Overexpressed α7 nicotinic acetylcholine receptor inhibited proinflammatory cytokine release in NIH3T3 cells(GENETICS, MOLECULAR BIOLOGY, AND GENE ENGINEERING)
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概要
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Alpha-7 nicotinic acetylcholine receptor (α7nAChR) played an important role during the process of inflammation, and was a potential therapeutic target for many diseases, including Alzheimer's disease, endotoxaemia, hypertension etc. However, it was difficult to express α7nAChR on commonly used host cells because of the lack of the chaperone protein RIC-3, which was indispensable to promote folding, assembly, and surface expression of α7nAChR. This work was designed to develop a cell line, which not only expressed α7nAChR highly and stably, but possessed biological activity. Full-length α7nAChR gene was extracted from rat peritoneal macrophages, recombinant plasmid pIRES2-EGFP-α7nAChR was constructed, and then transfected to the mouse fibroblast cell line NIH3T3, a cell line expressing RIC-3. Compared with the NIH3T3-pIRES2-EGFP, expression of α7nAChR was significantly higher in NIH3T3-pIRES2-EGFP-α7nAChR on both mRNA and protein levels. To evaluate the anti-inflammation activity of the over-expressed α7nAChR, TNF-α, IL-6, and IL-1β release induced by lipopolysaccharide was determined. No changes were found between NIH3T3-pIRES2-EGFP and NIH3T3-pIRES2-EGFP-α7nAChR culture supernatant under LPS challenge, however under nicotine pre-stimulation, pro-inflammatory cytokine release was significantly attenuated in α7nAChR transfected cell line. Moreover, in this established cell line, PNU-282987, a selective α7nAChR agonist, exerted a stronger ability to reduce TNF-α release than nicotine. We concluded that a functional NIH3T3-pIRES2-EGFP-α7nAChR cell line was developed, which might be useful for agonist screen and biological research.
著者
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SHEN Fu-Ming
Department of Pharmacology, School of Pharmacy, Second Military Medical University
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SU Ding-Feng
Department of Pharmacology, School of Pharmacy, Second Military Medical University
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Li Dong-Jie
School of Life Science and Technology, China Pharmaceutical University
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Tang Qiu
Department of Pharmacology, Second Military Medical University
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Duan Jun-Li
Department of Gerontology, Xinhua Hospital, Shanghai Jiaotong University
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Xi Tao
School of Life Science and Technology, China Pharmaceutical University
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Tang Qiu
Department Of Pharmacology Second Military Medical University
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Su Ding-feng
中華人民共和国
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Su Ding-feng
Department Of Pharmacology School Of Pharmacy Second Military Medical University
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Duan Jun-li
Department Of Gerontology Xinhua Hospital Shanghai Jiaotong University
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Li Dong-jie
School Of Life Science And Technology China Pharmaceutical University
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Shen Fu-ming
中華人民共和国
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Shen Fu-ming
Department Of Pharmacology School Of Pharmacy Second Military Medical University
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Xi Tao
School Of Life Science And Technology China Pharmaceutical University
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Shen Fu-ming
Dep. Of Pharmacology School Of Pharmacy Second Military Medical Univ. Chn
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Su Ding-feng
Dep. Of Pharmacology School Of Pharmacy Second Military Medical Univ. Chn
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