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As an important member of tool enzymes, exonuclease is a kind of hydrolytic enzymes without strict base sequence dependent. In recent years, by taking advantage of different hydrolysis ways of exonuclease and nanotechnology, cycle effect of enzyme digestion, aptamer, non Watson-Crick base pairing system by metal ions, fluorescent nucleic acid probes, electrochemical methods etc. , a series of exonuclease-assisted signal amplification strategies have been developed, which played a very key role in improving the sensitivity of detection methods. Therefore, exonuclease has been widely used in high sensitive detection of nucleic acids, proteins, ions, small molecules and so on. To understand it better and apply it well in the future, the application progress of exonuclease-assisted signal amplification strategies in biochemical analysis has been summarized in this review.
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Abstract A novel luminol electrochemiluminescence strategy based on titanium dioxide/carbon nanotubes ( TiO2/CNTs) nanocomposites for detection of glucose was developed. First, the TiO2/CNTs nanocomposites were prepared by a sol-gel method and modified on the glassy carbon electrode. The electrochemiluminescence ( ECL) signal could be greatly enhanced when the electrode was established by the nanocomposites, which finally resulted in the increased sensitivity. Glucose oxidase calalyzed the oxidation of glucose to form H2 O2 , and the H2 O2 reacted with luminol to produce the ECL signal. Thus the above system was proved to be efficient for glucose detection. The modified electrode exhibited excellent ECL signals and a good linear range of 1. 0í10-7-5. 0í10-6 mol/L with a detection limit of 5. 2í10-8 mol/L towards glucose detection. This strategy was successfully demonstrated as a sensitive, rapid, simple and cost-effective method to detect glucose. Meanwhile, the TiO2/CNTs nanocomposites offered a novel material for the signal enhancement in electrochemiluminescence sensor.
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Objective To estimate the influence of ADAR1 gene,which is considered to be responsible for the pathogenesis of dyschromatosis symmetrica hereditaria,on Wnt1 1 expression and tyrosinase activity.Methods Some cultured HaCaT cells were equally divided into four groups:control group remaining untreated,three experimental groups transfected with three different ADAR1-specific shRNAs respectively.Then,Western blot was performed to quantify the expression of Wnt11 protein in HaCaT cells so as to select the most potent shRNA.Some human A375 melanoma cells were cocultured with untransfected HaCaT cells (normally expressing ADAR1 and Wnt1 1 proteins) or HaCaT cells transfected with the selected specific shRNA (lowly expressing ADAR1 and Wnt11 proteins).Thereafter,cell appearance was observed using inverted microscopy at 24,48 and 72 hours,and tyrosinase activity was estimated at 48 hours.Results As Western blot showed,the expression of Wnt 11 protein was significantly lower in the three ADAR1-silenced experimental groups than in the control group.The number of dendritic protrusions at the junction sites between HaCaT cells and A375 cells was significantly decreased,together with a significant reduction in tyrosinase activity (absorbance value:0.0168 ± 0.0069 vs.0.0490 ± 0.0132,P ＜0.01),in A375 cells cocultured with transfected HaCaT cells compared with those cocultured with normal control HaCaT cells.Conclusion ADAR1 gene silencing in HaCaT cells can attenuate the expression of Wnt11 protein,and affect tyrosinase activity in A375 cells.
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Objective To observe the toxicity and adverse reaction of interleukin(IL) 2 gene modified allogenic gastric cancer cell line in far advanced gastric cancer patients. Methods A phase Ⅰ clinical trial was conducted for sixteen far advanced gastric cancer patients with IL 2 gene modified gastric cancer cell line. By gene recombinant technique, human IL 2 cDNA was transfected into human gastric cancer cell line MKN45 via retrovirus based vector.These cells were then inactivated by irradiation (100 Gy) and were cryopreserved for the vaccine. The immunization were administrated subcutaneously at the first, 8 th , 15 th , 29 th and 58 th day. The patients were divided into 4 dosage groups, which the dosage of the vaccine was administrated in each subsequent level. The toxicity and adverse reaction were evaluated by WHO criteria. Results Fifteen of the 16 patients completed the immunization. Side effects of treatment consisted of mild to moderate fever, redness and swelling at the site of injection, which were the most common symptoms. Only one patient abandoned after the third injection due to rapidly progressive disease. Other reactions including allergic shock, bone marrow depression and disturbance of hepatorenal function were not observed during the immunization.In some patients, the serum transferrin, humoral immune parameters such as IgG, IgA, IgM,IL 2 and cellular immune parameters such as CD + 3,CD + 4,CD + 8 had been improved after treatment.Conclusions This trial demonstrates the feasibility, safety and potential therapeutic effects of vaccination of gastric cancer patients with allogenic, gene modified gastric cancer cell line. The dosage of the vaccine recommended for phase Ⅱ clinical trail is 5?10 7 cells per time.
