Establishment of a peritoneal mesothelial cell line from a transgenic rat harbouring the temperature-sensitive simian virus 40 large T-antigen gene.
BACKGROUND
Establishing a peritoneal mesothelial cell (MC) line in which the native characteristics of primary MCs are constantly maintained in vivo is of great significance for investigating their morphological and functional changes in peritoneal dialysis. We established transgenic (Tg) rats that expressed the temperature-sensitive tsA58 mutant of the simian virus 40 large T-antigen (tsSV40T), which served as a source of immortalized rat cell lines. The cells were immortalized at a permissive temperature of 33 degrees C, although they were differentiated at a non-permissive temperature of 38 degrees C. In this study, we established a novel MC line from tsSV40T Tg rats and evaluated its characteristics.


METHODS
MCs were isolated from 8-week-old tsSV40T Tg rats and cloned. MCs from 8-week-old Wistar rats were used as controls. These cells were immunohistochemically and phenotypically evaluated by immunofluorescence, phase contrast and electron microscopy. The production of plasminogen activator inhibitor 1 (PAI-1) from MCs stimulated by tumour necrosis factor-alpha (TNF-alpha) was measured.


RESULTS
The tsSV40T MCs showed a cobblestone-like appearance at 33 and 38 degrees C, which was similar to normal primary cultured MCs. Microvilli-like structures were observed on the cell surface by a scanning electron microscope at 33 and 38 degrees C. Wilms tumour-1 and pancytokeratin, as MC markers, were expressed at 33 and 38 degrees C. Following TNF-alpha stimulation, PAI-1 production of tsSV40T MCs was similar to that of normal primary cultured MCs.


CONCLUSIONS
We established a novel, conditionally immortalized MC line that continuously retained the characteristics of primary cultured peritoneal MCs. This cell line might be a useful tool for various types of in vitro biological research on peritoneal dialysis.
Hotta
Yoko
Y
Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan.
Kaneko
Kayo
K
Inuma
Jiro
J
Inami
Yuko
Y
Aruga
Seiki
S
Shimaoka
Tetsutaro
T
Sekiguchi
Yoshimi
Y
Io
Hiroaki
H
Hamada
Chieko
C
Obinata
Masuo
M
Ueda
Masatsugu
M
Tomino
Yasuhiko
Y
D000818:Animals; D030801:Animals, Genetically Modified; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D049109:Cell Proliferation; D002472:Cell Transformation, Viral / Q000235:genetics / Q000276:immunology; D004247:DNA / Q000096:biosynthesis; D017931:DNA Primers / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D005814:Genes, Viral; D009154:Mutation; D010537:Peritoneum / Q000166:cytology* / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology*; D013696:Temperature; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D012367:RNA, Viral; D014409:Tumor Necrosis Factor-alpha; D004247:DNA