Abstract
Purpose
Vocal fold scarring is abnormal scar tissue in the lamina propria layer of the vocal fold. To facilitate investigation of vocal fold scarring, we established and characterized immortalized human vocal fold fibroblast (iHVFF) cell lines.
Methods
Human vocal fold fibroblasts were immortalized by introducing Simian virus 40 large T antigen (SV40TAg) by transfection. Successfully transfected fibroblasts were sorted using flow cytometry. Immunofluorescence cytochemistry and western blot were applied to analyze the expression of fibronectin, vimentin, alpha-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP). Cell proliferation rate was measured by CCK-8 assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA expression level.
Results
The iHVFFs continued to proliferate for more than 30 generations and appeared spindle-shaped. The expression of Vimentin and α-SMA were detected in both iHVFFs and primary fibroblasts, and enhanced expression of FAP was observed in iHVFFs. Furthermore, iHVFFs exhibited an increased proliferative capability compared with the primary fibroblasts. RT-qPCR results suggested that collagen type III alpha 1 chain (COL3A1), interleukin-6, cyclooxygenase 2 (COX2), hyaluronan synthase 2 (HAS2), hepatocyte growth factor (HGF) in the iHVFFs significantly increased, whereas transforming growth factor-β1 (TGF-β1), elastin and matrix metallopeptidase-1 (MMP-1) expression significantly downregulated. No differences in mRNA expression of α-SMA, fibronectin and collagen type I alpha 2 chain (COL1A2) were noted between iHVFFs and primary fibroblasts.
Conclusion
iHVFFs can be used as a novel tool cell for future researches on the mechanisms of pathogenesis and treatment of vocal fold scarring.
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Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
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The authors would like to appreciate all patients who participated in this study.
Funding
Financial support for this project came from the Youth Program of National Natural Science Foundation of China (Grant No. 82102863), the Science and Technology Commission of Shanghai Municipality of China (Grant Nos. 21ZR1480200 and 22ZR1409800), and the Shanghai Municipal Health Bureau (Grant No. 20204Y0055).
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by YC and YF. The first draft of the manuscript was written by YC and JC. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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The study was approved by the ethics committee of the Affiliated Eye and ENT Hospital of Fudan University (No. 2020014-1 and 2021155-1) and performed in line with the principles of the Declaration of Helsinki.
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Chu, Y., Fang, Y., Wu, H. et al. Establishment and characterization of immortalized human vocal fold fibroblast cell lines. Biotechnol Lett 45, 347–355 (2023). https://doi.org/10.1007/s10529-023-03350-6
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DOI: https://doi.org/10.1007/s10529-023-03350-6