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Experimental Study of the Interfering Effect on EJ Cell Line Induced by siRNA Targeting Survivin

Author: YangHuaAn
Tutor: GouXin
School: Chongqing Medical University
Course: Surgery
Keywords: EJ cells survivin Recombinant plasmid Transfection
CLC: R737.14
Type: Master's thesis
Year: 2008
Downloads: 42
Quote: 0
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Abstract


Objective To construct eukaryotic transcription vector pSilencer2.0 for two recombinant survivin gene interference vector (psilencer2.0-SVV1, psilencer2.0-SVV2), and bladder cancer cell lines transfected EJ cells by RNA interference (RNAi) EJ cells blocked the expression of survivin gene screening effective interference suppression suvivin recombinant vectors . And to detect its EJ cell proliferation, cell cycle , apoptosis and apoptosis-related gene expression . Method will be designed , synthesized DNA sense strand and antisense strand denaturation, annealing , to form linear double-stranded plasmid DNA and pSilencer2.0 T4 DNA ligase in order . Recombinant plasmid by digestion , DNA sequencing , the use of liposome transfection EJ cells by RT-PCR, Western blotting interference effect . MTT assay for cell growth and proliferation , flow cytometry cell cycle, apoptosis changes , RT-PCR detection caspase-3, bax and bcl-2 gene expression effects . Results ( 1 ) restriction enzyme digestion and sequencing confirmed that the design of synthetic DNA has been properly inserted into the vector . (2) RT-PCR and western blot analysis showed that : pSilencer2.0-SVV1, pSilencer2.0-SVV2 recombinant vector EJ cells can effectively block the expression of survivin gene , so that survivin gene at the mRNA and protein expression levels significantly reduced (P lt; 0.05), pSilencer2.0-SVV2 suppression better. ( 3 ) EJ cells after transfection significantly slower growth rate , in cell G2 / M phase increase in the number of apoptotic rate enhancement , caspase-3 and bcl-2 gene expression was not significantly change (P gt; 0.05), bax expression significantly increased (P lt; 0.05). Conclusion The recombinant plasmid psilencer2.0-SVV2 bladder cancer cell lines transfected after silencing targeting survivin gene expression , and can promote apoptosis of EJ cells , changing their biological behavior of bladder cancer gene therapy has a certain value .

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Urinary tumors > Bladder tumor
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