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ABSTRACT:Objective To construct a recombinant adenovirus vector harboring fusion gene NT4p53(C22)Ant and study its killing effect on HepG2 tumor cells.Methods Using molecular cloning technology,the rAVV-NT4p53(C22)Ant was produced by homologous recombination.Then we collected virus supernatant and measured its titer after it was amplified by PCR.The effect of this fusion gene on HepG2 tumor cells was evaluated by IHC, MTT assay,PI staining and flow cytometry.Results The recombinant adenovirus was successfully constructed. The p53 expression rate in rAAV-NT4p53(C22)Ant group was (44.88±2.45)%.MTT assay showed that rAAV-NT4p53(C22)Ant could strongly suppress the growth of HepG2 tumor cells.Flow cytometry showed that rAAV-NT4p53(C22)Ant could induce obvious apoptosis of HepG2 tumor cells.Conclusion The recombinant adenovirus vector encoding gene NT4p53(C22)Ant has been successfully constructed and expressed in this experiment,and it can inhibit proliferation and induce apoptosis of HepG2 tumor cells.
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Objective:To investigate the content change of EGF and NT-4/5 in the the frontal lobe,hippocampus and thalamus of rats after cerebral ischemia-reperfusion,in order to reveal the dynamic protection of EGF and NT-4/5 to cerebellum ischemia-reperfusion injury.Methods:Wistar senile rats were divided into the control group and the experiment groups at random.The control group included 5.The experimental groups were divided into cerebral ischemia 15 min group and ischemia-reperfusion 1 h,6 h,2 d,4 d,9 d groups after 15 min ischemia,5 rats per group.Results:There was all a little expression of EGF and NT-4/5 in the frontal lobe,hippocampus and thalamus of normal senile rats.The frontal lobe was shown evident increase of EGF expression in ischemia-reperfusion 2 d only.The hippocampus was shown continued increase of EGF expression during ischemia-reperfusion 6 h to 4 d and the EGF expression was shown a peak in ischemia-reperfusion 4 d.The increase of EGF expression in thalamus continued during ischemia-reperfusion 2-9 d too.The NT-4/5 expression of the frontal brain was clearly reduced after ischemia-reperfusion and returned to normal in ischemia-reperfusion 9 d.The NT-4/5 expression of hippocampus was shown one-off reduction after ischemia-reperfusion,but the expression quickly returned to normal and shows a little increase.The thalamus had only one-off reduction of NT-4/5 expression.Conclusion:The frontal lobe lacks of fast neuroprotective mechanism of EGF in early stage of ischemia-reperfusion and it has a limited neuroprotective effect of EGF in middle and advanced stage.The hippocampus has a fast and long-lasting neuroprotective mechanism of EGF.The thalamus has a delayed neuroprotective mechanism of EGF.The frontal brain and hippocampus have a delayed neuroprotective mechanism of NT-4/5.The neuroprotective mechanism of NT-4/5 is weaker in thalamus.
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OBJECTIVES: We examined the effects of neurotrophins and insulin-like growth factors on cell death induced by haloperidol, a typical anti-psychotic agent. METHOD: Neocortices from 14- or 15-daysold fetal mice for neuron-glia co-cultures were used for this experiment. RESULT: Twenty-four hours treatment of mouse cortical cell cultures with 30 M haloperidol-induced wide spread neuronal apoptosis characterized by cell body shrinkage, DNA fragmentation and condensation. Concurrent treatment with growth factors, BDNF, NT4/5, IGF-I and IGF-II, protect the neurons from the haloperidol-induced neuronal apoptosis(HINA) in a dose dependent manner(10-100ng/ml). CONCLUSION: The present study suggests the possibility that haloperidol toxicity can be hampered with growth factors. Further study about the mechanism underlying the protective capacity of the growth factors on HINA may lead to the development of the new protective strategy for tardive dyskinesia.
Subject(s)
Animals , Mice , Apoptosis , Brain-Derived Neurotrophic Factor , Cell Culture Techniques , Cell Death , Coculture Techniques , DNA Fragmentation , Haloperidol , Insulin-Like Growth Factor I , Insulin-Like Growth Factor II , Intercellular Signaling Peptides and Proteins , Movement Disorders , Neocortex , Nerve Growth Factors , Neurons , SomatomedinsABSTRACT
Objective:To construct a recombinant adenovirus vector harboring fusion gene NT4-p53(N15)-Ant,laying a foundation for gene therapy research of malignant tumors.Methods:The p53(N15)-Ant gene was obtained by T-vector method and was inserted in pBV220/NT4 vector after digested with restriction enzyme.The fusion gene of NT4-p53(N15)-Ant was subcloned into the shuttle plasmid of adenovirus;the products were cotransfered into HEK-293 cell line with helper plasmid PJM17.The recombinant adenovirus was produced by homologous recombination of above 2 plasmids in HEK-293 cells and its titer was measured by plaque-forming.The expression of Ad.NT4p53Ant in transfected 293 cells was confirmed by reverse transcription polymerase chain reaction(RT-PCR)procedure.The effect of Ad.NT4p53Ant on HepG2 cell line was measured by a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.Results:The p53(N15)-Ant gene was confirmed by restriction enzyme digestion and DNA sequencing.High titer of recombinant adenovirus was obtained by homologous recombination in HEK-293 cells(1?10 11pfu/ml).The expression of NT4-p53(N15)-Ant gene in 293 cells was confirmed by RT-PCR.Ad.NT4p53Ant had strong killing effect on HepG2 cells.Compared with Ad.GFP,Ad.NT4p53Ant significantly decreased the survival rate of HepG2 cells.Conclusion:The recombinant adenovirus vector encoding gene NT4-p53(N15)-Ant has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques,laying a foundation for further research of gene therapy of cancer.
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Objective To construct the expression box of fusion gene NT4-p53(N15)-Ant.Methods The gene of p53(N15)-Ant was obtained by PCR of two primers templating with each other and T-vector cloning method.The positive clone was identified and analyzed by the restriction enzymes and sequencing respectively.After digested with restriction enzyme,the interest gene of p53(N15)-Ant was subcloned into the plasmid pBV220/NT4.Results The gene of p53(N15)-Ant was confirmed by the digestion of restriction enzyme and sequencing.The recombinant plasmid pBV220/NT4p53(N15)Ant was identified by the digestion of restriction enzyme and agarose gel electrophoresis and the results conformed theoretical values.Conclusion The plasmid pBV220 containing the expression box of NT4-p53(N15)-Ant was successfully constructed by molecular cloning and recombination techniques in vitro,which will guide further study on gene therapy of cancer.
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Objective To investigate survivin as an anticancer therapeutic target by use of shepherdin [79-87],a novel peptide carrying the survivin sequence from Lys-79 through Leu-87,we constructed an recombinant vector containing fusion gene NT4-Ant-Shepherdin [79-87].Methods The gene of Ant-Shepherdin [79-87] was obtained by PCR and T-vector method.After cloned and digested with restricted enzyme,Ant-shepherdin [79-87] was inserted in PBV220NT4 vector.The recombinant vector was transformed into the competent cell,E.coli DH5?.The fusion gene of NT4-Ant-Shepherdin [79-87] was identified by agarose gel electrophoresis (AGE).Results DNA sequencing results verified that the sequence of Ant-Shepherdin [79-87] was consistent with what we had designed.After transformed E.coli DH5?,a fragment of 321 bp was confirmed.Conclusion The recombinant vector containing fusion gene NT4-Ant-Shepherdin [79-87] was successfully constructed in this experiment by molecular biology techniques,which provides the basis of further research of survivin for cancer gene therapy.