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Objective:To study the effects of down-regulated Smad4 expression on the proliferation and apoptosis of breast carcinoma MCF-7 cells,and to explore their mechanisms.Methods:The human breast carcinoma MCF-7 cells and MDA-MB-231 cells were cultured in vitro.RT-PCR method was used to detect the expression levels of Smad4 mRNA in MCF-7 cells and MDA-MB-231 cells.The Smad4-shRNA plasmid and Scramble-shRNA plasmid were respectively stably transfected into the MCF-7 cells with high expression of Smad4.The experiment was divided into non-transfected MCF-7 cells (normal control) group,Smad4 gene silencing group,Scramble(negative control) group.The proliferation abilities of the cells in various groups were detected by CCK-8 method.The apoptotic rates of the cells in various groups were detected by flow cytometry.Real-time PCR method wasused to detect the mRNA expression levels of the proliferation-related genes CDKN1A,CDK1 and CDK2 and the apoptosisrelated genes Suvivin,bcl-2,caspase 3 and caspase 9.Results:The proliferation abilities of cells had no statistical significance between various groups (P＞0.05).The mRNA expression levels of CDKN1A,CDK1 and CDK2 in the cells had no statistical significance between various groups (P＞0.05).Compared with normal control group and negative control group,the apoptotic rate of the cells in Smad4 gene silencing group was significantly decreased (P＜0.01),the expression levels of Suvivin and bcl-2 mRNA in Smad4 gene silencing group were significantly increased (P＜0.01),and the mRNA expression levels of caspase 3 and caspase 9 in Smad4 gene silencing group were significantly decreased (P＜0.05).Conclusion:Smad4 could induce the apoptosis of MCF-7 cells by downregulating the expressions of Suvivin and bcl-2 and up-regulating the expressions of caspase 3 and caspase 9.
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Objective:To study the effects of down-regulated Smad4 expression on the proliferation and apoptosis of breast carcinoma MCF-7 cells,and to explore their mechanisms.Methods:The human breast carcinoma MCF-7 cells and MDA-MB-231 cells were cultured in vitro.RT-PCR method was used to detect the expression levels of Smad4 mRNA in MCF-7 cells and MDA-MB-231 cells.The Smad4-shRNA plasmid and Scramble-shRNA plasmid were respectively stably transfected into the MCF-7 cells with high expression of Smad4.The experiment was divided into non-transfected MCF-7 cells (normal control) group,Smad4 gene silencing group,Scramble(negative control) group.The proliferation abilities of the cells in various groups were detected by CCK-8 method.The apoptotic rates of the cells in various groups were detected by flow cytometry.Real-time PCR method wasused to detect the mRNA expression levels of the proliferation-related genes CDKN1A,CDK1 and CDK2 and the apoptosisrelated genes Suvivin,bcl-2,caspase 3 and caspase 9.Results:The proliferation abilities of cells had no statistical significance between various groups (P＞0.05).The mRNA expression levels of CDKN1A,CDK1 and CDK2 in the cells had no statistical significance between various groups (P＞0.05).Compared with normal control group and negative control group,the apoptotic rate of the cells in Smad4 gene silencing group was significantly decreased (P＜0.01),the expression levels of Suvivin and bcl-2 mRNA in Smad4 gene silencing group were significantly increased (P＜0.01),and the mRNA expression levels of caspase 3 and caspase 9 in Smad4 gene silencing group were significantly decreased (P＜0.05).Conclusion:Smad4 could induce the apoptosis of MCF-7 cells by downregulating the expressions of Suvivin and bcl-2 and up-regulating the expressions of caspase 3 and caspase 9.
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Objective:The roles of HMGA2 in maintaining malignant phenotypes of the osteosarcoma U2OS cells was studied to explore the possibilities for it to be developed as a target for gene therapy.Methods:U2OS cells were stably transfected with a DNA based shRNA expression vector which targeted to HMGA2.The expression of HMGA2 mRNA was proved by RT-PCR;Cell growth,migration and apoptosis were determined with CCK8,hoechst33342 staining and Boyden ventricle,respectively.The mRNA levels of Caspase 3 and Caspase 9 were determined by real time quantitative RT-PCR.Results:The transfection with shRNA expression vector significantly decreased HMGA2 mRNA levels of U2OS cells.Cell growth and migration were decreased,but apoptosis and the mRNA levels of Caspase 3 and Caspas 9 were increased following the decrease of HMGA2 mRNA.Conclusion:The abnormal expression of HMGA2 plays an important role in maintaining the malignant phenotypes of U2OS cells.Gene therapy targeted to HMGA2 could be helpful in the treatment of human osteosarcoma.
