ABSTRACT
<p><b>OBJECTIVE</b>To establish a mice model of cisplatin-induced ototoxicity, and to investigate the effect of cisplatin on apoptosis of spiral ganglion cell and expression of caspase-3 in mouse cochlea.</p><p><b>METHODS</b>Terminal deoxynucleotidyl transferase-mediated nick end labeling method (TUNEL) was used to monitor the apoptosis of spiral ganglion cell. Envision method of immunohistochemistry was applied to detect the expression of caspase-3 in cochlea. Auditory brainstem response (ABR) was measured to observe the change of hearing.</p><p><b>RESULTS</b>The weight and hearing of mice in different dose of cisplatin groups were declined significantly as compared with those of control group (P < 0.05, P < 0.01), and the TUNEL positive cell number and expression of caspase-3 were greater remarkably with the more cisplatin injected.</p><p><b>CONCLUSION</b>A mouse model of cisplatin-induced ototoxicity can be established. Cisplatin can lead to the apoptosis of spiral ganglion cells, and caspase-3 has participated in this apoptosis process, which approves further that apoptosis might be one of the mechanisms of cisplatin ototoxicity.</p>
Subject(s)
Animals , Male , Mice , Antineoplastic Agents , Pharmacology , Apoptosis , Caspase 3 , Metabolism , Cisplatin , Pharmacology , Cochlea , Cell Biology , Metabolism , Mice, Inbred Strains , Spiral Ganglion , Cell Biology , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To construct effective RNA-interference plasmids targeting mouse HIF-1alpha gene and testify their effects and specificities in interfering HIF-1alpha expression.</p><p><b>METHODS</b>Three RNA-interference plasmids targeting mouse HIF-1alpha gene, pBS/U6/HIF-1alpha-siRNAI~III, were constructed and identified using double digestion method in the present study. RT-PCR, immunostaining and western blotting were employed to detect the expression alterations of HIF-1alpha in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1alphai-II in SH-SY5Y cell line was further investigated.</p><p><b>RESULTS</b>All the three RNA-interference plasmids, especially pBS/U6/HIF1alphai-II, showed significant inhibition in HIF-1alpha expression in 293T cell line. pBS/U6/HIF1alphai-II could also inhibit HIF-1alpha expression in SH-SY5Y cell line, in a dose-dependent way.</p><p><b>CONCLUSION</b>Plasmid pBS/U6/HIF1alphai-II constructed in our study can effectively and specifically inhibit HIF-1alpha expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/HIF1alphai-II plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and ischemia development.</p>
Subject(s)
Animals , Humans , Mice , Analysis of Variance , Cell Line, Tumor , Dose-Response Relationship, Drug , Gene Expression , Gene Silencing , Physiology , Green Fluorescent Proteins , Genetics , Hypoxia-Inducible Factor 1, alpha Subunit , Genetics , Metabolism , Plasmids , Genetics , RNA, Small Interfering , Genetics , Pharmacology , Transfection , MethodsABSTRACT
<p><b>AIM</b>To discover new drugs which may be applied to diseases of the immune system, hemogenesis system diseases and tumors, several high-throughput drug screening cell models based on JAK-STAT signal pathway have been established.</p><p><b>METHODS</b>Four repeats of STAT DNA binding conserved sequences were synthesized, subcloned into pGL-Luc reporter vector and stably transfected into cell lines in vitro. Cell clones with high copy numbers of STAT binding sites and reporter genes were chosen as high-throughput drug screening cell models. The cell models were tested with known anti-allergic drugs and anti-tumor drugs by determining luciferase activity. The reaction was performed in 96 well micro-plates with a final volume of 50 microL.</p><p><b>RESULTS</b>The cell models by performing rapid fluorescence assay were shown to be highly sensitive and stable after testing with cytokine and drugs. The modification of the expression plasmid simplified this method and made it more practical. It also provided good linear correlation, wide range of assay, highly sensitive and good reproducibility.</p><p><b>CONCLUSION</b>The method can be performed by high-throughput drug screening for effective extraction of Chinese traditional herbs.</p>
