ABSTRACT
<p><b>OBJECTIVE</b>To observe targeted expression of recombinant lentivirus-mediated (Lv)-hTERTp-TK and Lv-hTERTp-tumstatin in HepG2 cells, and explore the inhibitory effect of their combination on HepG2 cells both in vitro and in vivo.</p><p><b>METHODS</b>Lv-hTERTp-TK and Lv-hTERTptumstatin were used to infect HepG2 and L02 cells at different MOIs. Transfection efficiency was observed by fluorescence microscopy. Expression of TK and tumstatin mRNA was detected by reverse-transcriptase PCR. Proliferation and apoptosis were detected by MTT and flow cytometry, respectively. The HepG2 cells were examined by real time-PCR and western blotting to determine expression level of bcl-2 and VEGF mRNA and protein.A murine hepatocellular carcinoma model was established by injecting 1 * 10(7) HepG2 cells into 30 BALB/c nude mice. The modeled mice were randomly divided into a control group, mock group, Lv-hTERTp-tumstatin group, Lv-hTERTp-TK group, and combination group for four weeks of injections at regular intervals of PBS, Lv-hTERTp-null, Lv-hTERTp-tumstatin, Lv-hTERTp-TK, and Lv-hTERTp-tumstatin plus Lv-hTERTp-TK, respectively.Changes in tumor volume and weight, and cell morphology of tumor and major organs, were assessed by hematoxylin-eosin staining.Microvascular density of tumor tissue and cell apoptosis were assessed by immunohistochemical and TUNEL staining, respectively.</p><p><b>RESULTS</b>The Lv-infected HepG2 cells, and not the Lv-infected L02 cells, expressed TK and tumstatin. Lv-hTERTp-TK and Lv-hTERTp-tumstatin, alone or in combination, inhibited proliferation and increased apoptosis of the HepG2 cells, but the combination was more effective than either alone (P less than 0.05). None of the treatments affected proliferation or apoptosis of the L02 cells (P more than 0.05). The combination also led to a greater reduction of bcl-2 and VEGF than either alone (all, P less than 0.05). Tumor growth was significantly inhibited by the combination (P less than 0.05). In vivo, the combination treatment induced the greatest amount of apoptosis of the HepG2 cells. Cell morphology of major organs such as liver, spleen and kidney were similar to the control group. The combination also produced the most significant effect on tumor microvascular density (P less than 0.05) and the highest apoptosis index (P less than 0.05).</p><p><b>CONCLUSION</b>The HTERT promoter can induce targeted expression of TK and tumstatin in HepG2 cells. Lv-hTERTp-TK combined with Lv-hTERTp-tumstatin can significantly inhibit proliferation and induce apoptosis of human HepG2 cells in vitro and in vivo, which may be related to down-regulation ofbcl-2 and VEGF.</p>
Subject(s)
Animals , Humans , Mice , Apoptosis , Autoantigens , Genetics , Carcinoma, Hepatocellular , Therapeutics , Cell Line, Tumor , Collagen Type IV , Genetics , Down-Regulation , Flow Cytometry , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors , Hep G2 Cells , Lentivirus , Liver Neoplasms , Therapeutics , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Telomerase , Genetics , Transfection , Vascular Endothelial Growth Factor A , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To observe the targeted therapeutic effects of plasmid AF-pGL3-hTERT-TK on HepG2 cells.</p><p><b>METHODS</b>HepG2 cells were cultured and pGL3-hTERT-TK and AF-liposome were constructed. HepG2 and L02 cells were transfected with AF-pGL3-hTERT-TK. The growth, apoptosis of the cells and the bystander effects were studied using liquid scintillation analysis and tunnel and flow cytometry.</p><p><b>RESULTS</b>After the suicide gene was inserted into the downstream of hTERT, TK was effectively driven by the hTERT promoter, making the TK highly expressed in the HepG2 cells. The AF made the therapeutic gene enter the HepG2 cells more easily by recognizing and combining the ASGPR receptor protein on the HepG2 cell surfaces and induced their apoptosis and suicide with bystander effect. The apoptosis rate was 85%+/-3% in the HepG2 cells whereas in the normal L02 hepatic cells it was 16%+/-2%.</p><p><b>CONCLUSION</b>AF-pGL3-hTERT-TK can target and attack HepG2 cells and has almost no influence on normal L02 hepatic cells. AF-pGL3-hTERT-TK has a potential in the treatment of hepatocellular carcinomas.</p>