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Objective To study the effects of ethanol extract from Java Brucea Fruit on platelet-derived growth factor receptor ?(PDGFR?) mediated cell migration and to obtain the valuable messages on the characteristics of its active ingredient.Methods PAE cells was transfected with the vector expressing human PDGFR? with Transfectom 2000;After screening by G418 resisitance,RT-PCR was used to monitor the expression of PDGFR? in the cells;Wound healing of the cells was used to examine the lowest consistency of PDGFBB and inhibitory effect of ethanol extract of Java Brucea Fruit on cell migration after restoring 24 h.Results Human PDGFR? was stably expressed in PAE cells transfected with the expressing vector.The lowest consistency of exogenous PDGFBB which promoted PDGFR? mediated cell migration was 10 ?g?L-1.70% ethanol extract of Java Brucea Fruit which strongly inhibited PDGFR? mediated cell migration was dose-dependent(P10 ?g?L-1) mainly caused the death of the cell.Conclusion Ethanol extract of Java Brucea Fruit has a strongly inhibitory effect on the PDGFR? mediated cell migration which could play a major role in its effects against metastasis of malignant tumor,the active ingredients of it could be more dissolvable in the 70% ethanol.
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Objective To prepare active components from extract of Arca subcrenata Lischke and analyze its antioxidative activity.Methods The antioxidative component(P3)from Arca subcrenata Lischke was isolated by chromatography on Superdex-75 column followed by a Sephadex LH-20 column,and antioxidative activities were assayed using potassium ferricyanide and DPPH methods,respectively.The characters of this component were determined by ninhydrin reagent,anthrone reagent,Coomassie Brilliant Blue G250 reagent as well as thin-layer chromatography.Results and Conclusion A component with strong antioxidative activity was isolated and identified as glycosylated peptide.
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Objective To express and purify recombinant protease with thrombolysis activity of Perinereis aibuhitensis Grube and study on its characterization.Methods pMAL-PPA was in- troduced into E.coli DH5?to construct engineering bacteria and overexpression of the prote- ase of fused with maltose binding protein(MBP-PPA)was achieved with IPTG induction. The fusion protein was purified by affinity chromatography on amylose-resin column fol- lowed by chromatography on a DEAE-sepharose FF column.PPA cut with Factor Xa was assayed using casein plates supplied with plasminogen.Results Engineering bacteria express- ing maltose binding protein-thrombolytic protease of P.aibuhitensis were constructed and overexpression of MBP-PPA was achieved with IPTG induction.A recombinant fusion pro- tein of 51kD was purified,and PPA cut down from the fusion protein had a plasminogen-acti- rating activity.The protease showed a good thermal stability with an optimal pH of 8.0. This enzyme was also relatively stable in a pH range of 6.0～9.0 and still active after stored in organic solvents for 20d.Conclusion PPA was verified as a plasminogen activator,and might be a new thrombolytic medicine in the future.
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Objective To study antioxidant activity of extracts from Perinereis aibuhite.Meth- ods The extracts from Perinereis aibuhite were purified by using Sephadex LH-20 and its an- tioxidant activities were determined by the method of DPPH.Results and Conclusion The ex- tracts from Perinereis aibuhite showed strong free radical scavenging activity,and it was identified as a group of low molecular weight peptides by qualitative analyzing method.
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Spinosyns are secondary metabolites from the aerobic fermentation of saccharopolyspora spinosa Spinosyn is a kind of broad spectrum insecticide, which has contact and ingestion toxicity on insects On lepidopteran insects, spinosad (a mixture of spinosyn A and spinosyn D) is one of the most selective compounds ever discovered In this paper, the structure, biosynthesis, property and process of spinosyns are reviewed
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Perilla frutescens callus were induced from leave explants on MS medium suplemented with NAA and 2,4-D. The Rosmarinic acid (RosA) content of dried callus was 0.85%. The RosA was extracted from the callus with 80% alcohol and purified through extraction with ethyl acetate and a Sephadex LH-20 column chromatography. The parity of the final product was 95% as analyzed by HPLC RosA could inhibit the growths of Escherichia colt, Staphylococcus aueris and Rhizotonia with MICs of 300, 400, 800?g/mL, respectively.