Subject(s)
Humans , Anti-Allergic Agents , Pharmacology , Antineoplastic Agents , Pharmacology , Carcinoma, Hepatocellular , Pathology , DNA-Binding Proteins , Genetics , Metabolism , Drug Evaluation, Preclinical , Methods , Drugs, Chinese Herbal , Pharmacology , Janus Kinase 2 , Jurkat Cells , Metabolism , Liver Neoplasms , Pathology , Luciferases , Metabolism , Protein-Tyrosine Kinases , Genetics , Metabolism , Proto-Oncogene Proteins , STAT3 Transcription Factor , Signal Transduction , Trans-Activators , Genetics , Metabolism , Tumor Cells, CulturedABSTRACT
Dishevelled proteins are multifunctional and highly conserved. These proteins are also required for the specification of cell fate and polarity by secreted Wnt proteins. To investigate the molecular mechanism of Dishevelled in mediating Wnt signal transduction, a mouse 11.5dpc embryo library was screened by yeast-two-hybrid system to find mouse Dishevelled2 DEP domain and C-terminal interacting proteins. 15 possitive clones were identified from 4.1 x 10(6) transformants. The DNA sequences of the positive AD/library plasmids were determined. The BLAST results revealed that one of the positive clones contained N-terminus cDNA fragments (amino acids 6-122) of Gli3 protein. The interaction between Dv12 and Gli3 detected by yeast two-hybrid system suggests that Gli3 might play a role in some biological processes with Dishevelled.
Subject(s)
Animals , Mice , Adaptor Proteins, Signal Transducing , Chemistry , Genetics , Physiology , Dishevelled Proteins , Gene Library , Kruppel-Like Transcription Factors , Physiology , Nerve Tissue Proteins , Physiology , Phosphoproteins , Chemistry , Genetics , Physiology , Plasmids , Signal Transduction , Two-Hybrid System Techniques , Wnt Proteins , Physiology , Zinc Finger Protein Gli3ABSTRACT
Objective To investigate the effects of dominant-negative truncation mutant?NTCF4, lacking the N-terminal form of TCF4 gene,on biological characteristics of renal cancer cell line GRC-I and explore the molecular mechanisms.Methods GRC-I cell was transfected with pCDNA3-?NTCF4 eukary- otie expression plasmid,pCDNA3 empty vector to construct the stable cell line GRC-I/?NTCF4 and GRC-I/ Mock respectively.The morphological changes of stable cells were observed and the cells growth curve was detected through light microscope.The cellular proliferation activities were determined using the MTT assay. The protein expression of Wnt pathway downstream target gene C-Myc and Cox-2 was evaluated by immuno- cytoehemieal method and Western Blot analysis.Results After the dominant-negative?NTCF4 gene was permanently expressed,the GRC-I/?NTCF4 stable cells morphologically showed that appearance changed from circular to long-spindle shape,growth rate decreased with less karyosehisis found,malignant pheno- types reversed to normal renal tubular cells.MTT assay revealed that the proliferation activities of GRC-1/?NTCF4 cells were inhibited by 11.2%-35.5% compared with GRC-I cells (P＜0.05),while the GRC- I/Mock cells have no difference with the control cells.Immunocytochemical analysis and Western Blot showed that the C-Myc and Cox-2 protein expression level of GRC-I/?ANTCF4 cells were significantly sup- pressed in comparison with that of GRC-I/Mock and GRC-I cells.Conclusions The dominant-negative truncation mutant?NTCF4 could partially inhibit the growth of renal cancer cells and down-regulate the pro- tein expression of Wnt pathway target gene C-Myc and Cox-2.These findings provide a experimental founda- tion for applying cell signal therapy to renal cell cancer by blocking the Wnt signaling pathway.