Kailing Jiang1, Pengpeng Ma2, Xiaoqun Yang2, Liang Zhong2, Hui Wang2, Xinyu Zhu2, Beizhong Liu1. 1. Central Laboratory, Yongchuan Hospital, Chongqing Medical University, Chongqing 402160, Ministry of Education Key Laboratory of Laboratory Medical Diagnostics, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China. 2. Ministry of Education Key Laboratory of Laboratory Medical Diagnostics, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
Abstract
OBJECTIVE: To establish recombinant adenovirus carrying human neutrophil elastase (NE) gene using AdEasy system, over-express NE in K562 cell line and observe the effects of NE on K562 cell proliferation and apoptosis. METHODS: NE gene was amplified with RNA extracted from acute premyelocytic leukemia (APL) HL-60 cells as a template using reverse transcription-PCR. The coding sequence was cloned into shuttle plasmid pAdTrack-CMV to obtain the recombinant plasmid named pAd-NE. After digested with HindIII and EcoRV and sequenced, the pAd-NE was transformed to competent E.coli BJ5183 containing adenovirus backbone plasmid pAdEasy-1. The obtained recombinant adenovirus plasmid Ad-NE was digested with PacI and transfected into AD293 cells for packaging. Fourteen days later, primary recombinant adenovirus Ad-NE was harvested, and then subjected to five cycles of amplification, titer determination and PCR identification. K562 cells were infected by the recombinant adenovirus. The infection efficiency was observed under a fluorescence microscope and detected by flow cytometry. Western blotting was used to detect NE expression. The proliferation of K562 cells was detected by CCK-8 assay. Cell cycle and apoptosis was measured by annexin V/PI accompanied by flow cytometry. RESULTS: HindIII and EcoRV digestion and sequencing suggested that the recombinant vector Ad-NE was successfully constructed. The recombinant plasmid Ad-NE was packaged in AD293 cells as expected. Following five-cycle amplification, the viral titer was up to 1.64 × 10¹² pfu/mL. GFP expression observed by fluorescence microscopy and flow cytometry implied that the infection efficiency of Ad-NE in K562 cells reached about 80%. Western blotting showed that NE expression was up-regulated in K562 cells. CCK-8 assay revealed that the proliferation of K562 cells over-expressing NE was enhanced. Meanwhile, flow cytometry indicated that the K562 cells were arrested in S phase and the apoptosis rate was highly reduced. CONCLUSION: Over-expressed NE in K562 leukemia cells could promote cell proliferation, inhibit apoptosis and block cell cycle in S phase.
OBJECTIVE: To establish recombinant adenovirus carrying humanneutrophil elastase (NE) gene using AdEasy system, over-express NE in K562 cell line and observe the effects of NE on K562 cell proliferation and apoptosis. METHODS:NE gene was amplified with RNA extracted from acute premyelocytic leukemia (APL) HL-60 cells as a template using reverse transcription-PCR. The coding sequence was cloned into shuttle plasmid pAdTrack-CMV to obtain the recombinant plasmid named pAd-NE. After digested with HindIII and EcoRV and sequenced, the pAd-NE was transformed to competent E.coli BJ5183 containing adenovirus backbone plasmid pAdEasy-1. The obtained recombinant adenovirus plasmid Ad-NE was digested with PacI and transfected into AD293 cells for packaging. Fourteen days later, primary recombinant adenovirus Ad-NE was harvested, and then subjected to five cycles of amplification, titer determination and PCR identification. K562 cells were infected by the recombinant adenovirus. The infection efficiency was observed under a fluorescence microscope and detected by flow cytometry. Western blotting was used to detect NE expression. The proliferation of K562 cells was detected by CCK-8 assay. Cell cycle and apoptosis was measured by annexin V/PI accompanied by flow cytometry. RESULTS: HindIII and EcoRV digestion and sequencing suggested that the recombinant vector Ad-NE was successfully constructed. The recombinant plasmid Ad-NE was packaged in AD293 cells as expected. Following five-cycle amplification, the viral titer was up to 1.64 × 10¹² pfu/mL. GFP expression observed by fluorescence microscopy and flow cytometry implied that the infection efficiency of Ad-NE in K562 cells reached about 80%. Western blotting showed that NE expression was up-regulated in K562 cells. CCK-8 assay revealed that the proliferation of K562 cells over-expressing NE was enhanced. Meanwhile, flow cytometry indicated that the K562 cells were arrested in S phase and the apoptosis rate was highly reduced. CONCLUSION: Over-expressed NE in K562 leukemia cells could promote cell proliferation, inhibit apoptosis and block cell cycle in S phase.