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《Acta Academiae Medicinae Militaris Tertiae》 2006-21
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Construction of double expression retroviral vectors and its effect on phenotype of K562 cells

ZENG Jian-ming~ 1 , FENG Wen-li~ 1 , WANG Xiao-zhong~ 1 , ZHAO Shi-qiao~ 1 , BAI Wei-jun~ 1 , LUO Yun-ping~ 1 , WEN Jian-ping~ 2 , TU Zhi-guang~ 3 , HUANG Zong-gan~ 4 (~1Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, ~3Department of Clinical Biochemistry, ~4Department of Hematology, The First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China;~2Canadian Blood Services, Hamilton, ON L8S 1H8, Canada)  
Objective To construct double expression retroviral vectors targeting chronic myeloid leukemia (CML) b3a2-type mRNA and investigate its effect on the phenotype of K562 cells. Methods The eGFP coding sequence was inserted into the retroviral vector pMSCV-neo to construct pMSCV/GFP, then H1-RNA pol III-based transcription cassettes was subcloned into it to form pMSCV/GFP-H1-BCR/ABL40AS. Two control vectors pMSCV/GFP-H1-BCR/ABL40S pMSCV/GFP-H1-BCR/ABL80AS were constructed in addition. All these constructions were identified by restriction enzyme analysis and DNA sequencing. After that, the recombinant vectors were transferred into retrovirus packaging cell line PT67 by using lipofectamine2000, and G418 were used to select stable virus-producing cell lines. Viral titer was determined by infection of NIH3T3 cells sequentially. The cell-growth curve was assayed, cell apoptosis was checked with Annexin V-PE/7AAD double staining and flow cytometry analysis after 24-hour infection, the PKR phosphorylation was assayed by Western blotting. Results The plasmids were successfully constructed. Four cell lines, named as PT67-MSCV/GFP, PT67-40as, PT67-40s and PT67-80as were gained by G418 selection, and virus titers were 6.2×10~ 5 , 5.6×10~ 5 , 4.6×10~ 5 and 6.0×10~ 5 CFU/ml respectively. PT67-40as suspensions could induce K562 cell apoptosis by (22.54±3.19)%, significantly different from PT67-MSCV/GFP or PT67-40s (P0.05), except PT67-80as. The level of phosphorylated PKR was only increased in PT67-40as group and PT67-80as group (by 59.20% and 60.33%, respectively). Conclusion The double expression retroviral vectors have been successfully constructed and it can inhibit K562 cell growth and induce cell apoptosis. Therefore, a PKR-activating dependent method may be set up as a new strategy for cancer targeting therapy.
【Fund】: 国家自然科学基金资助项目(30470744)~~
【CateGory Index】: Q78;R733.72
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1 ZENG Jianming1,FENG Wenli1,ZHAO Shiqiao1,et al.(1.Faculty of Laboratory Medicine,Key Laboratory of Laboratory Medical Diagnostics,Ministry of Education,Chongqing Medical University,Chongqing400016,China;2.Canadian Blood Services,Hamilton,ON L8S 1H8,Canada;3.Department of Hematology,The First Affiliated Hospital,Chongqing Medical University,Chongqing400016,China);An experimental study on reconstruction of retrovirus vector in human leukemia K562 cell xenograft tumor of nude Mice[J];Chinese Journal of Clinical Laboratory Science;2008-02
Chinese Journal Full-text Database 1 Hits
1 WANG Xiao zhong, FENG Wen li, SHI Mei, ZENG Jian ming, TU Zhi guang, HUANG Zong gan. Chongqing University of Medical Sciences, Chongqing 400016, ChinaCorresponding author: FENG Wen li;Targeted blockage of STAT5 by a decoy oligodeoxynucleotide inhibits the growth and proliferation of K562 cells[J];Chinese Journal of Hematology;2004-12
【Secondary Citations】
Chinese Journal Full-text Database 1 Hits
1 Chen Gang, Li Jing, Li Fu Jun, Zhou Jian Feng, Lu Yun Ping, Ma DingCancer Biological Medicine Center of Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan, Hubei,430030,P.R.China;Construction of PTTGas Antisense Expression Vector and Its Inhibitory Effects on Human Ovarian Carcinoma Cell Line SK-OV-3[J];Chinese Journal of Cancer;2003-10
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