Subject(s)
Humans , Apoptosis , Asialoglycoproteins , Bystander Effect , Fetuins , Ganciclovir , Metabolism , Genes, Transgenic, Suicide , Genetic Therapy , Hep G2 Cells , Telomerase , Metabolism , Thymidine Kinase , Metabolism , Transfection , alpha-FetoproteinsABSTRACT
<p><b>OBJECTIVE</b>To evaluate the safety and efficacy of drug-eluting stent (DES) implantation in selective patients with left main coronary artery disease.</p><p><b>METHOD</b>From October 2002 to November 2007, 44 consecutive patients underwent percutaneous coronary interventions (PCI) on left main coronary artery lesions, including 5 patients with concurrent left ventricular dysfunction (ejection fraction<40%), 2 with chronic respiratory dysfunction and 5 with chronic renal failure. The findings in coronary angiography, procedural success rate, severe complications and the follow-up results of the patients were analyzed.</p><p><b>RESULTS</b>The immediate procedural success rate was 100% in these patients without any severe complications. No non-fatal acute myocardial infarction or emergency coronary artery bypass grafting (CABG) was performed and death occurred in none of the cases during hospitalization. In the follow-up period for 14.2-/+9.3 (6-65) months after PCI, no subacute or late thromboses were found. One patient died from heart failure 4 months after PCI, and 6 patients (13.6%) experienced recurrent angina. Thirty-seven patients (84.1%) were free of any major cardiovascular events (MACE) after the procedure. A repeat coronary angiography was performed in 35 patients (79.5%) within 6 months after PCI, and 3 (8.6%) of them were confirmed to have restenosis, including 1 patient with distal bifurcation restenosis who were subsequently treated with CABG and two patients with side-branch ostium restenosis managed with cutting balloon dilation.</p><p><b>CONCLUSIONS</b>Implantation of drug-eluting stents is safe and effective for management of left main coronary artery disease with good immediate and long-term outcomes.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Angioplasty, Balloon, Coronary , Coronary Angiography , Coronary Artery Disease , Therapeutics , Coronary Restenosis , Therapeutics , Drug-Eluting Stents , Follow-Up Studies , Treatment OutcomeABSTRACT
<p><b>OBJECTIVE</b>To observe the effects of puerarin on activity of dimethylarginine dimethylaminohydrolase (DDAH) in human umbilical vein endothelial cells (HUVECs) cultured with oxidized free radical (OFR), to explore the effect of puerarin on metabolic mechanism of asymmetric dimethylarginine (ADMA).</p><p><b>METHODS</b>HUVECs of the 3rd - 6th passage cultured with modified Jaffe's method were divided into 4 groups, the blank control group cultured with DMEM medium, the OFR group cultured with DMEM medium containing 0.1 mmol of OFR per liter, the puerarin group 1 and 2 cultured with DMEM medium containing 0.1 mmol of OFR per liter as well as 0.5 mg/ml and 1.0 mg/ml of puerarin respectively. After being incubated for 24 h, activity of nitric oxide synthase (NOS), contents of nitric oxide (NO), ADMA, endothelin (ET), and L-citrulline (L-cit) in the supernate were measured, and DDAH protein expression in the lysate was detected by Western blotting.</p><p><b>RESULTS</b>Compared with those in the blank control group, ADMA and ET contents were higher, while the levels of NO and L-cit and the activity of NOS were lower markedly, but the DDAH expression changed insignificantly in the OFR group. These abnormalities were restored significantly in the puerarin groups.</p><p><b>CONCLUSION</b>The increase of ADMA in OFR injured HUVECs was correlated with the reduction of DDAH activity and irrelevant to DDAH expression. Puerarin could promote ADMA metabolism through increasing DDAH activity, and improve NOS activity, thus to reduce the impairing of OFR on endothelial function.</p>
