Prolonged down regulation of specific gene expression in nucleus pulposus cell mediated by RNA interference in vitro.
To investigate the efficacies and the longevity of RNA interference in nucleus pulposus cells from rat and human, two reporter luciferase plasmids (Firefly and Renilla) were used. These plasmids were cotransfected with siRNA targeting Firefly luciferase to the nucleus pulposus cells extracted from Sprague Dawley rats and scoliosis patients. The inhibitory effects were evaluated by dual luciferase assay for 3 weeks. Proliferation activity of fibroblast-like cells extracted from the subcutaneous tissue of Sprague Dawley rats and the nucleus pulposus cells were measured by proliferation assay (WST-8 assay) every 2 days after plating. The expression of Firefly luciferase was drastically inhibited both in rats (94.7%) and in humans (93.7%). The inhibitory effects were maintained for 2 weeks and had disappeared completely by 3 weeks. The proliferation activity of nucleus pulposus cells was significantly lower than fibroblast-like cells. We have shown, for the first time, siRNA-mediated gene silencing in rat and human disc cells for a relatively sustained period, probably due to the stability of the nucleus pulposus cells in terms of cell proliferation. The demonstration of this study may allow further exploration of the use of siRNA for scientific research and the treatment of disc degenerative diseases.
Kakutani
Kenichiro
K
Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan.
Nishida
Kotaro
K
Uno
Koki
K
Takada
Toru
T
Shimomura
Takatoshi
T
Maeno
Koichiro
K
Kurosaka
Masahiro
M
Doita
Minoru
M
D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D015536:Down-Regulation; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007403:Intervertebral Disc / Q000201:enzymology* / Q000473:pathology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D034743:RNA-Induced Silencing Complex / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012600:Scoliosis / Q000378:metabolism / Q000473:pathology; D014162:Transfection
D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D049452:Green Fluorescent Proteins; D008156:Luciferases