE staining and transmission electron microscopy.RESULTS: The results showed that the proliferation of human osteosarcoma MG-63 cells was inhibited and apoptotic rate was 37.92% when treated with c-myc antisense oligonucleotide at the does of 10.0 μmol/L for 48 h. c-myc antisense oligonucleotide (10.0 μmol/L) also inhibited the expression of c-myc protein.CONCLUSION: c-myc antisense oligonucleotide is able to induce apoptosis in human osteosarcoma MG-63 cells. 相似文献
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AIM:To investigate the effects of thrombospondin 1 on transforming growth factor β1 induced rat cardiac fibroblasts (CFs). METHODS: CFs of neonatal Sprague -Dawley (SD) rats were isolated with the method of digestion and differential anchoring velocity. The proliferation and collagen synthesis of rat CFs were observed with MTT and hydroxyproline. The expression of TSP-1mRNA was analyzed by RT-PCR.RESULTS: The dose and time-dependent effects of TGF-β1 were observed. Expression of TSP-1 was increased significantly (P<0.01). Stimulation of CFs with TGF-β1 (20 μg/L, 24 h) significantly increased CFs proliferation and collagen synthesis (P<0.01). TSP-1 antisense oligonucleotide effectively inhibited TGF-β1 induced CFs proliferation and collagen synthesis (P<0.01).CONCLUSION:The proliferation and collagen synthesis of CFs induced by TGF-β1 are inhibited by TSP-1 antisense oligonucleotide, which may exert helpful effect on anti-fibrosis. 相似文献
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DENG Hui LUO Huan-min GAO Qin LI Xiao-guang XIAO Fei ZHANG Pei-fen WENG Wen 《园艺学报》2004,20(6):981-984
AIM: To investigate the inhibitory effect of Tanshinone ⅡA on U251 glioma cell line and its mechanism. METHODS: MTT was used to measure the levels of the proliferation in U251 cultured with Tanshinone ⅡA at different concentrations. The effects of Tanshinone ⅡA on cell cycle of U251 were observed by FCM. The expression of proto-oncogene c-myc was measured by RT-PCR. RESULTS: The proliferation of U251 was obviously inhibited by Tanshinone ⅡA in a dose dependent manner. The inhibitory rate came to the peak at (54.2±0.9)%, when cultured with Tanshinone ⅡA at 0.10 g/L. The outcome of FCM showed that the proportion of G0/G1 phase cells was increased and the proportion of S phase cells was reduced obviously, when cultured with Tanshinone ⅡA at 0.10 g/L for 3 days. The RT-PCR experiment showed that the expression of proto-oncogene c-myc was notably decreased, when the dose of Tanshinone ⅡA was increased. CONCLUSION: Tanshinone ⅡA inhibited the proliferation of U251 and the expression of proto-oncogene c-myc. 相似文献
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AIM: To down-regulate expression of c-myc through antisense therapy and to investigate its effect on the sensitivity of osteosarcoma MG-63 cells to cisplatin-induced apoptosis. METHODS: The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed and transfected into osteosarcoma MG-63 cells in vitro in order to down-regulate the expression of c-myc, and the change in the sensitivity to cisplatin-induced apoptosis was observed. MTT, Western blot, RT-PCR, flow cytometry (FCM) and electron microscope were used to evaluate tumor cell proliferation in vitro, genes expression related to apoptosis regulation and effects on the sensitivity of osteosarcoma MG-63 cells to cisplatin-induced apoptosis. RESULTS: Ad-Asc-myc down-regulated the expression of c-myc protein after transfected MG-63 cells for 48 h, combined with the treatment of 2.0 mg/L cisplatin for 2 h inhibited tumor cell proliferation in vitro by 38.0%. RT-PCR revealed that Ad-Asc-myc down-regulated the expression of Bcl-2 and up-regulated the expression of Bax. No appreciable change was observed in the expression of E2F-1. FCM showed that Ad-Asc-myc induced apoptosis in intransfected cells, and rendered it more sensitive to cisplatin. CONCLUSION: Antisense c-myc is able per se to induce apoptosis and sensitize osteosarcoma cells to cisplatin-induced apoptosis. 相似文献
