关键词:  , 巩膜；成纤维细胞；永生化；转录组

Abstract:  Objective To establish an immortalized human scleral fibroblast cell line and analyze the effect of immortalization using transcriptome sequencing. Design Experimental study. Participants The experimental group was immortalized human scleral fibroblasts, and the control group was primary human scleral fibroblasts. Methods Lentiviral particles were obtained by transfection of HEK293T using calcium phosphate transfection method using plasmid carrying SV40 T gene and lenti-Mix lentivirus packaging plasmid. The obtained lentiviral particles were transfected with primary cells and exposed to puromycin containing 1 μg/ml for 2 days, and the surviving cells were continued to be cultured for 25 generations to obtain immortalized human scleral fibroblast cell lines. Afterward, total RNA of primary human scleral fibroblasts and immortalized human scleral fibroblasts were extracted and sequenced to analyze the transcript characteristics of immortalized cells and primary cells. Differential genes were analyzed using the DESeq package and gene ontology (GO) analysis was performed using Clusterprofiler.  Main Outcome Measures Cell morphology, collagen-related gene expression and transcript correlation. Results Immortalized human scleral fibroblasts maintained the morphology of the primary cells and showed significantly enhanced proliferation ability and were successfully cultured for more than 20 generations. The transcriptomes of the immortalized and primary cells were highly similar, and the correlation between the two was r2=0.995 obtained by RNA sequencing. The transcriptome yielded 22903 genes and 33691 isoforms, and the eifferential gene analysis based on DESeq package revealed that 2063 (3.6%) genes were up-regulated and 2776 (4.9%) genes were down-regulated. GO functional analysis showed that the up-regulated genes were mainly clustered in DNA replication, cell division and other basic cellular metabolic activities and interactions with viruses, while the down-regulated genes were mainly enriched in extracellular matrix regulation and intercellular junction pathways. As for the extracellular matrix-related gene pathways, transcription of genes encoding collagen and fibronectin was slightly downregulated (log2 fold change=-0.4~-3.1), and COL1A1 transcription was downregulated 3.34-fold. Conclusion In this study, an immortalized human scleral fibroblast cell line was constructed by introducing SV40T exogenous gene using lentiviral transfection method, with high similarity to primary cells and good stability, which can provide sufficient research material for sclera-related experiments. (Ophthalmol CHN, 2023, 32: 387-391)

Key words: sclera, fibroblast, immortalization, transcriptome