Subject(s)
Humans , Amidohydrolases , Metabolism , Arginine , Metabolism , Blotting, Western , Cells, Cultured , Culture Media , Endothelial Cells , Cell Biology , Metabolism , Endothelins , Metabolism , Free Radicals , Chemistry , Pharmacology , Isoflavones , Pharmacology , Nitrates , Metabolism , Nitric Oxide , Metabolism , Nitric Oxide Synthase , Metabolism , Nitrites , Metabolism , Oxidation-Reduction , Umbilical Veins , Cell BiologyABSTRACT
Objective: To study the effect of intracellular-free calcium and the expression of Fas and Fas L on the process of pancreatic carcinoma cell apoptosis. Methods: Apoptosis induced by 2 μmol/L arsenic trioxide in pancreatic cancer cell lines SW-1990 was investigated.Concentration of intracellular-free calcium （［Ca2+］i） was determined by Fura-2a fluorescein load technique. Fas and FasL were determined by flow cytometry. Results: Pancreatic cancer cells treated with 2 μmol/L arsenic trioxide presented apoptotic features: intact cell membrane, chromatin condensation, nucleic fragmentation and apoptotic body formation; agarose electrophoresis showed marked ladder; flow cytometery analysis showed a sub-G1 cell peak. In the process of pancreatic carcinoma cell apoptosis Fas and FasL and the ［Ca2+］i were significantly higher than that in the control. Conclusion: The pancreatic cancer cell apoptosis induced by arsenic trioxide is related to Fas and FasL expression by the cancer cells and the ［Ca2+］i increase in the cancer cells.
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Objective: To select the telomerase positive cancer cell lines of gastrointestinal tract and to provide a convinced methodology for future telomerase study. Methods: Fifteen cancer cell lines (carcinoma of stomach 4, of liver 6, of pancreas 2, of colon 3) were cultured and telomerase activity were detected by TRAP-ELISA. The normal hepatic cells were taken as control. Results: Thirteen cell lines were telomerase positive in the 15 lines(86.7%). Conclusion: Most of gastrointestinal tract cancer lines express telomerase, indicating the detection of telomerase activity has clinical potential.
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Objective: To study the effect of intracellular-free calcium and the expression of Fas and Fas L on the process of pancreatic carcinoma cell apoptosis. Methods: Apoptosis induced by 2 μmol/L arsenic trioxide in pancreatic cancer cell lines SW-1990 was investigated.Concentration of intracellular-free calcium （［Ca2+］i） was determined by Fura-2a fluorescein load technique. Fas and FasL were determined by flow cytometry. Results: Pancreatic cancer cells treated with 2 μmol/L arsenic trioxide presented apoptotic features: intact cell membrane, chromatin condensation, nucleic fragmentation and apoptotic body formation; agarose electrophoresis showed marked ladder; flow cytometery analysis showed a sub-G1 cell peak. In the process of pancreatic carcinoma cell apoptosis Fas and FasL and the ［Ca2+］i were significantly higher than that in the control. Conclusion: The pancreatic cancer cell apoptosis induced by arsenic trioxide is related to Fas and FasL expression by the cancer cells and the ［Ca2+］i increase in the cancer cells.
ABSTRACT
Objective: To select the telomerase positive cancer cell lines of gastrointestinal tract and to provide a convinced methodology for future telomerase study. Methods: Fifteen cancer cell lines (carcinoma of stomach 4, of liver 6, of pancreas 2, of colon 3) were cultured and telomerase activity were detected by TRAP-ELISA. The normal hepatic cells were taken as control. Results: Thirteen cell lines were telomerase positive in the 15 lines(86.7%). Conclusion: Most of gastrointestinal tract cancer lines express telomerase, indicating the detection of telomerase activity has clinical potential.