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AIM: To investigate the effect of chloride channel CLCN2 antisense oligonucleotide on the cell injury of malignant U251 glioma cells induced by cisplatin (DDP). METHODS: The experiment was divided into 4 groups: control group (nonsense oligonucleotide), CLCN2 antisense oligonucleotide group, DDP group (DDP＋nonsense oligonucleotide), DDP＋CLCN2 antisense oligonucleotide group. The viability of U251 cells was measured by MTT assay, CLCN2 mRNA level was determined by RT-PCR, cell apoptosis was measured by TUNEL assay. RESULTS: Compared to the control group, the cell viability, CLCN2 and cyclinD1 mRNA decreased in CLCN2 antisense oligonucleotide group, DDP treated group and CLCN2 antisense oligonucleotide with DDP treated group, cells apoptosis increased. Compared to DDP group, the cell viability (P<0.05) and CLCN2 mRNA decreased in CLCN2 antisense oligonucleotide with DDP treated group, and cells apoptosis increased (P<0.01). Compared to CLCN2 antisense oligonucleotide group, CLCN2 mRNA significantly decreased (P<0.01) in CLCN2 antisense oligonucleotide with DDP treated group. CONCLUSION: CLCN2 antisense oligonucleotide inhibits the expression of CLCN2 mRNA in U251 cells. Inhibition of CLCN2 mRNA facilitates the cell injury of U251 cells induced by DDP. The decrease in CLCN2 mRNA is involved in the mechanism of cell injury by DDP. 相似文献
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AIM: To explore the effect of SOCS3 gene on the expression of c-myc mRNA and proliferation of rat pulmonary arterial smooth muscle cells (PASMCs) under hypoxia conditions. METHODS: PASMCs was cotransfected with pEFSOCS3 and pSV2neo by lipofectamine, and positive cell clones were obtained after being screened with G418. Expressions of SOCS3 protein in PASMCs before and after transfection were detected by immunocytochemistry, respectively. Before and after transfection, PASMCs were exposed to normoxic and hypoxia conditions at various time points, respectively, and the expressions of c-myc mRNA were assessed by semi-quantitive RT-PCR. ［3H］-TdR incorporation method was used to detect the cell proliferation. RESULTS: The expression of SOCS3 protein was confirmed by immunocytochemistry in PASMCs transfected with SOCS3 gene. c-myc mRNA level in the SOCS3 gene-transfected cells exposed to hypoxia were remarkablely lower than that in the control cells, respectively (P<0.01). Compared with the control groups at the same time points, ［3H］-TdR incorporation in SOCS3 gene-transfected cells was significantly low. CONCLUSION: SOCS3 protein may inhibit the proliferation of PASMCs by downregulating the c-myc gene expression under hypoxia conditions. 相似文献
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AIM: To investigate the role of GATA-3 in the pathogenesis of airway inflammation in a Wistar rat asthma model. METHODS: The Wistar rat asthma model was made with conventional method and animals were divided into five groups (10 rats in each group): asthma group (A group), dexamethasone group (D group), antisense oligonucleotide group (AS group), nonsense oligonucleotide group (NS group) and normal control group (N group). Antisense, nonsense oligonucleotide were administered intranasally, and the dexamethasone was injected intraperitoneally. The airway inflammation was observed with HE staining method. The GATA-3 positive cells were stained immunohistochemically. The GATA-3 mRNA expression in pulmonary tissue was investigated with RT-PCR. The GATA-3 protein in pulmonary tissue was detected by Western blotting. RESULTS: In contrast to N group, the expression of GATA-3 mRNA, protein and the amount of inflammatory cells in pulmonary tissue in group A were increased significantly (P<0.01) and were decreased evidently in group AS and D (P<0.01). The expression of GATA-3 mRNA, protein and the amount of inflammatory cells in NS group were obviously increased compared with those in gropu AS and D (P<0.01). The expression of GATA-3 was related to the amount of eosinophils (r=0.995). CONCLUSION: GATA-3 antisense oligonucleotide blocks the expression of GATA-3 gene and the infiltration of eosinophils. GATA-3 plays an important role in the effector phase of allergic airway inflammation in a Wistar rat asthma model. 相似文献
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AIM: To observe the effect of antisense locked nucleic acid (anti-LNA) blocking the translation initiation region of c-myc exon 2 on the viability and apoptosis of hepatocellular carcinoma cells.METHODS: The anti-LNA that was complementary to the translation initiation region of c-myc exon 2 was designed, synthesized, and introduced into the HepG2 cells by cationic liposome-mediated transfection. The mRNA and protein levels of c-Myc in the cells were determined by RT-PCR and Western blot. The change of cell apoptosis was analyzed by flow cytometry, and the toxicity of anti-LNA to the cells was detected by MTT assay.RESULTS: Five days after transfection, the mRNA level of c-Myc in anti-LNA group was 0.335±0.016, and the protein level was 0.448±0.037, significantly lower than those in control group (both P<0.05). The ratio of apoptotic cells in anti-LNA group was 32%±6%, which was higher than that in control group (P<0.05).CONCLUSION: Antisense locked nucleic acid targeting at the translation initiation region of c-myc exon 2 shows strong inhibitory effects on the apoptosis of hepatocellular carcinoma cells. 相似文献
