ABSTRACT
Objective To investigate the feasibility of green fluorescent protein (GFP) as a marker to trace the transplanted um-bilical cord mesenchymal stem cells (ucMSCs) in rats with cerebral ischemia-reperfusion .Methods ucMSCs were transfected by GFP-adenovirus .The rats were subjected to left middle cerebral artery occlusion using suture method .1 × 106 GFP-ucMSCs were transplanted with cerebral stereotaxic technique .Frozen sections of brain tissue were made at 7 d after cerebral ischemia .The ex-pression of GFP was observed by fluorescence microscope .Results In vitro ,the morphology of GFP in ucMSCs was fibrous ,and when fused ,the clusters were arranged in a radial or whirlpool shape ,The morphological feature of transfected ucMSCs was similar to that un-transfected ucMSCs .Under the fluorescence microscope ,the positive rate of GFP was more than 80% .In addition ,GFP could spread to the whole cells and show the completed form .No rejection was observed in the rats after transplantation ,and the GFP was found near the site of transplantation after 7 d ,and the fluorescence was not attenuated .Conclusion GFP is an effective tracer maker for ucMSCs transplantation in the treatment of ischemia-reperfusion ,which could provide experimental method for fur-ther study .
ABSTRACT
Objective To investigate the feasibility of green fluorescent protein (GFP) as a marker to trace the transplanted um-bilical cord mesenchymal stem cells (ucMSCs) in rats with cerebral ischemia-reperfusion .Methods ucMSCs were transfected by GFP-adenovirus .The rats were subjected to left middle cerebral artery occlusion using suture method .1 × 106 GFP-ucMSCs were transplanted with cerebral stereotaxic technique .Frozen sections of brain tissue were made at 7 d after cerebral ischemia .The ex-pression of GFP was observed by fluorescence microscope .Results In vitro ,the morphology of GFP in ucMSCs was fibrous ,and when fused ,the clusters were arranged in a radial or whirlpool shape ,The morphological feature of transfected ucMSCs was similar to that un-transfected ucMSCs .Under the fluorescence microscope ,the positive rate of GFP was more than 80% .In addition ,GFP could spread to the whole cells and show the completed form .No rejection was observed in the rats after transplantation ,and the GFP was found near the site of transplantation after 7 d ,and the fluorescence was not attenuated .Conclusion GFP is an effective tracer maker for ucMSCs transplantation in the treatment of ischemia-reperfusion ,which could provide experimental method for fur-ther study .
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Objective To compare the transfection efficiency of cationic polyethylenimine(PEI)with Lipofectamine 2000TM by using the plasmid DNA encoding vascular endothelial cell growth factor (VEGF165 )gene in human embryonic kidney cell line 293T.Methods PEI of different N/P ratio and Lipofectamine 2000TM were used to deliver the vector containing VEGF165 to 293T cells,respectively.Green fluorescent protein(GFP)gene was inserted into the vector as a report gene.Evaluation of cytoactive was performed by CCK-8 assay 24 h after transfection.The cells were observed by fluorescent microscope and the presence of VEGF165 in cell supernatant was detected by ELISA 48 h after transfection.The transfection efficiency was calculated and com-pared.Results Similar cytoactive and best transfection efficiency could be obtained when N/P ratio was 9,the transfection efficien-cy was around 70%.Furthermore,the presence of VEGF165 increased significantly after transfection(P <0.05),but there was no significant difference between the two groups in which different transfection methods were adopted.Conclusion PEI as a novel oli-gofectamine reagent could mediate more efficient transfection compared with lipofectamine.It also has low cell-toxicity and low price and could be an ideal vector in gene delivery technology.
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Objective To construct a recombinant interfering RNA(miRNA)plasmid vector targeting annexinA3 and observe its impact on the growth and apoptosis of human gallbladder cancer cell line SGC-996.Methods Four small fragments of miRNA and one negative control were sequenced and cloned into vector pcDNATM 6.2-GW/EmGFPmiR.The recombinant plasmids were then transfected into gallbladder cancer cell line SGC-996 by positive liposomal transfection.The transcription and translation level of annexinA3 expression were detected by RT-PCR and Western Blot.Meanwhile, the proliferation and apoptosis of transfected tumor cells were determined by MTT and flow cytometry.Results The recombinant plasmids containing annexinA3 miRNA were successfully constructed.AnnexinA3 expression was significantly knocked down after miRNA plasmid transfection.Transfection of annexinA3 gene miRNA could effectively inhibit the proliferation of gallbladder cancer cell and promote apoptosis.Conclusion AnnexinA3 gene silenced by miRNA can induce the apoptosis of human gallbladder cancer cell line SGC-996.The process might serve as a potential approach for cancer gene therapy.
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<p><b>OBJECTIVE</b>To observe the effect of grape seed extract (GSE) on breast cancer cell MCF-7 about the of proliferation and the gene expression of survivin, and to investigate the molecular biological mechanism of inhibition by GSE.</p><p><b>METHOD</b>MTT was used to measure the role of proliferated inhibition of GSE at the dosage of 40, 80, 120, 160, 200 mg x L(-1) with different time. To calculate the IC50 of GSE and to select the suitable concentration and treatment time. The change of cell cycle was detected by flow cytometry. mRNA expression of Survivin was observed by RT-PCR. Luciferase kit was used to observe the change of core promoter of survivin which was inserted in the flourescence report vector. And further to analyse the bind side of transcription factor on the sequence of core promoter of survivin was further analysed by using the microsoft of bioinformatics.</p><p><b>RESULT</b>GSE could inhibit the proliferation of breast cancer cell MCF-7 with time and dosage-dependent (P < 0.05). GSE could arrest the cell cycle in S periods (P < 0.05) and also inhibit the mRNA expression of survivin effectively (P < 0.01); GSE could down-regulate the activity of core promoter of survivin significantly (P < 0.01).</p><p><b>CONCLUSION</b>GSE can inhibit the proliferation of breast cancer cell MCF-7 through arresting the cell cycle in S periods. The mechanism may be that GSE regulate the activity of transcription factor which are related to the activity of core promoter of survivin and decrease the gene expression of survivin.</p>
Subject(s)
Female , Humans , Antineoplastic Agents, Phytogenic , Pharmacology , Breast Neoplasms , Genetics , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Cloning, Molecular , Down-Regulation , Gene Expression , Gene Expression Regulation, Neoplastic , Genetic Vectors , Inhibitor of Apoptosis Proteins , Microtubule-Associated Proteins , Genetics , Metabolism , Transfection , Xenograft Model Antitumor AssaysABSTRACT
Objective Investigate the effect of TrKA variation on the expression of neucleoprotein NF-?B P65 and apoptosis.Methods To construct the expression vector of TrKA small interfering RNA,the recombinant was transfected into MCF-7 cells.the stable cell line expressing TrKA small interfering RNA were selected by G418.The mRNA and protein of TrKA were tested by real-time PCR,Western-blot and Immunohistochemistry.The change of neucleoprotein NF-?B P65 was detected by WB,Flow cytometry was used to observe the cell apoptosis.ResultsThe expression vector of TrKA-siRNA was successfully constructed.The mRNA and protein of TrKA were decreased by 74.7% and 80.5% respectively(P