ABSTRACT
Objective To investigate the effect of over-expression of human ribonuclease inhibitor suppresses invasion and migration of transplanted bladder cancer .Methods The T24 cells were stably transfected with pIRES2-EGFP-RI and pIRES2-EG-FP plasmid respectively .Using the cell transfected with pIRES2-EGFP and untransfected cell as controls .and the positive clones were screened by G418 ,respectively ;Tumor cells of the three groups at 2 × 106 were respectively injected into the back of BALB/C nude mice to establish the xenograft models .Change of micro-blood vessels in tumor tissue and expression of CD31 were detected by Immunohisto-chemical and HE staining .Immunohisto-chemical assay was used to detect the expression of RI ,MMP-2 ,MMP-9 ,E-cadherin ,N-cadherin ,Vimentin ,Snail ,Slug ,Twist in the tumors .Results Animal experiment showed that the T24-RI cells group significantly inhibited the growth of bladder cancer compared with the other two control groups .Compared with the T24 and T24 vector cells groups ,the microvessel density in tumor tissue of T24-RI group was notably reduced and the expressions of MMP-2 , MMP-9 ,N-cadherin ,Vimentin ,Snail ,Slug ,Twist significantly were decreased simultaneously ,while the expressions of RI and E-cadherin were increased .Conclusion up-regulation RI can inhibit the growth of transplanted bladder cancer in nude mice by decrea-sing the expression of invasion protein and EM T protein .
ABSTRACT
Objective:To identify the expression of egfp-hri fusion on NIH3T3 cells which is carried by recombinant retrovirus. Methods:The egfp-hri fusion gene was integrated intoNIH3T3 cells by the infection of recombinant viral supernatant from the G418 resistent PA317 cells transfected stably with the plasmid of pLNCX-EGFP-C1-hri. The expression effect of egfp-hri fusion was visualized under the fluorescent microscope, while the expression levels of egfp-hri fusion protein by Western blot. Results:The observation from fluorescent microscope showed the green fluorescent was obviously observed in the cytoplasm , western-blotting showed the expression level of egfp-hri fusion gene increased by 83%and 81%, as compared with those uninfected cells and in the cells infected with empty vector, respectively. Conclusion:The infected NIH3T3 cells can express egfp-hri fusion protein successfully.
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Objective:To identify the expression of plasmid pLNCX-EGFP-C1-hri targeting the gene of Human ribonuclease inhibitor (hri) in PA317 cells which is capable of expression in mammalian cells.Methods: The vector of pLNCX-EGFP-C1-hri was transfected into PA317 cells by Lipofectamine 2000 and then the expression of recombinated plasmid was verified in living cells by observing the transcription level of egfp-hri fusion gene mRNA with RT-PCR method and the expression level of egfp-fusion hRI protein with western blotting method respectively.Results: Both RT-PCR and western blotting showed the egfp-hri fusion gene was obviously expression in PA317 cells.Conclusion: The plasmid of pLNCX-EGFP-C1-hri targeting hRI is successfully constructed and the protein of hRI can be expressed in PA317 cells correctly.
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Objective To develop and optimize a new method to extract miRNAs from plasma.Methods miRNAs were extracted from plasma by mixing it with the extraction solution that contained surfactant and by heating .Then the ribonuclease inhibitor was added into the extraction to prevent RNAs from degradation .The expression level of each miRNA was detected by real-time quantitative PCR in oder to evaluate the feasibility of this method .Results A method which extracted miRNAs from plamsa in just one step was established .The specificity , reproducibility and stability of this method have been demonstrated by real-time quantitative PCR .Conclusion The one-step method is simple , inexpensive , and plasma-saving.It seems like a new method for clinical examination of miRNAs from plasma .
ABSTRACT
Purpose:To clone and construct an eukaryotic expressive vector of ribonuclease inhibitor (RI) gene ,as well as to observe the effects of the transfected pLNCX-ri on the growth of C6 glioma cells.Methods:A segment of RI gene of 1.4 kb was obtained by Nde I/Xho digestion and cloned into pLNCX. Transfective agent and selective antibiotic were lipofect AMINE and G418 respectively. The expression of pLNCX-ri in C6 glioma cells was detected by Western blotting. And SD rats were inoculated by the transfected C6 glioma cells.Results:An eukaryotic expressive vector of RI gene was constructed successfully. RI content was remarkably higher in the transfected cells than that of in the untransfected cells. After SD rats were inoculated by the transfected C6 glioma cells,the tumorigenic time was prolonged, the tumor weight was reduced and the density of tumor vessels was notably decreased. Conclusions:These results indicated that RI gene powerfully inhibited the growth of C6 glioma cells via decreasing tumor vessels formation.