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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2015, Vol. 35 Issue (10): 1-12    DOI: 10.13523/j.cb.20151001
    
Enhancement of the Expression and Responsiveness of TRPV1 and TRPV4 Channels on HepG2 Cells with Micropillar Arrayed Substrate Topography
SONG Ming-li1, LIN Yu1, LUO Nan-shu1,3, FENG Quan-yi1,4, HUANG Qi-ping1, ZHANG Hwan-you2, ZHANG Yi-guo1, WU Ze-zhi1
1. Key Laboratory of Biorheological Science and Technology under the State Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China;
2. Institute of Molecular Medicine and Department of Medical Science, National TsingHua University, 101Section2, Kuang-Fu Road Hsinchu 300, Taiwan, China;
3. College of Biology and Environmental Sciences, Jishou University, Jishou 416000, China;
4. School of Life Science, Southwest University, Chongqing 400715, China
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Abstract  

Polydimethylsiloxane (PDMS) micropillar arrayed topographic substrates were fabricated, with dimensions of 4 and 10μm in nominal pillar diameter, 4 and 7μm in spacing and 4μm in height, to investigate the expression and responsiveness of transient receptor potential (TRP) V1 and TRPV4 channels of HepG2 cells cultured on the substrates. Quantitative real-time PCR (qRT-PCR) analysis revealed that the mRNA levels of TRPV1 and TRPV4 were significantly up-regulated in the cells grown on all four topographic substrates, when compared with the cells grown on the flat PDMS substrates. The presence of TRPV1 and TRPV4 proteins in HepG2 cells was confirmed with Western blotting, and similar up-regulation of these two channel proteins by the substrate topography also revealed by stronger immunofluorescence staining. Subsequently, the channel responsiveness of TRPV1 and TRPV4 was quantified by using the calcium inflow-responding magnitudes and percentages of cells having a defined responding magnitude upon stimulation by the channel agonists capsaicin and 4-α-phorbol-12, 13-didecanoate(4αPDD), respectively. Herein, Calcium Green-1 as a fluorescent indicator was employed in its dynamic assessment by confocal laser scanning microscopy. The results displayed that upon stimulation by the channel agonist, the calcium influx through TRPV1 exhibits a dynamic characteristic of rapid desensitization with a transient within 25 seconds, and either the relative fluorescence responding amplitudes or the percentages of responsive cells on the topographic substrates are greater than those observed from the cells on the flat substrates. By contrast, stimulation by the TRPV4 agonist caused a calcium response with much slower recovery within 5 minutes, and both the relative fluorescence responding amplitudes and the percentages of responsive cells on the topographic substrates are higher than those of the cells on the flat substrates. Collectively, these data indicate that the TRPV-mediated ion signaling elicits an important role in the cellular phenotypic and functional regulation by the substrate topography.

Key words： Micropillar arrayed topographic substrates      TRPV1 channels      HepG2 cells      Laser Confocal Scanning Microscopy      TRPV4 channels     
Received: 10 April 2015      Published: 25 October 2015
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SONG Ming-li, LIN Yu, LUO Nan-shu, FENG Quan-yi, HUANG Qi-ping, ZHANG Hwan-you, ZHANG Yi-guo, WU Ze-zhi. Enhancement of the Expression and Responsiveness of TRPV1 and TRPV4 Channels on HepG2 Cells with Micropillar Arrayed Substrate Topography. China Biotechnology, 2015, 35(10): 1-12.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20151001     OR     https://manu60.magtech.com.cn/biotech/Y2015/V35/I10/1

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