Immortalization Of Porcine Oral Mucosal Epithelial Cells And Proliferation Characteristics Of Porcine Circovirus Type 2 And Foot-and-Mouth Disease Virus

Oral mucosal epithelium is not only an important physiological barrier but also a main target or approach for many viruses and bacteria invasion.Normal tissue cells have a limited lifespan.Primary porcine oral mucosa epithelial cells usually die after subcultured12 passages because of senescence.Immortalized cell lines provide significant cell models for study of virus-related diseases in vitro.Porcine circovirus 2?PCV2?is a major etiological agent of porcine circovirus-associated diseases and causes enormous economic losses in swine industry.However,there are currently no suitable cell models to study the cytopathic effects?CPE?of PCV2 in vitro,which severely restricts the study of PCV2pathogenesis.Foot-and-mouth disease virus?FMDV?attacks susceptible animals?particularly cloven-hoofed livestock including pigs,cattle and sheep?,causing fever and painful fluid-filled vesicles?blisters?on mucosa or skin.The lack of suitable animal cell line seriously limits the research on its pathogenesis.In the present study,we established an immortalized porcine oral mucosal epithelial cell line?hTERT-POMEC?by introducing the hTERT gene into primary porcine oral mucosal epithelial cells?POMECs?derived from a neonatal,unsuckled piglet,and a spontaneous immortalized porcine oral mucosal epithelial cell line?Sp-POMECs?was obtained in the process of successive subcultivation of primary POMECs.The basic characteristics of PCV2 or FMDV infection on hTERTPOMECs and Sp-POMECs were preliminarily studied and the results were shown as follow:1.Pure porcine oral mucosal epithelial cells?POMECs?were successfully obtained.Tissue culture method,trypsin digestion method,Dispase?processing incorporating with tissue culture method and Dispase?processing incorporating with trypsin digestion method were used to isolate POMECs,and we found that Dispase?processing incorporating with tissue culture method is the most quick and efficient way to get primary POMECs.Morphology and indirect immunofluorescence assays showed that primary POMECs had a homogeneous paving-stone-like morphology,positively staining pan-CK and negatively staining vimentin.Cell proliferation assay and cell cycle analysis indicated that early passage primary POMECs had good proliferation ability,but they would rapidly undergo replicative senescence after a certain number of divisions.2.An immortalized POMEC cell line?hTERT-POMECs?was successfully established and had been subculured for 150 passages.Eukaryotic expression plasmid pCI-neo-hTERT was transfected by lipidosome into primary POMECs and hTERT-POMECs were obtained through G418 resistance screening.Telomerase activity detection and western blotting assays indicated that hTERT gene was successfully introduced into primary POMECs.Cell proliferation assays and cell cycle analysis suggested that hTERT enabled primary POMECs to have a good proliferate ability.Indirect immunofluorescence assay indicated that hTERT-POMECs had the molecular characteristics of lamellated squamous epithelial cells,positively expressing keratin pan-CK,CK14 and CK13,negatively expressing CK18and vimentin.Karyotype analysis showed that 100^th passage hTERT-POMECs had a diploid chromosome complement of 2n=38,consisting of one pair of sex chromosome?X,Y?and 18 pairs of autosomes and no chromosome abnormality was found.Nude mice assays suggested 80^th passage hTERT-POMECs had no tumorigenicity.3.A spontaneously immortalized POMEC cell line?Sp-POMECs?was established and had been subculured for 150 passages.Continuous subcultured POMECs broke the Hayflick limit and occurred into spontaneous immortalization.Sp-POMECs presented paving-stone-like features,with good proliferating ability and positively expressing pan-CK,CK14 and CK13,negatively expressing CK18 and vimentin.Even after subcultured for 100 passages,Sp-POMECs remained the basic morphological and physiological characteristics of primary POMEC cells.4.Either PCV2 or FMDV could effectively infect and well replicate in porcine oral mucosal epithilial cells,resulting in cytopathic effects and infectious progeny virus.Immunofluorescence or PCR assays indicated that PCV2 could efficiently infect hTERT-POMECs and Sp-POMECs.Quantitative real-time PCR and TCID₅₀ assays suggested that the replication capacity of PCV2 or FMDV in hTERT-POMECs and Sp-POMECs was higher than that in PK-15 cells.In conclusion,primary POMECs were isolated and cultured in vitro and two immortalized POMEC cell line?hTERT-POMECs and Sp-POMECs?were eatablished by hTERT exogenous transfection or successive subcultivation in this study.Virus infection assays showed that PCV2 or FMDV could effectively infect and well replicate in hTERT-POMECs and Sp-POMECs with cytopathic effects.All these results indicated that porcine oral mucosal epithelial cell is a good cell model for studying porcine diseases.hTERT-POMECs and Sp-POMECs could provide important experimental materials for the study of pathogenic mechanism of PCV2 and FMDV.