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AIM: To elucidate the co-transfection of platelet derived growth factor B(PDGF-B) antisense oligonucleotide and tissue-type plasminogen activator gene to prevent vascular anastomotic restenosis after coronary bypass.METHODS: A dog model of vascular anastomotic restenosis after coronary bypass was constructed.A constructed tissue-type plasminogen activator(tPA) gene plasmid and a designed PDGF-B oligonucleotide were used to transfect into the dog cardiomyocytes and anastomotic vascular smooth muscle cells(VSMCs) at the same time of coronary bypass,using a therapeutic ultrasound for the gene delivery.Effects of these two genes on thrombosis in local anastomotic vessels,the expressions of proliferating cell nuclear antigen(PCNA) and PDGF-B mRNA by VSMCs and the proliferation of vascular intima were observed with the methods of routine pathological,immuno-histochemical staining,in situ hybridization and morphometry.RESULTS: PDGF-B antisense oligonucleotide and tissue-type plasminogen activator gene were succesfully transfected.These two genes significantly inhibited the expressions of PCNA and PDGF-B mRNA in intimal VSMCs with the inhibitory rates of 65.01% and 81.75%,respectively.The local intimal thickness and area also reduce markablely and the thrombosis of the anastomosis was prevented followed by the reduction of the anastomotic restenotic rate of 62.63%.CONCLUSION: Co-transfection of PDGF-B antisense oligonucleotide and tissue-type plasminogen activator gene inhibits the dog experimental anastomotic restenosis after coronary bypass. 相似文献
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AIM: To explore the role of Akt/NF-κB pathway in immune-complexes-induced monocyte chemoattractant protein-1 (MCP-1) and colony stimulating factor-1 (CSF-1) expression in Mesangial Cells. METHODS: Primary murine glomerular mesangial cells were cultured in vitro and divided into control group, stimulation group and antisense, sense and mismatched oligodeoxynucleotide group. In control group, the cells were stimulated with monomeric IgG after treatment with 0.5% lipofectin for 8 h. In stimulation group, the cells, which had been treated with 0.5% lipofectin for 8 h, were stimulated with aggregated IgG. In antisense, sense and mismatched oligodeoxynucleotide group, being transduced antisense, sense and mismatched oligodeoxynucleotide respectively with 0.5% lipofectin 8 h, the cells were stimulated with AIgG. MCP-1 and CSF-1 in supernatant were deteced with ELISA. In addition, RT-PCR was used to determine MCP-1 and CSF-1 mRNA expression, and EMSA to investigated the activation of NF-κB. RESULTS: Mesangial cells cultured in vitro had a low level NF-κB activation and a low level constitutive expression of MCP-1 and CSF-1. Stimulated with AIgG, activation of NF-κB was markedly increased(0.35±0.06 vs 0.75±0.16, P＜0.01), expression of MCP-1 and CSF-1 mRNA (0.48±0.03 vs 0.72±0.02, P＜0.05; 0.44±0.01 vs 0.59±0.02, P＜0.05), MCP-1 and CSF-1 levels in supernatant(15.52±1.81 vs 43.05±3.18, P＜0.05; 389.06±13.75 vs 764.22±31.78, P＜0.05) were markedly increased. Akt1 antisense oligodeoxynucleotide markedly inhibited immune-complexes-induced NF-κB activation, MCP-1 and CSF-1 mRNA and protein expression. CONCLUSION: Akt/NF-κB pathway mediates immune-complexes-induced MCP-1 and CSF-1 expression in mesangial cells. It suggests that Akt/NF-κB pathway may be a new therapy target for macrophage recruitment and activation in immune complexes nephritis. 相似文献
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“中国园艺学会热带南亚热带果树分会”是由广东省农业科学院果树研究所、福建省农业科学院果树研究所、仲恺农业技术学院园艺系、中国热带农业科学院南亚热带作物研究所等单位联合申请,并经中国园艺学会第九届六次常务理事会扩大会议讨论同意成立的。经筹委会讨论决定,将于200 相似文献
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HUA Ping LIU Jia-liang YANG Song-ran JIANG Hui-qi WANG Meng TAO Jun YANG Yan-qi 《园艺学报》2013,29(8):1502-1507
AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs. 相似文献