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Objective To study the relationship between the expression of p53 and its downstream effecter p21~(waf1)protein and multi-drug resistance(MDR) of Tca8113/BLM cell line.Methods The cDNA of wildtype p53 gene was introduced into the drug-resistance cell line Tca8113/BLM by Lipofectamine-mediated transfection.The expression of p53 mRNA in Tca8113/BLM and its parental counterpart Tca8113 cell lines were analyzed using molecular beacons.The expression of p53,p21~(waf1),P-gp,and MRP proteins in Tca8113/BLM cell line transfected by wt-p53 cDNA,the untransfected control Tca8113/BLM cell line,its parental counterpart Tca8113 were analyzed by Western blotting.MTT assay was used to determine the drug-sensitivity of different cell lines.The cell cycle distribution and apotosis of different cell lines were also observed by flow cytometery(FCM) and laser confocus microscope(LCM),respectively.Results The expression of p53 protein was significantly lower,and no p21~(waf1) protein was detected in Tca8113/BLM cells.The protein expression of P-gp and MRP were significantly higher in Tca8113/BLM cells than in Tca8113 cells.By Western Blotting,the expression of p53 and p21~(waf1)proteins was significantly increased,while the expression of P-gp and MRP proteins was significantly decreased in Tca8113/BLM/p53 cells.Comparing to Tca8113/BLM cells,the sensitivity of BLM treatment in Tca8113/BLM/p53 cells was 10.1 fold increased.Tca8113/BLM/p53 cells decreased at G1 and S phase,and increased at G2 phase.LCM results showed that the apoptosis of Tca8113/BLM/p53 cells was more obvious than Tca8113/BLM cells when treated with the drug BLM.Conclusion The multi-drug resistance in Tca8113/BLM cell line is involved in down-regulation of p53 and p21~(waf1) protein levels,and up-regulation of P-gp and MRP protein levels.
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Objective To explore the reverse effect and mechanism of progesterone(Prog) on a human tongue cancer drug-resistant cell line Tca8113/BLM in vitro. Methods The reverse effect of Prog on Tca8113/BLM was determined by MTT. The effects of Prog on the intracellular ADM congregation, cell cycle distribution and the expression of P-gp in the cell membrane were detected by flow cytometry (FCM). Results The value of IC50 of Tca8113 and Tca8113/BLM cells without Prog was 9 56 and 120 1,respectively. After treated with Prog(2umol/L),The value of IC50 of Tca8113 and Tca8113/BLM cells was 9 56 and 12 1,respectively. The potent fold of BLM in Tca8113/BLM cells was 9 92, the concentration of intracellular ADM in Tca8113/BLM cells significantly increased(P
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?-tumor necrosis factor can enhance tumor cells autolysis and the expression of HLA antigenes. So we infected Human Hepatoma Cell Line (HHCL)with recombinant retroviral vector containing an insert of human tumor necrosis factor-? cDNA. The transduced tumor cells were isolated by G418 selection. After being irradiated by different doses of 60Co, their expression of TNF in 24 hours were tested. Observations of the relationship between the doses of irradiation and the expression levels of TNF were done. We also preserved the irradiated HHCL-TNF cells into liquid nitrogen, after recovering, tested their expressions of TNF again. This paper shows that: (l)following irradiation by 60Co, the production of ?-TNF persisted for about four weeks, and there is no difference among the different irradiated doses(40G~100G); (2)After being irradiated and kept into liquid nitrogen, the recovering HHCL-TNF cells constitutively secrete ?-TNF for about three weeks.These findings provide a logical basis for designing protocols for active immunotherapy in humans.