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《Anhui Medical Journal》 2018-01
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Construction of DDX46 lentiviral vector and its expression identification in human bladder cancer cells

LIU Xi;ZHANG Chong;HUANG Denggao;Department of Central Laboratory,Haikou Hospital Affiliated to Xiangya Medical School of Central South University;  
Objective To construct lentiviral vector of low expression of DDX46 gene and detect its expression in human bladder cancer cells to explore the effect of DDX46 gene on bladder cancer cells. Methods The expression levels of DDX46 m RNA in 5637 cells and T24 cells were detected by real-time fluorescence to find suitable cell lines to go on. DDX46 gene and universal negative sequencewere used as template to design target sequence to synthesize oligonucleotide duplexes and be cloned into plasmid GV115,then the recombinant plasmids sh DDX46 and sh RNA were synthesized,and all of them combined with pHelper 1 and pHelper 2 were transfected into 293 T cells to package lentivirus particles to infect 5637 cells and T24 cells. The DDX46 m RNA expression levels of 5637 cells and T24 cells transfected by recombinant plasnids were detected by real-time fluorescence quantitative PCR to judge the interference efficiency. Results A total of 5637 cells and T24 cells expressed high abundance of DDX46 m RNA,and their results(expressed by delta Ct values) were(6. 53 + 0. 08) and(8. 48 + 0. 11). After recombinant lentiviral infection trasfecting bladder cancer,the expression level(represented by a ΔCt value)(0. 32 ±0. 01) of DDX46 m RNA in the experimental group in 5637 cells was lower than that in the control group(1. 00 ± 0. 10) in 5637 cells,the difference was statistically significant(P 0. 05),and the knockdown rate was 67. 70%; the expression level(represented by a Ct value)(0. 11 ± 0. 01) of DDX46 m RNA in the experimental group in T24 cells was lower than that in the control group(1. 00 ± 0. 03) in T24 cells,the difference was statistically significant(P 0. 05),and the knockdown rate was 89. 00%. Conclusion The lentiviral vector of low expression of DDX46 gene issuccessfully constructed to lay the foundation for studying the role of DDX46 gene in bladder cancer.
【Fund】: 国家自然科学基金(项目编号:81460450);; 海口市重点科技资助项目(项目编号:2012-073);; 海南省自然基金项目(项目编号:813256);; 海南省重点科技项目(项目编号:ZDXM2014076);; 海南省社会发展科技专项(项目编号:SF201409)
【CateGory Index】: R737.14
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