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| Construction of a Cellular Model for the Study of Autophagy in Human Skin Keratinocytes |
| SHI Ji-hong, HU Da-hai, BAI Xiao-zhi, ZHANG Zhan-feng, ZHU Xiong-xiang, DONG Mao-long, HAN Jun-tao, SU Ying-jun, CAI Wei-xia, ZHU Hua-yu, TANG Chao-wu |
| Xijing Hospital, the Fourth Military Medical University, Xi'an 710032, China |
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Abstract Autophagy has been reported to contribute to wound repair and regeneration, but in vivo, it is infeasible to detect autophagy of human patient's skin. HaCaT cell line is spontaneously immortalized human keratinocytes from human skin. To establish a stable GFP-LC3-expressed HaCaT cell line.The pcDNA3.1-GFP-LC3 plasmid was constructed and transfected into HaCaT cell with transfection reagent.The stable transfectants were screened by G418.The GFP-LC3 protein expression was analyzed by Western blot.The fluorescent signals were detected by inverted fluorescence microscope.Rapamycin-induced autophagy was detected by confocal microscope, Western blot and transmission electronic microscope (TEM).Selected by G418,3 transfected cell lines showed high expression level of GFP-LC3,as demonstrated by Western blot analysis.More than 95% cells showed positive fluorescent signals under inverted fluorescence microscope.The formation of autophagosomes and the increases in the conversion of LC3-I to LC3-II was observed in the constructed cells when treated with Rapamycin.Rapamycin-induced autophagy in HaCaT-LC3 cell line was detected by TEM.A HaCaT cell line stably expressing GFP-LC3(named HaCaT-LC3)was constructed successfully. The HaCaT-LC3 cell line may act as a cellular model to study the autophagy in epidermic cell exactly.
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Received: 10 June 2011
Published: 25 December 2011
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Cite this article:
SHI Ji-hong, HU Da-hai, BAI Xiao-zhi, ZHANG Zhan-feng, ZHU Xiong-xiang, DONG Mao-long, HAN Jun-tao, SU Ying-jun, CAI Wei-xia, ZHU Hua-yu, TANG Chao-wu. Construction of a Cellular Model for the Study of Autophagy in Human Skin Keratinocytes. China Biotechnology, 2011, 31(12): 79-85.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2011/V31/I12/79
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