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AIM: To observe the proliferation and apoptosis of ovarian cancer cells by silencing the expression of human pituitary tumor-transforming gene 1 ( hPTTG1 ) using RNA interference technique.METHODS: The chemically synthesized siRNA targeting hPTTG1 was transfected into ovarian cancer cell line A2780 in vitro. The expression levels of hPTTG1 and c-myc were examined by RT-PCR and Western blotting. Cell proliferation was measured by MTT colorimetric assay and -TdR incorporation test. Cell apoptosis was detected by flow cytometry with annexin V/PI and TUNEL labeling.RESULTS: The expression of hPTTG1 at mRNA and protein levels was inhibited after transfection of hPTTG1 siRNA. The inhibitory efficiency was 70.5%±3.9% and 63.8%±4.5%, respectively. The absorbance began to decrease 24 h after transfection of hPTTG1 siRNA,and the highest inhibitory rate was 42.9%±5.2% at 48 h post-transfection. Radioactive incorporation of -TdR in hPTTG1 siRNA group was lower than that in normal and negative groups. The survival rate declined while the apoptotic rate and necrotic rate increased in hPTTG1 siRNA group. Apoptotic index in hPTTG1 siRNA group was higher than that in normal and negative groups. The expression of c-myc at mRNA and protein levels was down-regulated.CONCLUSION: Cell proliferation is inhibited and cell apoptosis is induced by hPTTG1 siRNA through down-regulating the expression of c-myc. hPTTG1 can be regarded as a candidate gene for ovarian cancer gene therapy. 相似文献
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AIM: To study the role of c-myc oncogene in L6565 leukemia oncogenesis and the effects of therapy by inhibition of its expression with antisense c-myc. METHODS: A recombinant retroviral vector containing antisense c-myc of the murine (pGNCas) was constructed and then transfected into PA317 cells by the method of calcium phosphate precipitation. L6565 clone cells were infected with retrovirus particles. Stable integretion of antisense c-myc was shown by PCR. The change of the malignance and phenotype of L6565as were detected by the examination of the growth, morphology, cells cycle, agar assay and expression of c-myc. RESULTS: The shape of most L6565as cells became spherical. The growth of L6565as was inhibited compared to control cells. The analysis of cells cycle: L6565as cells were arrest in G0/G1 phase, decreased in S phase. The ability of L6565as cells to form colony in soft agarose was significantly suppressed. c-myc in L6565as cells was lowly expressed. CONCLUSION: (1)c-myc plays a critical role in L6565 leukemia oncogenesis; (2)Inhibition of expression of c-myc makes partly reversion of malignant phenotype of L6565 murine leukemia clone cells. 相似文献
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AIM:To investigate whether the bcl-2 antisense oligonucleotide increases the sensitivity of HL60 and K562 cell lines to daunorubicin.METHODS:IC50 for HL60 and K562 was determined with MTT method, the expression levels of Bcl-2 protein were assayed by immunofluorescence using fluoresce isothiocyanate labeling. In addition, apoptosis was detected by morphological observation and flow cytometric analysis of DNA fragmentation.RESULTS:It was found that the two oligonucleotides directed against the coding region and the translation initiation of bcl-2 mRNA, combined respectively with daunorubicin, inhibited expression of bcl-2 protein, increased apoptosis in HL60 and K562 cells, and decreased IC50 of daunorubicin significantly (P＜0.05). Compared to the antisense oligonucleotide directed against the translation initiation of bcl-2 mRNA, the antisense oligonucleotide directed against the coding region showed stronger effects in the aspects of increasing the sensitivity of HL60 cells to daunorubicin (P＜0.05).CONCLUSIONS:These two antisense sequences in the translation initiation and the coding region of bcl-2 mRNA increased the sensitivity of HL60 and K562 cell lines to daunorubicin in a sequence-specific manner. 相似文献
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ZHOU Zhen-hai LI You-ji CHEN Yun-xian LI Xiao-ying YU Xue-qing LI Juan LUO Shao-kai YUAN Yao-guang 《园艺学报》2003,19(9):1257-1260
AIM:To study the effect of Bene Jones protein (BJP) from multiple myeloma(MM) patient and TGF-β1 on cultured renal proximal tubular cell(PTC) proliferation.METHODS:[H3]TdR incorporation was used to study the effect of λBJP and TGF-β1 on cultured rat NRK.52E PTC proliferation, the expression of TGF-β1 in the supernatant of PTC cultured with BJP was assessed with ELISA.RESULTS:① [H3]TdR incorporation of PTC was inhibited by BJP in a dose-dependent manner, when co-cultured with 100-800 μmol/L BJP and 2.0 μg/L TGF-β1, the [H3]TdR incorporation was lower than that of BJP alone, especially when BJP≥400 μmol/L;②The expression of TGF-β1 in the supernatant of PTC cultured with BJP was increased, especially when BJP≥400 μmol/L(P＜0.05);③ The [H3]TdR incorporation of PTC was also inhibited by exogenous TGF-β1 in a dose-dependent manner.CONCLUSION:λBJP has antiproliferative effect on rat PTC in vitro, The effect is related with stimulating the PTC to produce excessive TGF-β1, which also has antiproliferative effect on PTC in some degree. 相似文献